1. Arabidopsis seed, development, germination Flashcards
Amen 1968, The New York Botanical Garden
-** Seed dormancy ** is considered as an aspect of growth cessation.
- It is characterized by *partial metabolic arrest *with its inception and termination under endogenous hormonal control
- preserving a potential for growth without loss of biologic integrity
- preserve the capacity for growth while circumventing death
Baskin 2004, Seed Science Research
: A classification system for seed dormancy
- Primary dormancy: A freshly matured dormant seed has primary dormancy. Primary dormancy develops during seed maturation on the mother plant
- Seed development -> induction of primary dormancy -> mature seed -> conditional/relative dormancy -> non-dormancy -> conditional/relative dormancy -> secondary dormancy -> conditional/relative dormancy.
- Conditional/relative dormancy: seed not capable of germinating in as wide a range of physical environmental conditions as is a non-dormant seed
- **Secondary dormancy: **the re-entrance of the non-dormant seed into dormancy
- ABA (produced by the embryo) induces dormancy during seed development, and GA promotes germination of non-dormant seeds.
-Ethylene breaks dormancy and / or stimulates germination in the seeds of many species -
physiological dormancy
- deep (GA does not promote germination, seeds require 3-4months of cold stratification to germinate)
- intermediate (GA promotes germination, requires 2-3 months for cold stratification)
- non-deep (GA promotes germination, cold and warm stratification breaks dormancy)
- **- morphological dormancy **
- embryo is small (underdeveloped)
- need time to grow to full size and then germinate (radical protrusion)
**- morphophysiological dormancy **
- needs time + dormancy-breaking pretreatment
**- physical dormancy **
- caused by one or more water-impermeable layers of palisade cells in the seed or fruit coat
- needs the formation of an opening (‘water gap’) in a specialized anatomical structure on the seed coat, through which water moves to the embryo
**- combinational dormancy **
- physical dormancy + physiological dormancy
- the seed (or fruit) coat is water impermeable and the embryo is physiologically dormant
**- evolution: **
- morphological/morphophysiological dormancies are basal for angiosperms
- physical, physiological, and combinational dormancies are derived.
- physical and combinational dormancies are the most phylogenetically restricted classes of seed dormancy and are the only ones not found in gymnosperms
- physiological dormancy is the most evolutionarily advanced and phylogenetically widespread class or dormancy
- The (low) embryo size to seed size ratio (ES) has increased between ancestral and derived angiosperms (and gymnosperms).
- the underdeveloped embryo (morphological/morphophysiological dormancy) is primitive among angiosperms (and gymnosperms), and that the other classes of dormancy and of non-dormancy are derived conditions. The most primitive class of dormancy is morphological dormancy.
- Seeds of Arabidopsis winter annuals: type1 non-deep physiological dormancy: come out of primary dormancy during the high termperatures of summer and seeds that do not germinate in autumn are induced into secondary dormancy by low temperatures during winter
- seeds of summer annuals, common ragweed, have type 2 non-deep physiological dormancy: come out of dormancy during winter (cold stratification), and seeds that do not germinate (while buried in soil) in spring are induced into secondary dormancy by the increasing temperature of late/spring/early summer.
Bentsink 2008, American Society of Plant Biologists
: Seed Dormancy and Germination
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Seed is the dispersal unit of the plant, which is able to survive the period between seed maturation and the establishment of the next generation as a seedling after it has germinated
- For this survival, the seed, mainly in a dry state, is well equipped to sustain extended periods of unfavourable conditions.
- To optimize germination over time, the seed enters a dormant state.
- dormancy prevents pre-harvest germination as well
-Seed development: has 2 phases - embryo development and seed maturation.- embryogenesis (morphogenesis) - starts with the formation of a single-cell zygote and ends in the heart stage when all embryo structures have been formed.
- followed by a growth phase during which the embryo fills the seed sac
- at the end of embryo growth phase, cell division in the embryo arrests.
- hereafter, the seed, containing a full sized embryo, undergoes maturation during which food reserves accumulate and dormancy and desiccation tolerance develops.
- Dormancy in Arabidopsis: physiologically non-deep, the embryos released from surrounding structures grow normally, and that dormancy is lost through moist chilling (stratification) or after-ripening).
- germination: events that commence with the uptake of water by the quiescent dry seed and terminate with the elongation of the embryonic axis
** 1st signs of germination: ** the resumption of essential processes, including transcription, translation and DNA repair followed by cell-elongation and eventually at the time of radical protrusion, resumption of cell division. - germination (physically) - a two-stage process, where testa rupture is followed by endosperm rupture. Following rupture of the micropylar endosperm by the emerging radicle, germination is complete.
-** Maternal effect:** Since tissues from both maternal (testa) and zygotic origin (embryo and endosperm) contribute to seed germination, genetic analyses of seed dormancy have to take into account these different tissue origins. Maternal effects, in contrast to zygotic factors are maternally inherited and might be due to the genetic make up of the testa surrounding the embryo, but can also be due to genetic differences related to factors that are transported into the seed from the mother plant. Maternal inheritance can be deduced from the germination of seeds obtained after reciprocal crosses, where the parental genotypes are used both as female and as male parent. The endosperm is the product of fertilization. However, the genomic contribution of the female parent is twice that of the male parent in this triploid tissue. - **Seed dormancy in Arabidopsis can be overcome **by germination promoting factors such as after-ripening, light, cold treatment (stratification).
- ABI3, FUS3, and LEC2 genes encode related plant-sepcific transcription factors containing the conserved B3 DNA binding domain whereas LEC1 gene encodes a HAP3 subunit of the CCAAT binding transcription factor. All four abi3, lec1, lec2, and fus3 mutants are severely affected in seed maturation and share some common phenotypes, such as decreased dormancy at maturation and reduced expression of seed storage proteins. Specific phenotypes - the absence of chlorophyll degradation in the dry seed (abi3 mutant), a reduced sensitivity to ABA (abi3 mutant and, to a lesser extent, lec1 mutant ), the accumulation of anthocyanins (fu3, lec1 mutants, and to a lesser extent, lec2 mutant), and intolerance to desiccation (abi3, fus3, and lec1 mutants), or defects in cotyledon identity (lec1, fus3, and lec2 mutants).
- The LEC1 gene is required for normal development during early and late phases of embryogenesis and is sufficient to induce embryonic development in vegetative cells. LOF of LE1 gene leads to germination of excised embryos (between 8 -10 days after pollination).
- LEC2 directly controls a transcriptional program involved in the maturation phase of seed development. Induction of LEC2 activity in seedlings causes rapid accumulation of RNAs normally present primarily during the maturation phase, including seed storage and lipid-body proteins. Promoters of genes encoding these maturation RNAs all possess RY motifs (cis-elements bound by B3 domain transcirption factors).
- LEC1 gene regulate the expression of both ABI3 and FUS3 genes. FUS3 and LEC2 genes act in partially reudundant manner to control gene expression of seed specific proteins, and LEC2 gene was shown to locally regulate FUS3 gene expression in regions of the cotyledons.
- **Fatty acids **are stored in oil bodies as triacylglycerol (TAG), which are hydrolysed by lipases. The released fatty acids are passed to glyoxysomes (a peroxisome in which the glyoxylate cycle occurs) via an ABC transporter encoded by the CTS (comatose) protein. In here beta oxidation takes place converting fatty acids, activated to acyl-CoA esters to acetyl Co-A, which is subsequently converted to four-carbon sugars. These sugars are then transported to the mitochondria from where they are converted to malate and transported to the cytosol for gluconeogenesis or used for respiration. Many mutants of genes of enzymes in beta-oxidation have defects in hypocotyl elongation in darkness and seedling establishement that can be rescued by sugars.
- Sugars arrest early seedling establsihment. some ABA insensitive mutants are sugar insensitive. ABI4 gene encodes an AP2 domain containing TF that binds a CE-1 like element present in many ABA and sugar regulated promoters linked sugar regulation to ABA signalling.
Berger 2006, Current Opinion in Plant Biology
: Endosperm: an integrator of seed growth and development
Mutations in the HAIKU (IKU) **genes decrease endosperm size and eventually embryo and seed size.
- The HAIKU genes IKU2 and MINISEED3 (MINI3) encode a leucine-rich-repeat kinase and a WRKY TF, respectively.
- These genes are expressed in endosperm immediately after fertilization.
- **The decreased endoperm size of iku mutants is accompanies by a decrease cell elongation in the seed integuments, indicating communication between two genetcially distinct seed components. **
- Similarly, reducing the degree of cell elongation in the seed integuments reduces endosperm growth.
- Conversely, increasing the number of seed integument cells causes a symmetrical increase in endosperm growth.
- In Arabidopsis, the **final size of the seed **is determined before the embryo initiates the major phase of cell proliferation after the heart embryo stage
- These results indicate the capacity of seed integuments to regulate endosperm growth by a maternal sporophytic effect
- The central cell as progenitor of the endosperm contributes much cytoplasm to the endosperm.
- The endosperm could inherit maternally derived mRNA and proteins that are located at specific sub-domains in the central cell
- another origin of maternal influence: activation of maternal alleles and silencing of paternal alleles, resulting in maternal genomic imprinting
- i.e., DNA methylation is maintained by the methyltransferase MET1 during vegetative development and male gametogenesis. At the end of female gametogenesis, however, FIS2 gene silencing is relieved in the central cell by the DNA glycosylase DEMETER (DME). Hence, a transcriptionally acitve maternal FIS2 allele is provided to the endosperm whereas the paternal FIS2 allele remains silenced by MET1.
Bewley 1997, The Plant Cell
: Seed Germination and Dormancy
- It may not be advantageous for a seed to germinate freely, even in seemingly favorable conditions.
-For example, germiantion of annuals in the spring allows time for vegetative growth and the subsequent production of offpsring, whereas germination in similar conditions in the fall could lead to the demise of the vegetative plant during the winter. - Seed dormancy is generally an undesirable characteristic in agricultural crops, where rapid germination and growth are required. However, some degree of dormancy is advantageous, at least during seed development. -** This is particularly true for cereal crops because it prevents germination of grains while still on the ear of the parent plant (pre harvest sprouting), a phenomenon that results in major losses to the agricultural industry.**
- Extensive domestication and breeding of crop species have ostensibly removed most dormancy mechanisms present in the seeds of their wild ancestors, although under adverse environmental conditions, dormancy may reappear. By contrast, weed seeds frequently mature with inherent dormancy mechanisms that allow some seeds to persist in the soil for many years before completing germination.
- germination = events that commence with the uptake of water by the quiescent dry seed and terminate with the elongation of the embryonic axis. The visible sign that germination is complete is usually the penetration of the structures surrounding the embryo by the radicle -> visible germination. Then -> mobilization of the major storage reserves during the seedling growth
- dormancy = the failure of an intact viable seed to complete germination under favourable conditions
- radicle extnesion through the structures surrounding the embryo is the event that terminates germination and marks the commencement of seedling gorwth
Daszkowska-Golec 2011, A Journal of Integrative Biology
: Arabidopsis Seed Germination Under Abiotic Stress as a Concert of Action of Phytohormones
- Seed development is divided into embryo and endosperm development and seed maturation.
- Seed maturation is completed when storage compounds have accumulated, water content has decreased, desiccation tolerance has been developed, phytohormones level has been established and, in the case of dormant seeds, primary dormancy have been esablished.
- The Arabidopsis seed is composed of the embryo surrounded by a single layer of endosperm cells and a testa
- After the seed shape and size are determined, germinating seeds start to uptake water in order to set the metabolic events essential for germination in motion
-** This water uptake by seeds is triphasic**
- During phase I, dry seeds absorb water rapidly.
- Initially, imbibition results in the hydration of the cell walls and reserve polymers within the cells.
- During phase II, which is called the plateau because of the stable water content, testa rupture begins.
- Duing phase III of water uptake, the endosperm rupture and a protrusion of radicle is observed. - Differences in seed dormancy between Landsberg erecta (Ler) and Cape verdi (Cvi) led to the generation of a recombinant inbred line population and the identification of DOG1 (DELAY OF GERMINATION) gene, which controls seed dormancy. The transcription of DOG1 gene, which belongs to a small gene family, is eed specific and drops during seed imbibition.
- JA ans SA are negtiave regulators of seed germination
- Auxins are regulators of the seed germination process in a crosstalk with GAs, ABA, and ET.
- The brassinosteroids signal leads to a reduced sensitivity to ABA and stimulates germination
- Salt stress can be explained by two levels: ionic toxicity and osmotic stress
- At the cellular level, salt tolerance mechanisms function to reduce sodium accumulation in the cytoplasm. This is achieved by the active transport of sodium out of the cell through plasma membrane Na+/H+ antiporters, that is, SOS1 (Salt Oversensitivite1) or addressing it into the vacuole by tonoplast antiporter NHX1.
- In order to cope with dehydration and osmotic stresses, plant have evolved complicated machinery to synthesize and accumulate metabolites (osmoprotectants) which help to withstand osmotic pressure and maintain turgor and the driving gradient for water uptake.
- Osmoprotectants include low molecular weight compounds such as amino acids, polyols, sugars, and methylamines.
- These compatible metabolites stabilize the enzyme structure, cellular membranes, and other cellular components during any exposure to sress.
- In response to stress, de novo synthesis of dehyrins, osmotins, and LEA (Late Embryogenesis Abundant) proteins occurs.
- Osmotins and dehydrins stabilize the integrity of the cellular membranes and protein structure, whereas LEA are able to sequestrate ion and water in order to protect cellular components against stress. - ABA and its role in response to abiotic stress during seed germination
- ABI3 - (B3), ABI4 - (APETALA2), ABI4 - (bZIP); insensitive to ABA, defects in response to glucose, NaCl during germination and seedling growth
- ABI3 and ABI4 genes are expressed from globular stage embryogenesis and their products can regulate ABI5 gene expression, which is activated at the heart stage
Debeaujon 2000, Plant Physiology
1. : Influence of the Testa on Seed Dormancy, Germination, and Longevity in Arabidopsis
- At maturity, seeds consist of a whitish embryo surrounded by a hyaline layer of remaining central endosperm and a single layer of peripheral endosperm cells (aleurone layer) containing storage reserves and associated with brown seed coat or testa
- The seed coat derives from ovular tissue and is therefore a maternal origin.
- The aleurone layer of mature seeds is physiologically active, in contrast to the testa layers, whose cells died during late seed maturation after having encountered considerable developmental changes
- Mature Arabidopsis seeds exhibit primary dormancy when freshly released from the mother plant, which means that seeds are unable to germinate under the appropriate envrionmental conditions without the help of dormancy-breaking agenets such as stratification, after-ripening, or gibberellins.
- Germination begins with the uptake of water by dry seed and ends with the elongation of the embryonic axis.
- The visible consequence of germination is the protrusion of the radicle tip through the seed envelops
- In wheat, the strongest dormancy is associated with a red coat color, whereas the lines with white seed coats are non-dormant or weakly dormant and therefore are susceptible to pre-harvest sprouting damage
- Arabidopsis seed coat is composed of a mucilaginous epidermal layer, a palisade layer with thickened tangential walls, and a pigmented inner layer
- The brown pigmenets of wild-type (WT) Arabidopsis seeds are mainly condensed tannins of the procyanidin type and derivatives of the flavonol quercetin, which are end-products of the flavonoid biosynthetic pathway
- ## One group, affected in flavonoid pigmentation, is represented by the transparent testa (tt) and transparent testa glabra (ttg) mutants
Dekkers 2013, Plant Physiology
: Transcriptional Dynamics of Two Seed Compartments with Opposing Roles in Arabidopsis Seed Germiantion
- Seeds are important in the plant life cycle, since they reppresent the link between two successive generations.
- They are stress-resistant structures that help to bridge unfavorable periods and allow dispersal
- Seed formation starts with a double fertiliation event, and it takes about 20 d to form a mature dry seed.
- At maturity, three major seed compartments can be distinguished: the testa (seed coat), a dead tissue that forms a protective outer layer; the endosperm, a single cell layer of tissue positioned directly underneath the testa; and the embryo (enclosed by the testa and endosperm), which emerges to become the fugure plant.
- A dry seed is a unique structure in the sense that it allows severe dehydration (desiccation tolerance) adn enters a phase of quescence, bring processed occurring in “living” organisms to a halt wihtout affecting viability
- Upon imbibition of water, the dry mature seed swells and metabolic activity resumes, marking the start of seed germiantion and the end of the quiescent state
- Arabidopsis germination consists of two sequential events
- 1st, the testa splits (testa rupture) due to underlying expansion of the endosperm and embryo
- increase in embryo growth potential leading to the elongation of the proximal embyronic axis (hypocotyl and radicle) that overcomes the restratin tof the covering tissue - 2nd, the radicle (embryonic root) protrudes through the endosperm, completing germination
- the weakeneign of these covering layers (includign the micropylar endosperm, positioned over the radicle tip) to ease the protrusion of radicle.
Fang 2012, The Plant Journal
: Maternal control of seed sizez by EOD3/CYP78A6 gene in *Arabidopsis thaliana *
- Seedlings of large-seeded plants are better able to tolerate many of the stresses encountered during seedling establishment, whereas small-seeded plants are considered to have superior colonization abilities because they produce large numbers of seeds
- In angiosperms, seed development invovles a double-fertilization process in which one sperm nucleus fuses with the egg to produce the diploid embryo, whereas the other sperm nucleus fuses with two polar nuclei to form the triploid endosperm
- The seed coat differentiates after fertiliastion from maternally derived integuments
- The embryo is surrounded by the endosperm, which, in turn, is enclosed within the maternal seed coat
- IKU, IKU2, and MINI3 genes function int he same pathway to promote endosperm growth in Arabidopsis
- Arabidopsis TRANSPARENT TESTA GLABRA (TTG2) promotes the seed growth by increasing ecll expansion in the integuments
Finch-Savage 2006, New Phytologist
: Seed dormancy and the tcontrol of germination
- Germination commences with the uptake of water by imbibition by the dry seed, followed by embryo expansion.
- The uptake of water is triphasic with a rapid initial uptake (phase I, i.e., imbibition) followed by a plateau phase (phase II).
- A further increase in water uptake (phase III) occurs as the embyro axis elongates and breaks through the covering layers to ocmplete germination
- In typica angiosperm seeds, the embryo is surrounded by two covering layer: the endosperm and testa (seed coat)
- Cell elongation is necessary and is generally accepted to be sufficient for the completion of raidcle protrusion
- dormancy: a characteristic of the seed that determines the conditiosn required fro germination
- Once primary dormancy is lost in response to prevailing environmental conditions, secondary dormancy will soon start to be induced if the conditions required to terminate dormancy and induce germination are absent (light and/or nitrate). Secondary dormancy can be lsot and re-introduced repeatedly as seasons change until the required germination conditions become available.
- A classification system for seed dormancy
- Physiological dormancy (PD) is the most abundant form and is found in seeds of gymnosperms and all major angiosperm clade. I
- It is the msot prevalent dormancy form temperate seeed banks
- PD deep: Embryos excised from these seeds either do not grow or will produce abnormal seedligns; GA treatment does not break their dormancy; and several months of cold or warm stratification are required before germiantion can take place
- PD non-deep: Embyros excised from these seeds produce normal seedlings; GA treatment can break this dormancy; dormancy can be broken by scarification, after-ripening in dry storage, and cold or warm stratification - Morphological dormancy (MD) is evident in seeds with embryos that are underdeveloped (in terms of size), but differentiated (into cotyledons and hypocotyl-radical). These embryos are not physiologically dormant, but simply need time to grow and germinate; i.e., celery (Apium)
- Morphophysiological dormancy (MPD) is evident in seeds with underdeveloped embryos, but in addition they have a physiological component to their dormancy. These seeds therefore require a dormancy-breaking treatment, for example a defined combination of warm and/or cold stratification which in some cases can be replaced by GA application.
- Physical dormancy (PY) is caused by water-impermeable layers of palisade cells in the seed or fruit coat that control water movement. Mechanical or chemical scarification can break PY dormancy
- Combinational dormancy (PY + PD) is evident in seeds with water-impermeable coatas (as in PY) combined with physioloigcal embryo dormancy
- In angiosperm phylogeny, ‘embryo to seed’ (E:S) values increases.
- in mature seeds of primitive angiosperms a small embryo is embedded in abudnat endosperm tissue. Such seed types prevail among basal angiosperms
- The general evolutionary trend within the higher angiosperms is via the LA seed type (embro linear axile and developed, endosperm abudnance medium to high) towards the FA seed types (embryo foliate axile developed, often storage cotyeldonss, endosperm abudnace low or endosperm obliterated) with storage cotyledons.
- MD is thoguht to be the ancestral dormancy type among seed plants and is the most primitie dormancy class.
- The dispersal of seeds with an underdeveloped embryo that need time to grow mgiht have evovled as an ancient strtegy to disperse germination over time.
- MD and MPD are typical not only for primitive angiosperms but also for primitie gymnosperms sucha s Ginkgoaceae.
- Evolution of larger embro size in MD seeds reulsted in non-dormant (ND) seeds.
- Concurrently, the gain of physioloigcal dormancy mechanisms(s) led from seeds with MD to seeds with MPD, which upon gain in embryo size led to PD seeds
- PD is the most phyologenetically widespread dormancy class.
- The most phyologenetically restircted and derived dormancy classes are PY and PY + PD
- The occurence of an impermeable seed or fruit coat combined with a ND embryo (PY) or a PD (PY + PD) is probably an adaptation to specialized life strategies or habitats.
- PY and PY + PD are the only dormancy classes not found in gymosperms
- ABA induces dormancy during maturation, and GA plays a key role in dormancy release and in the promotion germiantion
- ABA:GA ratio, not the absolute hormone contents, that controls germiantion
- While dormancy maintenenace also depneds on high ABA:GA ratios, dormancy release invovles a net shift to increased GA biosynthesis and ABA degradation resulting in low ABA:GA ratios
-winter annual: germination in the autumn
- summer annual: germiatnion in the spring
-
Garcia 2003, Plant Physiology
: Arabidopsis *haiku *mutants reveal new controls of seed size by endosperm
- in flowering platns, the two female gametes, the egg cell and the central cell, are fertilized by one of the two male gametes delivered by the poleen tube.
- The zygotic pdorudct of the fusion of one male gamete with the egg cell develops into the embryo of the daughter plant
- The fertilized central cell develops as the endosperm that nurtures emnryo development.
- In most species, endosperm development is initiated by a proliferative syncytial phase accompanied by cell growth that generates a large multinucleate cell.
- This syncytium is partioned into individual cells by a specific type of cytokinesis called cellularization
- Because the embyro is surrounded by the endosperm, which, in turn, is enclosed within the ovule integument, these three structures must coordinate their development to produce a mature seed of the appropriate size
Garcia 2005, The Plant Cell
: Maternal control of integument cell elongation and zygotic control of endosperm growht are coordinated to determine seed size in Arabidopsis
-2 distinct phases of seed development
1st: the active proliferation and grwoth of the endosperm -> a large increase of size
2nd: growth of the embryo occurs during the second phase at the expense of the endosperm
- the final seed size is mainly attained during the initial phase
- During early endosperm development, nuclear division is not followed by cytokinesis and produces a synctium
- the mulinucleate endosperm cell enlarges as pseudosyncrhonous nuclear division take place until the 8th mitotic cycle when cellulariation begins.
- Increase in size of the syncytial endosperm is prevented by haiku (iku) mutations, leading to precocious endosperm cellularization, reduced embryo proliferation, and decreased seed size
- Although iku gene mutations affect primarily endosperm growth, the overall seed size is decreased, including the size of the integment from the reduction of cell elongation in integuments
Iwasaki 2022, Annual Review of Plant Biology
: Parental and Environmental Control of Seed Dormancy in *Arabidopsis thaliana *
- Plant terrestrialization, the evolutionary process that produced a phototrophic lineage on land from algal ancestors, started around 540 million years ago (Mya) and changed the Earth’s landscape while exerting a profound influence on animal evolution and human history
- Seeds, produced by gymnosperms and angiosperms, initiated their evolution ~350 Mya (Late Devonian).
- During the Cretaceous (~140 Mya), angiosperm spread reapidly around the Earth and underwent an astonishing diversification
- **Unlinke in nonseed plants, the female gametophyte of seed-bearing plants is physically associated with the mother plant by means of a specialized tissue, the ovule, where it is protected and nourished. **
- the male gametophyte (pollen) protects the male gametes while enabling the fertilization of distant female gametes
- **Unlike nonseed plants, the pollen tube delivers the male gametes directly to the female gametes, thus avoiding the need for external water for fertilization **
- Seed arise from the fertilized ovule, which harbors and protects the developing embryo
- Upon completion of embryogeneis, emnryos undergo a maturation program in which they enter a highly resistant, metabolically inert, desiccated state.
- **Seed longevity is illustrated by a 2,000-year-old Judean date palm seed, unburied from the palace of Herod the Great in Israel, that sprouted and produced Methuselah, a male palm tree that produces functional pollen **
- Thus, seed-bearing land plants achieved less-hazardous reproduction, expanded the physical range where fertilization could take palce, and enclosed their embyros in highly resilient capsules, enabling colonization of new habitats.
- Seeds can be dormant, a trait whereby the embyro-to-seedlign transition is withheld even under favorable conditions
- dormancy provides seeds with an opportunity to be dispersed away from their mother plant, influencing a given species’ distribution while enabling plants to experiment with a wider variety of environments, thus promoting diversification.
- In addition, dormancy is important for seasonal plant behavior and thus influences the environment in whcih other plant traits are expressed- Once isolated from their mother plants, seed start losing dormancy in their dry state, a poorly understood process referred to as dry after-ripening, or else lose their dormancy after exposure to certain environmental conditions, such as imbibition under cold temperatures (cold stratification).
- In Arabidopsis, the unfertilized ovule consists of a seven-celled female gametophyte, of the *Polygonum *type present in approximately 70 % of angiosperms, that is completely surrounded by inner and outer integuments except for the micropylar openeing.
- The pollen tube enters the micropylar opening and delivers two sperm cells in close proximity to the female gametes: the haploid egg and homodiploid central cells.
- Gamete fusion, which produces the diploid zygote and triploid endosperm, initiates seed development by triggering specific developmental programs of the three genetically distinct tissues composing the seed: integuments, endosperm, and embryo.
- The maternal integuments differentiate and and die to produce the seed coat of the mature seed. The endosperm proliferates alongside the embryo and serves as a nourishing tissue for the embyro.
- Endosperm is gradually consumed by the embryo after embryogenesis as the embryo enters the maturation hase, where it accumulates nutrients, expands in size, and eventually desiccates.
- In the mature seed, food is stored mainly in the embryo, whereas the endosperm persists as a single layer of cells surrounding the embryo.
- Seed dormancy is establisehd during maturation, and repression of germination of dormant seeds upon imbibition critically require the endosperm.
- The maternal seed coat plays an importnat role
- The inner integument layer 1 accumulates proanthocyanidins (tannins), a type of flavonoid, and transparent testa (tt) mutants, deficient in proanthocyanidin synthesis, have low dormancy.
- Tannins are antioxidants, and their absence in the seed coat likely promotes the releaes of dormancy by accelerating oxidation in seeds or by increasing seed permeability to oxygen.
- Accordingly tt mutant seeds also have low seed viability
- Furthermore, the inner integument layer 1 produces a cuticle and tannic cell walls tightly associated with the endosperm that limit seed oxidation and promote dormancy
- High ABA levels first appear in seeds at the onset of seed maturation and are largely maternal in origin.
- Maternal ABA is gradually replaced by ABA synthesized by zygotic tissues as maturation progresses, and this zygote-derived ABA is more important for maintenance of primary dormancy before and after imbibition
- ABA levels fall upon dry seed imbibition irrespective of seed dormancy levels; however, repression of seed germination in imbibed dormant seeds is due to sustained high ABA accumulation over time.
- ABA accumulatio upon imibition activates ABA signaling to block seed germination and maintain the embryonic state
- This process includes stimulation of the expression of ABI3 and ABI5, which encode a B3 transcription factor and a basic leucine-zipper (bZIP) transcription factor, respectively, promoting the seed maturation protective program.
- Notably, it also leads to expression of LEA genes, which encode osmotolerance proteins, and the blockade of food store consumption in the embryo by blocking triacylglycerol catabolism.
- ABA signaling invovles the PYR/PYL/RCAR family of ABA receptors, group A type 2C protein phosphatases (PP2Cs), and Snf1-related protein kinases, group 2 (SnRK2s).
- ABA signals by binding to PYR/PYL/RCAR, enabling the sequestration of PP2Cs by direct interation with them.
- In turn, this sequestration enables SnRK2 activation through autophosphorylation, as SnRK2s are inhibited by PP2C-dependent dephosphorylation in the absence of ABA.
- Activated SnRK2s phosphorylate downstream targets, such as ABI3, that repress seed germination
- ABA synthesized by both the endosperm and the embyro contribute to repress seed germination.
- However, in the Arabidopsis mature seed, the endosperm is essential to enable seed dormancy because its removal triggers the growth of the embyro even in the most dormant accessions.
- Upon dormant seed imbibition, the endosperm releases ABA toward the embryo.
Joosen 2010, The Plant Journal
: GERMINATOR: a software package for high-throughput scoring and curve fitting of Arabidopsis seed germination
Completion of germination is defined as the protrusionof the radicle through the endosperm and seed coat.
- The uptake of water of dry seed durign imbibition is triphasic and consists of a rapid initial uptake (phase I) followed by a plateau phase (phase II) and a further increase in water uptake (phase III).
- During phase II the embyro axis elongates and breaks through the testa.
- In Arabidopsis the testa is dead tissue whereas the endosperm layer is living tissue
Koornneef 2002, Current Opinion in Plant Biology
: Seed dormancy and germination
Quntitative trait loci (QTL) analysis for seed dormancy requires permanent or immortal mapping populations, such as recombinant imbred lines (RILs), because these allow the testing of a large number of genetically identical seeds (i.e., seed sfrom the same RIL) in different environmental conditions.
- QTL analyssis of seed dormancy has been reported for Arabidopsis, barley, rice, and wheat.
- It appears that QTL identified for wheat co-locate with barely QTL but not with rice QTL.
- Wild species often show stronger dormancy than cultivated genotypes, making crosses between wild and cultivated genotypes useful for QTL analysis.
- QTL analysis can be followed by the study of individual genes (or chormosome regions) containing specific dormancy QTL and by fine mapping.
-
Kucera 2005, Seed Science Research
: Plant hormone interactions during seed dormancy release and germination
Germination commences with the uptake of water by imbibitionof the dry seed, followed by embryo expansion.
- This usually culminates in rupture of the covering layers and emergence of the radicle, generally considered as the complettion of the germination process
- Radicle protrusion at the completion of seed germination depends on embryo growth driven by water uptake
- Uptake of water by a seed is triphasic, with a rapid intial uptake (phase I, i.e., imbibition) followed by a plataeu phase (phase II).
- A further increase in water uptake (phase III) occurs only when germination is completed, as the embryo axis elongates and breaks through its covering structures.
- RGL2 mRNA decline occured after radicle emergence (after gemriantion had been completed).
Li 1997, Trends in Plant Science
: Genetic and molecular control of seed dormancy
Seeds shed from the mother plant in a dormant state are in primary dormancy
-induced dormancy in mature, partially or fully after-ripened (nondormant) seeds is termed secondary dormancy
- The transition of many seeds from a dormant to nondormant state (after-ripeneing) is accomplished by exposing the seed for a period of time to specific environmental conditions
- Wheat is an allo-hexaploid (2n = 6x = 42). As in barely, a predictable and adequate level of seed dormancy in wheat varieties is beneficial because it prevents preharvest sprouting.
- Dormancy in white-kernelled wheat is a quantitative trait
- Two recombinant-inbred-line populations of white-kernelled wheat were used for QTL analyses to identify the genomic regions associated with resistance to preharvest sprouting
- Eight loci (four from each population) were signficantly associated with resisatnce to preharvest sprouting.
- These loci accounted for 44 % (NY6432-18 x Clark’s Cream - both non-dormant varities) of the genetic variance for preharvest sprouting.
Li 2015, Journal of Experimental Botany
: Maternal control of seed size in plants
- Large seeds accumulate sufficient nourishing substances for germination and have better tolerance to abiotic stresses, whereas small seeds are efficient at dispersing and colonizing
- In crops, seeds are the major products for consumption, and seed size is one of the most important traits of seed yield
- Since the beginning of agriculture, crop plants have undergone selection for larger seed size during domestication
- Seed develpment begins with double fertilization, which leads to the development of the embryo and the endosperm
- One of the two sperm cells fuses with the egg cell to form the diploid zygote
- The zygote undergoes elongation and a progressive transition to establish the basic embryo pattern
- The embryo possesses a shoot meristem, cotyledon(s), hypocotyl, root, and root meristem
- The other sperm cell fertilizes the diploid central cellt to generate the triploid endosperm
- Endosperm development in flowering plants progresses through four phases: syncytial, cellularization, differentiation, and death
- In monocots such as rice (Oryza sativa) and wheat (Triticum aestivum), the endosperm constitues the major part of the mature seed
- In most dicots such as Arabidopsis thaliana and Brassica napus, the endosperm grows rapidly initially and is eventually consumed in later stages
- Thus, the embryo occupies most of ht emature seed
- The maternal integuments surroudnging the developing embryo and endosperm form the seed coat after fertilization
- Integument primordia initiate from the flanks of the chalaza during the early stage of megasporegenesis
- These two primordia grow and enclose the functional megagametophyte and become the inner and outer integuments, respectively
- After fertilizastion, the integuments undergo cell differentiation, accumulate pigments, mucilage, and starch granules, and eventually form mature seed coat
- Therefore, seed development is influenced by the co-ordinated growth of the diploid embryo, the triploid endosperm, and the maternal integuments
- Growth of plant seeds up to their species-specific size is predominantly determined by the internal developmental signals from maternal sporophytic and zygotic tissues, although the growth of seeds is affected by environmnetal cues
- During the early stages of seed development, the endosperm grows faster than the embryo, and the increase of seed volume is in concordance with the growth of the endosperm
- Several factors that regulate endosperm growth have been identified to control seed size in Arabidopsis
- For example, HAIKU (IKU1), IKU2, and MINISEED3 (MINI3) function in the same genetic pathway to promote endosperm growth
- SHORT HYPOCOTYL UNDER BLUE1 (SHB1) binds to the promotoers of MINI3 and IKU2 and regualtes their transcription to promote endosperm growth
- Loss-of-function mutants in these genes formed small seeds
- The small seed size phenotype of these mutatns is determined by the genoype of zygotic tissues rather than the genotype of maternal tissues, indiciating that these genes act zygotically to regualte seed grwoth
- The maternal integuments surrouding the ovule form the seed coat after fertilization, which provides the cavity for the growth of the embryo and the endosperm.
- The seed coat has been proposed to set an upper limit to the final seed size
**Maternal control of seed size by the ubiquitin pathway ** - Ubiquitin is a conserved 76 amino acid protein that is covalentaly attached to target proteins by the sequential action of three enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3).
- The Arabidopsis da1-1 (DA means large in Chinese) mutant produced large leaves, flowers, and seeds
- Recipirocal crossing experiments showed that seeds produced by a da1-1 mother plant, regardless of the gentoype of the pollen donor, are consistentantly larger than those produced by maternal wild-type plants, indicating taht DA1 acts maternally to control seed size
- DA1 regulates seed growth by limiting cell proliferation in the maternal integuments of developing ovules and seeds
- DA1 encodes a ubiquitin receptor with two ubiquitin-interacting motifs (UIMs) and a single zinc-binidng LIM domain.
- UIM domains of DA1 have ubiquitin-binding activity in vitro
- UIM-containing proteins are characterized by coupled ubiquitin binding and ubiquitination, which usually result in monoubiquitination of the ubiquitin receptors.
- The monoubiquitinated receptors are active forms and initiate signal cascades.
- DA2 (E3 ubiquitin ligase) act materanlly to restrict cell proliferation in the integuments of ovules and developing seeds, indiciating that they may negatively affect the level or the activity of positive factors by the proteasomal degradation pathway
- ubiquitin reeptor DA1 and the E3 ligase DA2 form a complex to mediate the degratation of their substrates.
- Likely that DA2 interacts with DA1 to help DA1 recognize the specific substrate(s) for proteasomal degradation.
**- Maternal control of seed size by transcription factors ** - The Arabidopsis AINTEGUMENTA (ANT) gene was originally identified as a regulator of integument growth.
- The ant mutants showed defects in integument initiation and ovule development
- *ANT *encodes an APETALA2-like TF.
- Further analysis revelaed that ANT plays important roles in organ growth control
- The ant mutants exhibited small leaves and flowers due to decreases in cell number, whereas plants overexpressing ANT produced large leaves and flowers by promoting cell proliferation
- APETALA2 (AP2) is a member of the AP2/EREBP (Ethylene Responsive Element Binding Protein) family of transcription factors.
- AP2 plays an importnat role in the specification of floral organ identity in *Arabidopsis *
- Loss of function of AP2 resulted in increased seed size and reduced fertility in Arabidopsis, and suppresion of AP2 activity in transgenic plants increased seed size
- Seeds produced by the ap2 mutant plants are larger than wild-type seeds, regardless of the genotype of the pollen donor, indicating that AP2 acts maternally to control seed size
- ## In the ap2 mutants, the cell size in the outer integument was increased, and the cell shape was irregular, suggesting that AP2 might control seed size by limiting cell elongation in the maternal integuments
Luo 2005, PNAS
: MINISEED3 (MINI3), a WRKY family gene, and HAIKU2 (IKU2), a leucine-rich repeat *(LRR) KINASE *gene, are regulators of seed size in *Arabidopsis *
MINI3 promoter::GUS fusions show the gene is expressed in pollen and in the developing endosperm from the two nuclei stage at ~ 12 hr postfertilizastion to endosperm cellularization at ~96 hur.
- MINI3 is also expressed in the globular embryo but not in the late heart stage of embryo development
- Seed development invovles a complex of processes, includign the expansion and growth of the maternal integuments of the ovule and the development of the diploid zygote after the union of the maternal egg cell with one of the two sperm cells delivered to the embryo sac by the pollen tube.
- It also involves the development of a triploid endosperm after the union of the two nuclei of the homodiploid central cell of the embryo sac with the second sperm cell
- In eudicots such as Arabidopsis, endosperm development progresses through phases of synctial growth, cellularization, and cell death
- The syncytial phase is characterized by successive division of the triploid nuclei without cytokineses
- The endosperm cytoplasm is intially compartmentalized into nuclear cytoplasmic domains, and subsequently cellularization occurs after the eighth round of syncital mitoses, intiallly in the region surrounding the embryo, and proceeding toward the chalazal region.
- Viable seed formation results from the integrated growth and development of the genetically diverse integument, embryo, and endosperm tissues
- Major seed controls are provided by genes that define the development of the maternal integument and the new-generation embryo and endosperm
- A number of mutation shave been described that impair integument development, and genes disrupting embryo pattern formation have also been described.
- Endosperm development controls are represented by the FIS loci, MEA, FIS2, and FIE, as well as MSI1.
- These genes conde for proteins that are components of a chromatin-associated polycomb complex that prevents endosperm development before double fertilization
- In both monocots and idcots, when the relative dosage of maternal and paternal genomes is perturbed, the endosperm and seed size are affected
- Arabidopsis, a diploid plant pollinated with tetraploid pollen, produces large seeds.
- This cross generates tetraploid endosperm (2 maternal: 2 paternal) with paternal genome excess rather than the normal triploid endosperm produced by diploid parents
- A different endosperm and seed result is generated when a tetraploid plant is pollinated with diploid pollen generating maternal genomic excess (4 maternal: 1 paternal), and the pentaploid endosperm results in smaller seeds than normal.
- Apart from the ploidy level and parental genome representation, endosperm development is also subject do differetial expression of many genes that depends on their parent of origin.
- At the genetic level, Garcia described two mutants in Arabidopsis, haiku1 (iku1) and *haiku2 (iku2), whcih shows a recessive sporophytic mode of action causing reduction in endosperm growth accompanied by precocious cellularization and reduced seed size.
- Combinations of the iku mutants with the transparent testa glabra 2* (ttg2) mutant, which has defective seed integuments, cause even greater reduction of seed size, indicating the integument and endosperm growth are both regulators of seed size.
O’Neill 2019; The Plant Journal
: The onset of embryo maturation in *Arabidopsis *is determined by its developmental stage and does not depend on endosperm cellularization
- The fertilization of the female gametohyte leads to the fomration of two tissues: the embryo and the endosperm
- The development of the embryo is usually divded into two phases, morphogenesis and maturation
- Morphogenesis, lasting until the late heart or torpedo stages, establishes the basic body plan of the embryo
- Embryo maturation in turn, leads to a dry, resilient, nutrient-filled seed
- This latter process was a key innovation leading to the evolutionary success of the angiosperms.
- During early maturation, also known as ‘seed filling’, the embryos turn green and accumulate storage products which, in the case of Arabidopsis and realted oilseed plants, are proteins (cruciferins/12S globulins and arabins/2S albumins) and oils.
- Late maturation invovles the loss of water (desiccation) and the establishment of a dormant state
- Early maturation in Arabidopsis emrbyos becomes first apparent around the early heart stage
- At this time, chloroplast development and accumulation of chlorophyll start in the protoderm and then expand centripetally.
- Color becomes apparent at the late heart stage
- In oilseeds like Arabidopsis, photosynthesis is necessary for the accumulation of storage lipids and therefore appears to be an integral part of the seed maturation process.
- The onset of seed filling itself is first evidenced by the expression of the genes encoding the seed sotrage proteins (SSPs), enzymes invovled in the synthesis of storage lipids, and proteins associated with oil bodies. This occurs at the late heart to early torpedo stages
- L1L , LEC1, and *ABI3 *gene exspression start very early, followed by LEC2 at the early/midglobular stage, and FUS3 at the late globular/early heart stage
- LOF mutations in the LAFL genes lead to the reduction or elimination of SSPs and lipids.
- These mutants have significant defects in later maturation processes such as growth arrest, desiccation tolerance and dormancy
- The hormone ABA synthesized both in fruit and the embryo has been proposed to be a maternal signal that triggers early maturation
- A hypothetical signal for maturation that is endogenous to the seed - a redistribution of sugars resulting from the cellularization of the endosperm
- Studies in legumes led to the proposal that early seeds have a high hexose/sucrose ratio that promotes embryo cell division, while a later shift to low hexose/sucrose ratio below a certain threshold (‘sugar switch’) triggers maturation and accumulation of storage produts.
- Only about half of plant spceis have cellularizing endosperm, but the sugar switch was nonetheless proposed as a general mechanism for the control of maturation.
- In Arabidopsis the endosperm develops first as a syncytium, and contains a large central vacuole
- This vacuole has been suggested to serve as a sink for sucrose and accumulates high levels of hexoses.
- The endosperm starts cellularizing at the early heart stage, consuming the vacuole in the process and transforming the embryo into the main sucrose sink, lowering its hexose/sucrose ratio.
- This infusion of sucrose is necessary for embryo growth and overall viability
- The lack of significant additive effects in double and triple mutants shows that this process (embryo greening), like others regulated by the LAFL genes, is controlled by these products acting together int he same pathway
- LEC2 expression does not appear in the embryo proper at detectable levels until the early-to-mid-globular stages and FUS3 expression not until the late globular to early heart stages.
- Endosperm cellularization is ilikely to be required for embryonic cell division and growth, since all mutants that fail to cellularize the endosperm show embryo arrest. Endosperm cellularization is not required for the onset of maturation.
- The final size of the embryo (and seed) appears to be influenced by the timing of endosperm cellularization, by the proliferation and expansion of the seed coat, and by interactions between all these tissues and the embryo. The timing of maturation, conversely appears to be regulated mostly by the LAFL genes.
Orozco-Arroyo 2015, Plant Reprod
: Networks controlling seed size in *Arabidopsis *
-Seed development starts after a double-fertilization event:
- During the first event, the zygotic embryo is generated by the fusion of the egg cell and one sperm cell.
- The second fertiliazation event, which triggers the development of the triploid endosperm, starts with the fusion of the central cell of the embryo sac with the second pollen sperm cell
- The two biparentally derived fertilization products (the embryo and the endosperm) are encased by the maternal sporophyte tissue (the seed coat), which is derived from the ovule integuments (seed coat).
- The seed coat represents a protective layer that prevents damage from external factors such as UV radiation, toxic chemicals, and pathogenes, as well as impeding germination until conditions are favorable
- In Arabidopsis, final seed size is mainly attained during the rapid proliferation of the endosperm and the seed coat cells.
- These events span from fertilizastion to 6 days after pollination (DAP) of seed development.
- From 7 to 13 DAP, there is a residual increase in seed volume occurring when the embryo expands at the expense of the endosperm.
- At hthis point, seed growth is limited by the seed coat that acts as a constrainign physical barrier.
- Endosperm development has four phases: synctial, cellularizsation, differentiation, and death.
- The syncytial phase is characterized by a series of divisions of the triploid nuclei without cytokinesis and parallels the maximal phase of seed growth
- After eight rounds of syncytial mitoses, the cellularization process starts, initially from regions surrounding the embryo and proceeding toward the chalazal region.
- Cellularization is folowed by a differentiation of functional tissues, and eventually most endosperm cells die during seed maturation.
- The timing of endosperm cellularizaion correlates with the end of the main stage of seed growing; therefore, the size attained by the endosperm syncytium appears to be a major determinant of seed size.
- Consequently, precocious endosperm cellularizastion results in small seeds, while delayed endosperm cellularization causes the formation of enlarged seeds.
- 3 pathways that control endosperm cellularization
- 1st pathway - AP2 and MADS-box TF AGL62
- 2nd pathway:- Polycomb group (PcG) proteins and their targets
- 3rd pathway - IKU pathway
-1st pathway - AP2 encodes the plant-specific family of TFs that contain AP2/EREBP (ethylene-responsive element binding protein) DNA-binding domain.
- ap2 mutant seeds undergo an extended endosperm proliferation stage, associated with a delay in cellularization
- Additioanlly, the abnormal endosperm development in ap2 mutants resulted in other seed defects, such as enlarged embryos, that show increased cell number and cell size
- AP2 has also been associated with seed coat development and integument-endosperm cross talk
- the agl62 mutants have precocious endosperm cellularization and a small seed phenotype, while increased AGL62 expression correlates with a delay or a complete absence of cellularization
- 2nd pathway: Genomic imporinting and parent-of-origin effects
In plants, genomci imprinting has been observed parimarily in the endosperm and rarely on the embryo
- Imprinting of a specific allele depends on the presence of an epigenetic mark on the corresponding locus
- It has been proposed that imprinted genes regulate the transfer of nutrients from sporophyte to the developing progeny.
- Maternally expressed genes (MEGs) function to equally allocate nutrients to all seeds, while on the other hand, paternally expressed genes (PEGs) function as growth factors that allow their own offspring to extract the maximum amount of nutrients from the mother
- Therefore, increased PEGs activation determines the formation of larger seeds
- Epigenetic modifications performed on genetically identic alleles lead to parent-of-origin specific expression.
- Of particular importance is the balance of methylation between maternal and paternal alleles in the central cell
- Removal of DNA methylation relies on the enzymatic activity of DEMETER (DME) and DNA methylation depends on the enzyme DNA methyltransferase 1 (MET1).
- DME is exprssed in the central cell in embryo sac and in the vegetative cell of the pollen grain.
- This leads to specific DNA hypomethylation of the maternally inherited genome
- Altering DNA methylation in a parental-specific manner via MET1 resulted in variation in seed size
- When crossing MET1::RNAi pistilas with wild-type pollen, the result is production of enlarged F1 seeds.
- Meanwhile, reciprocal corsses generated smaller F1 seeds, as expected from the presence of hypometylated paternal genome
- Thus, the methylation status of both the maternal and paternal genome directly influences seed size
- The 2nd major mechanism invovled in imporinted expresseion of a subset of genes relies on PcG proteins.
- pcG proteins are pivotal regulators of cell ideneity that act as transcriptional repressors in multimeric complexes
- Among these, the PRC2-complex catalyzes the trimethylation of histone H3 on lysine 27 (H3K27me3) and has been implicated in controlling endosperm development.
- Specifically, the FIS-PRC2(fertilization-independent seed-Polycomb repressive complex), which comprises MEDEA (MEA), FERTILIZATION INDEPENDENT SEED 2 (FIS2), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) AND MULTICOPY OF SUPPRESOR OF IRA1 (MIS1), acts in the cnetral cell of the female gametophyte and in the endosperm, targeting DNA hypomethylation sites.
- The FIS-PRC2 mainly represses the expression of maternally inherited (and hypomethylated) alleles.
- Seeds with mutations in mea, fis2, or fie2 show endosperm proliferation even in the absence of fertilization, but also prolonged endospermal proliferation and absent or delayed cellularization if fertilization occurs.
- The phenotypes of these mutants imply that PRC2 complexes promote fast endosperm differentiation after fertilization, thus directly acting on a pathway that greatly influences seed size
-3rd pathway: IKU pathway - HAIKU1 (IKU1) and IKU2 have been shown to be key regulators of seed size in Arabidopsis via control of the transition from syncytial phase to the cellularization phase of the endosperm.
-* IKU1* encodes a protein containing a VQ motif, while IKU2 encodes a leucine-rich repeat kinase.
- iku1 or iku2 mutant plants show reduced proliferation of the endosperm, as well as precocious cellularization process, leading to reduced seed size
- MINISEED3 (MINI3), a WRKY TF that regulates endopserm cellularization process
- mini3 mutant platns phenocopy iku1 and iku2 small seed phenotypes, due to precocious cellularization of the endosperm
- In addition, the small seed phenotype of mini3 mutant is ascribable to reduced cell expansion in the seed coat and reduced cell proliferation that results in a smaller embyro compared with wild type
- IKU1 regulating both MINI3 and IKU2, and MINI3 regulating *IKU2
- MINI3 could positively regulate IKU2* by binding to the putative W-box identified in the *IKU2 *promoter.
- Seed size of the double mutants iku2-1 mini3-1 is similar to the seed size of homozygous mutant alleles of each single locus
- The Arabidopsis seed coat derives from the ovule integuments, formed by a set of five cell layers in mature ovules.
- Two cell layers derive from the outer integument (oi) and three from the inner one (ii).
- The outer integument consists of two cell layers (oi1 and oi2), and the inner integuments consists of three cell layers (ii1, ii1’, and ii2).
- The innermost layer of the inner integument, ii1, named the endothelium, is in direct contact with the endosperm cells
- The seed coat deeply influences seed size, highlighting a fundamental role of seed matneral tissues in teh control of this aspect of seed yield.
- The seed cavity (space enclosed by the seed coat) increases in volume after fertilisation, partly due to the independent developmental plan of the seed coat and partly as the result of the interplay between the seed coat and partly as the result of the interplay between the seed coat and the endosperm
- After fertilization, the cells belonging to the differnet seed coat layers predominantly experiment intense expansion activity but still undergo division activity
- Both cell division and expansion cease at 6 DAP
Factors controlling integuments cell proliferation
- A key player in the control of cell cycle and expansion in Arabidopsis is AUXIN RESPONSE FACTOR 2 (ARF2), which encodes a B3-type TF of the ARF family.
- ARF genes take part in auxin-related responses and recognize specifci AuxRE (auxin response elements) consensus elements on target genes.
- ARF2 is thought to act as transcriptional repressor, exercising a negative control over cell proliferation and expansion
- *arf2 *loss-of-function mutants exhibit abnormal flower morphology and enlarged seeds in comparison with the wild type, a phenotype characterized in detail in the case of arf2-9, which presented more cells in the seed coat compared with wild-type seeds.
- Another negative regulator of cell division is the TF AP2,
- The increased cell proliferation observed in *ap2 *is under materanl control and affects both the seed coat and the endosperm.
Factors controlling integuments cell elongation - A reduction in cell elongation is observed in the LOF mutant transparent testa GLABRA 2 (TTG2).
-In the *ttg2 *mutant, cell elognation in the integuments is affected, possibly because of the increased physical constraint of the cell walls, or possibly because of disruption of the developmental pathways for elongation - Endosperm development is also affected, probably as a consequence of the defects in integument cells.
- Developing seeds produced by the double mutant* ttg2* iku2 display extremely reudced size in comparison with the single mutants *ttg2 *and iku2 seeds.
- The combination of ttg2 and *iku2 *mutations prevents integument cell elongation and growth of the endosperm more severely than in each single mutant
- The doubel homozygous mutant displays a cumulative phenotype combining the maternal effects of* ttg2* with the endospermal effect of *iku2 *
- The additive reduction in integument cell division and elongation, endosperm grwoth and seed size when iku2 and ttg2 mutations are combined, indicateas that each mutation acts in distinct genetic pathways, but has common effectros.
- *TTG2 *would modulat ethe competence of the integument cells to elongate via a maternal integument elongation-dependent pathway.
-**
Orozco-Arroyo 2015 #2, Plant Reprod
: Networks controlling seed size in *Arabidopsis *
Penfield 2017, Current Biology
: Seed dormancy and germination
- dormancy can be imposed by the formation of a simple physical barrier around the seed through which gas exhance and the passage of water are prevented.
- Seeds featuring this so-called ‘physical dormancy’ often require either scarification or passage through an animal gut (replete with its associated diggestive enzymes) to disrupt the barrier and permit germination
- In other types of seeds with ‘morphological dormancy’ the embryo remains under-developed at maturity and a dormant phase exists as the embryo continues its growth post-shedding, eventually breaking through the surrounding tissues.
- By far, the majority of seeds exhibit ‘physiological dormancy’ - a quiescence program initiated by either the embryo or the surrounding endoserpm tissues.
- Physiological dormancy uses germination-inhibiting hormones to prevent germination int he absence of the specific environmental triggers that promote germination.
- During and after germination, early seedling growth is supported by catabolism of stored reserves of protein, oil, or starch accumulated during seed maturation.
- These reserves support cell expansion, chloroplast development and root growth until photoauxotrophic growth can be resumed
- The phenomenon of seed dormancy is best understood at three levels.
- At the population level, seed dormancy enables the formation of a soil seed bank from which plants can emerge at different times of year or in response to ecosystem disturbances
- At the single-plant level, individual mothers have evovled mechanisms for maintaining control of progeny seed-germination bejaviour and generating heterogeneity in progeny seed properties.
- This enables mothers to hedge their bets by producign seeds with different propensities to germinate or that are likely to germinate at different times and palces.
- Finally, at the level of the individual progeny seed, mechanisms exist to maintain and break dormancy, often in response to environmental stimuli that limit germination to specific annual time windows, or enable seeds to wait for gap sin the canopy to appear
Control of seed dormancy and germination by the mother:
Although it is tempting to think of germiantion as a process that starts and ends after seed shedding, in reality, many of the most important aspects of seed germination behaviour are determined during seed maturation and are closely linked to the control of seed dispersal.
In plants the mother benefits from diversity in seed properties.
She favours wider dispersal of progeny, spreading individuals between safer zones closer to the motehr herself but also to increasingly risky locations ever further away.
Increasing disperal has the duel benefits of discovering new locations suitable for plant reproduction and reducing resource conflict between individual progeny seeds.
However, the fact that the chances of successful reproduction decreases with distance of germination from the mother plant creats a conflict among individual progeny seeds whoa re best served by avodiing the fate of being included int eh most risky and sacrificial cohort.
This optimum maternal strategy therefore contrasts with the optimum strategy for any one individual seed, challenge in the design and execution of experiments analysing germination behaviour.
- Seeds can be sensitive even to 1 C changes in temperature, and can use natural noisy environmental-temperature variation as an importnat way to generate diversity in germination propensity in seeds on the same inflorescence.
- In this way, the mother produces cohorts of seeds with varying strategies, for immediate germination or to popualte the soil seed bank
- Several mechanisms through which maternal signals control seed properties
- Firstly, they affect the developmental and metabolic processes in teh fruit- and seed-coat tissues during seed development and maturation, resulting in changes to fruit morphology (affecting disperal), seed-coat thickness and the deposition of seed-coat polymers such as tannin and suberin that in turn affect the depth of seed dormancy.
- Secondly, through specific regulation of the maternal copies of genes in the endosperm, the mother can control gene expression patterns in the endosperm during seed maturation and even after seed shedding and imbibition, affecting dormancy loss, storage-rpotein breakdonw and germination rate.
The induction of dormancy during seed maturation
-When embryogenesis is complete, the maturation programme commences and the seed begins to accumualte reserves of carbon and nitrogen that will fuel seedling establishment after germinatino
- During the maturation phase, seeds of many species also acquire the means to survive desiccation, and enter primary dormancy.
- In the dormant state, the cells lack hydrated vacuoles, do not break down stored reserves and the cell cycle is suppressed.
- This developmental state is promoted by a group of B3-family transcription factors known from Arabidopsis as LAFL. This same group is also responsible for the homeotic conversion of leaves to cotyledons, and when any of these genes are compromised desiccation tolerance is reudced or absent, dormancy is lost, and accumumulation of storage reserves is incomplete.
- A key function of the LAFL genes in dormancy imposition is the induction of a high abscisic acid (ABA) state, which is necessary for the prevention of germiantion-related processes.
- If ABA signalling in developing seeds is insufficient, they can begin to germinate on the mother platn before shedding, a properting known as vivipary.
- Vivipary is common in major crops that have been bred for vigorous stand establishment, as is worse in envrionmental conditions that lead to low dormancy, suh as warn, wet conditions.
- In cereals, this vivipary is known as pre-harvest sprouting, and is accompanies by premature preoduction of alpha amylase and a sharp reudction in grain quality.
- Pre-harest sprouting can be a major probelm for cereal production in areas o fth world with higher humidity and rainfall.
- The mechanism of dry after-ripening is unclear, but after imbibition a signal specific to after-ripneed seeds activates ABA catabolism
**Functions of different seed tissues during decision to germination **
- In cereal seeds, it is especially clear that during germination GA is produced by the embryo, and its arrival in the aleruone layer of the endosperm initiates the breakdown and mobilization of stored endospermic carbon reserves.
- Metabolic acviation, vacuolizastion, and reserve mobilizastion of endosperm cells are the first visible signs that a seed has transitioned to germination
- These events are followed by expansion of embryo cells that lead to testa rupture, endosperm rupture, and radicle emergence.
- In Arabidopsis, the root apical meristem appears to be an important site of GA production in the embryo
- GA-derived signal moves from the root to activate water uptake and metabolism in teh basal cells of the hypocotyl
- The subsequent expansion of hypocoyl cells driven by the GA-dependent regulation of homeodomain TFs, and is the principle cellular event in the embryo that provides the mechanical force for radicle emergence.
- At this time, ribosome synthesis increases and an apparent tranlsational block is released, allowing translation of the key proteins at the heart of basic metabolic processses and cell growth
- The endosperm also reponds by secreting cell wall-loosening enzymes from the micropylar pole that weaken the attachemnts between micropylar endosperm cells to faciliate radicle emergence.
- All these events are under the inhibitory control of ABA in dormant seeds and are promoted by GA in germinating seeds.
-
Sohn 2021, International Journal of Molecular Sciences
: Seed Dormancy and Pre-Harvest Sprouting in Rice - An Updated Overview
- PYR-like/ regulatory PYR-like / AB receptor components are found in seeds and vegetative organs, and they internalize and regulate protein phosphatase 2C when ABA is present
- As protein phosphatase 2C in inactive, this permits SNF-related kinase-2 to become activated, whihc then causes SNF1-related response elements to bind to their promoter regions.
- DELAY OF GERMINATIO-1 (DOG-1) is a master regulator of primary dormancy taht acts in concert with ABA to dealy germination
- DOG-1 boosts ABA signaling thorugh interacting with the protein phosphatase 2C, where DOG-1 inhibits its activity to elevate ABA sensitivity and imposes primary dormancy
- Even though the shorter dormancy period is believed to have enhanced the commercial productivity of cereals such as *O. sativa, H. vulgare, T. aestivum, Z. mays, *the rapid germination percentage has led to pre-harvest sprouting in places with more rainfall, leading to economic consequences.
- Pre-harvest sprouting, which occurs when embryos with less or no dormancy are exposed to external variables (a rain event) before harvest and germination on the spikeletes, is an important evolving problem that impacts the enduse quality among several cereals
- One of the most common methods of describing pre-harvest sprouting in model species and crops is the disruption of primary dormancy.
- In many crops, the absecence of dormancy has resulted in lower productivity because seeds germinate too early before harvest.
- Due to the excessive rainfall during grain maturation, pre-harvest sprouting is widespread in rice, expecially in southwest Asian countries
Sun 2010, Current Opinion in Plant Biology
: Transcriptional and hormonal signaling control of Arabidopsis seed development
- In angiosperms, a double-fertilizastion event leads to the fomration of a diploid embryo and a triploid endosperm.
- In Arabidopsis and many dicots, seed development undergoes an initial phase of active endosperm proliferation followed by a second phase in which embryo grows to full size and replaces most of the endosperm volume at its maturity
- Since the seed coat and endoseprm growth in Arabidopsis preceds embryo growth, the major volume of the mature seeds is largely attained before the enlargement of the embryo.
- Therefore, the seed size is coordinately regulated by the growth of the triploid endosperm, the diploid maternal ovule, and the diploid embryo
- In angiosperms, a double-fertilization event leads to the fomration of a diploid embyro and a triploid endosperm.
- The endosperm arises from the central cell that contains two identical haploid genomes, and constitutes the major volume of the mature seed in monocots and some idcots.
- However, seed development is marked by two distinct phases in Arabidopsis and many other dicots, and the embryo grows to full size and replaces most of the endosperm volume at its maturity
- In the first phase, a rapid proliferation and expansion of the endosperm occurs to generate a large multinucleated cell dfrined as the syncytial phase and results in a large increase in the size or volume of a seed cavity
- This syncytium is then partitioned into individual cells by a speicific type of cytokinesis called cellularizastion.
- Cellularization is first initiated in the micropylar pole and occurs after the eighth mitotic cycle in the peripheral endosperm
- By contrast, the posterior pole of the endosperm does not udnergo cellularizsation and contains a multinucleated pool of cytoplasm called chalazal endosperm cyst
- The chalazal endosperm cyst is located just above the placentaochalazal area of the seed integument whereas vascular elements terminate and is importnat for the transfer of maternal nutrients to the developing seed
- In the first phase, the initial endosperm development and enlargement may affect seed size by enforcing a space limitation
- Accordingly, endosperm may initiate a signal to regulate the subsequent embryo development or the initial endosperm cavity may simply impose a physical restriction for subsequent embryo enlargement
- In the second phase, embryo growth takes place at the expense of endosperm and endosperm is essential to provide nutrient supplies for embryo development
- At maturity, the seed contains only a single layer of endosperm cells in Arabidopsis, and the maternal integument ultimately becomes the seed coat
- Whether the embryo passively grows larger when a cooresponding space is available or an additional set of regulatory mechanism exists to regulate cell proliferation and expansion of the embryo at the post-endosperm stage still remians largely unknown.
- Since the seed coat and endosperm growth in Arabidopsis precedes embryo growth, the seed retains almost its final size before the enlargement of the embryo
- Therefore, the seed size is determined by the coordinated growth of the triploid endosperm, the diploid maternal ovule, and the diploid embryo
Regulation of endosperm development by AP2 and MADS-box TF
The original apetala2 or ap2 mutant alleles were isolated for their disturbed meristem and floral organ identity phenotypes
AP2 encodes a plant specific TF and AP2 has a DNA binding motif of 68 amino acids named AP2 domain.
- Different ap2 mutant alleles all maternally set large seeds and show an increase in both embryo cell number and cell size
- In ap2 mutant, the seed coat epidermal cells are longer, the endosperm central vacuole is larger, and the cellularization process is delayed and prolonged to result in a larger embryo sac
- Interestingly, embryo development is initially slower in ap2 mutant at 8 - 9 days after pollination, and embryo then grows for a longer period to finally fill up the enlarged embryo sac
- Seeds of ap2 mutants accumulate more seed oil and proteins, and more hexose and less sucrose
- Sucrose i the major form of translocating carbohydrate from source to sink tissue
- The overconsumed scurose may account for the increased carbon accumulation in the enlarged ap2 seeds
- MADS-box gene family is divided into five functional clades from A to E and AGAMOUS is the first MADS-box gene cloned in Arabidopsis for its homeotic transformation of floral organs.
- AGL61 was identified as MADS-box protein that is expressed in central cell and endosperm
- The AGL61/80 heterodimers probably regulate the expression of their target genes invovled in central cell development
- YEH also identified another AGL80 interactor, AGL62
- In agl62 mutant, the endosperm has less nuclei, undergoes early cellularization and collapse of seed structure
- The function of AGL62 could be interpreted as promoting nuclear proliferation and suppressing cellularization during early endosperm developlment
Regulation of endosperm development by WRKY transcriptioanl factor and IKU pathway
- mutations in either HAIKU2 (IKU2) or MINISEED3 (MINI3) reduce seed size, and the mutant seed phenotypes depend on the genotype of the embryo and endosperm but not on the genotype of the maternal ovule
- MINI3 encodes a WRKY family TF, and is not expressed in the unfertilized ovule but is expressed after fertilzation in both the endosperm and the embryo
- The mini3 mutant is marked with a precocious cellularization of the endosperm, a possible cause of the final smaller seed size
- Two haiku (iku) mutations cause a similar phenotype as mini3, including early endosperm cellularization, reduced proliferation of endosperm, and reduced embryo development.
- IKU2 encodes a leucine-rich repeat (LRR) receptor kinase, and is expressed in the endosperm but not in the embryo or elsewhere of the plant
- The expression of both MINI3 and IKU2 was decreased in the iku1-1 mutant
Epigenetic regulation of endoserpm development and paternal imprinting
A large group of polycomb (PcG) protines are involved in endosperm development and include FERTILIZATION INDEPENDENT SEED 2 (FIS2), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE/FIS3), MEDEA (MEA/FIS1), MULTICOPY SUPPRESOR OF IRA (MSI1), AND SWINGER (SWN). The 5 proteins from polycomb repressive compelxes that suppress gene expression through histone methylation
- In female gametophyte, proper FIS complex function even before fertilization plays on importnat role in regulating seed development after fertilizastion
- In the corresponding mutants, the female gametophytes initiate endosperm overproliferation without fertilziation but lacks endosperm cellularization.
- After fertilizastion, theri embryo development is arrested and the mutatn embryo displays different defects in cell proliferation and morphogenesis
- AGL61 is one of the candidate genes that mediate FIS protein function
- AGL62 expression drops right before endosperm cellularization and in fis mutants, its expression is consistent until seeds collapse and in fis mutatns, its expression is consistent until seeds collapse.
- Since AGL62 has been described to suppress endosperm cellularizaion, the misexpression of AGL62 in fis mutants may account for the cellularziation phentoype of the fis mutants.
- The paternal imprinting of MEA and FIS2 is mediated by METHYL TRANSFERASE1.
- The MET1 is a homolog of mammalian DNA methyltransferase Dnmt1 and maintains CpG DNA methyaltion in Arabidopsis
- The human homolog of MET1 directly interacts with polycomb protein complex in vivo.
- In Arabidopsis, loss of MET1 activity causes a genome wide DNA hypomethyaltion at CpG dinucleotides
- When the maternal genome carries the met1 mutation, larger seeds were produced
- By contrast, smaller seeds were produced when the parternal genome carries the met1 mutation
- The smaller seed phenotype caused by paternal hypometylation was probably due to an early cellularizsation of the endosperm, whereas the larger seed phenotype by maternal hypomethyaltion was a result of a delayed endosperm cellularization and a larger endosperm volume
Regulation of integument or seed coat development by WRKY TF
TTG2 (TRANSPARENT TESTA GLABRA 2) encodes a WRKY family transcription factor and is mainly expressed in the endosperm and the integument
-ttg2 mutant has smaller seeds, which is correlated with a precocious endosperm cellularization, a smaller endosperm, and inhibition of cell elongation in the integument.
The IKU pathway directly controls the size of endosperm, and might trigger the elongation of the integument cells to coordinate the growth of the endosperm.
-By contrast, TTG2 might directly control cell elongation in the integument to shape the size of the endosperm
Regulation of embryo prolfieration by bHLH TFs
- RETARDED GROWTH OF EMBRYO (RGE1) is a member of the basic helix-loop-helix (bHLH) gene family, some of which are known to function as TFs.
- The emgryo of the LOF RGE1 mutant shows retarded emrbyo growth after the heart stage but with normal morphogenesis and pattern formation to result in small and shriveled seeds.
- By contrast, its endosperm development is normal
Verdier 2008, Plant Cell Physiol
: Transcriptional regulation of storage protein synthesis during dicotyledon seed filling
- Sequence comparison of the SSP rpomoters in combination with DNA-protein binding assays revealed two major conserved factor-binding sties, the RY/G motif and the B-box, which act in synergy
- RY/G motifs are composed of two RY elements (CATGCA), with which B3 domain proteins interact, and a G-box (CACGTG), which binds basic zipper (bZIP) or basic helix-loop-helix (bHLH) TFs, and is responsible for the ABA response
- The B-box is composed of DistB (GCCACTTGTC) and ProxB (CAAACACC) elements and mediates a strong ABA response in seeds
- The B-box constitues an ABA response element (ABRE) domain whose elements interact with various factors such as bZIP or MYB
- AtbZIP10 and AtbZIP25 are co-expressed during seed filling with Arabidopsis SSPs, but are also expressed, at highe rlevels, in vegetative tissues.
- These bZIPs bidn to the G-box, and both interact with the B2 domain of ABI3 in two-hybrid assays
- LEC1 encodes a protein with sequence similarity to the HAP3 subunit of CCAAT-binding factors (CBF or NF-Y factors). In plants, CBF is a hetertrimer composed of HAP2, HAP3, and HAP5 subunits.
- In Arabidopsis, the HAP3 subunit genes can be divided into the LEC1-type HAP3 subunits and the non-LEC1-type.
- LEC1- and non-LEC1-types differ by 16 amino acid residues that serve as signatures of their conserved domains
- LEC2 is a member of the B3 domain-containing TF family, closely related to FUS3 and ABI3.
- ## It is also capable of activating FUS3 and ABI3, and of directly binding RY motifs to activate SSp genes
Verma 2022, Planta
: Transcriptional control of Arabidopsis seed development
In most angiospemrs, the seed is an outcome of a double fertilization process, in which one of the two sperm nuclei fuses with the egg cell to produce a diploid zygote, and the second sperm nucleus fuses with the binucleate central cell to generate the triploid endosperm
-Subsequently, the single cellular zygote undergoes highly coordinated cell divisions and cellular differentiation to develop into a multicellular embryo in a process termed embryogenesis
- In many dicots, including Arabidopsis, the endosperm develops as a coenocyte (series of mitosis without cytokinesis) followed by endosperm cellularization
- Later, in Arabidopsis, the endosperm is absorbed in part by the growing embryo.
- The seed coat is developed from the ovule’s integuments and consists of five cell layers.
- Two cell layers are derived from the outer integuments (OI): OI2 and OI1, and three cell layers are derived from the innter integuments (II): II2, II1’ and II1.
- During the development of the seed, cells of the outermost layer, i.e., OI2, produce mucilage which accumulates specifically at the outer conrners of the cell
- Besides, both outer integument cell layers (OI1 and OI2) produce starch granules, falvanols, and suberin (a lipophilic polymer).
- In contrast, cells of the innermost layer accumulate proanthocyanidins (PAs) which later oxidize and give the characteristic brown colour to the seed coat.
- In the end, the mature seed contains a filial embryo, a single-cell endosperm layer, and a protective covering, i.e., a seed coat developed from the ovule’s integuments
- Overall, embryo development can be classified into two distinct phases, (i) morphogenesis and (ii) maturation
- The morphogenesis phase begins immediately after fertilization and lasts until the late-heart stage.
- This phase is characterized by highly coordianted cell divisions and differentation, during which the basic body organization of the embryo is established
- By contrast, cell division and proliferation are ceased during embryo maturation.
- However, cell expansion occurs along with the accumulation of seed storage reserves such as carbohydrates, proteins, and fatty acids.
- Embyro maturation partially overlaps with the morphogeneiss phase.
- It begins around the early-heart stage and lasts until the seed is fille dwith nutrients, becomes dry, and acquires a dormant state (e.g., seed maturation).
- It has been observed that the developmental stage, not the time elpased since fertilization, determines the onset of maturation
- Seed devleopment depends on the spatiotemporal expression of various genes invovled in different processes occurring during this period, including cell division, differentaition, seed filling, desiccation, and dormancy
- Therefore, control of spatiotemporal patttern and expression levels of such genes is crucial, which results from the regulation of their transcription by different TFs.
- TFs are critical regulatory proteins that act in a combinaotorial manner together with other proteins to orchestrate this transcriptional regulation
- They work through binding to specific DNA sequences (cis-regulatory elements) present over the promoters of their target genes.
- These regulatory proteins are themselves regulated by otehr TFs, hormones, thereby creating multiple layers and networks of regulation to control a particular developmental aspect
Transcriptional control of early embryogenesis (morphogenesis)
Embryogenesis begins when the zygote undergoes an assymetric division that generates a small apical cell and a large basal cell
- The apical cell gives rise to the spherical embryo proper (proembryo) that will create most of the mature embryo
- Two rounds of longitutinal and one round of transverse divisions convert the apical cell into an eight-celled embryo.
- In this stage, two domains, termed the upper tier and lower tier, are distinguished
- The basal cell divides transversely and gives rise to a 7 - 9 celled filamentous suspensor
- Later, the uppermost suspensor cell is specificed as hypophysis, which ultimately protrudes into the embryo.
- The upper tier gives rise to the cotyledons and shoot apical meristem (SAM)
- The lower tier and hypolysis generate the cotyledon’s abaxial part, hypocotyl, root apical meristem (RAM), and embryonic root.
- After a periclinal division, a 16-celled embryo results from the first visiable cell differentation whehn the outermost layer is specified as protoderm
- Subsequent divisions give the embryo a globular appearance
- At this stage, precursor cells of ground tissue and vascular tissue are specified.
- Later, with the development of cotyledon primordia, the embryo takes the shape of a heart
- At this stage, SAm is established, cotyledons are formed, and they begin to elongate, marking the beginning of the maturation phase
**Transcriptional regulation of seed maturation **
- In oilseeds, like Arabidopsis, embryo greening appears necessary for photosynthesis and eventually accumulation of storage lipids.
- In Arabidopsis seeds, lipids as triacylglycerols (TAGs) and seed storage proteins (12S globulins and 2S albumins) are the major constitutients of seed storage reserves
- ABI3, FUS3, and LEC2 (B3-AFL) are the members of the B3 domain-containing TFs, whereas LEC1 encodes the member of the NF-YB protein family(NF-YB9, HAP3 subunit of the CCAAT box-binding factors)
- LEC1 and LEC2 have a distinct role in initiating and maintaining embryonic fate
- Their ectopic expression is sufficient to induce somatic embryogenesis in vegetative tissues
- And their loss-of-function mutants exhibit trichomes and anthocyanin accumulation on cotyledon’s surface
- Recently, it has been observed that the expression of LEC1 in the embryo is not sufficient to initiate the maturation process.
- The endosperm–produced LEC1 protein, trafficked to the embryo through the suspensor, performs this function
- ABI3 and FUS3 reuglate each other’s expresison and can autoactivate themselves
- Furthermore, their expression is positively regulated by LEC1 and LEc2
- B3-AFL AFL directly regulate maturation genes by binidng to the RY element (CATGCA)
- LEC1 does not exhibit specifci DNA-binding activity
- It interacts with NF-YA and NF-YC subunits to form the NF- complex, recognizing the CCAAT motif to regulate the transcription of its target genes
- ABI3 cooperates with the members of bZIP TFs such as bZIP10, 25 in regulating SSP (At2S) gene expression
- Likewise, LEC1 and its paralogs LEC1-like (LIL) inteact with an NF-YC subunit and bZIP67
- This trimeric complex (LEC1/LIL-NFYC2-bZIP67) binds to ABRe/G-box elements through bZIP67 to activate the expression of genes invovled in storage, such as CRUCIFERIN and FATTY ACID DESATURASE3 (FAD3)
- WRI1 acts downstream to LEC1 and LEC2
- Previously, FUS3 has been shown to affect the expressino of fatty acid biosynthetic genes, probably by acitvating WRI1 expression
- WRI has appeared as the direct target of LEC1 and FUS3
- ABI3 directly induces WRI and other fatty acid biosynthesis-related genes such as FAD3 and SSI2
- The plant hormone abscisic acid (ABA) plays a central role in inducing and maintaining seed dormancy
- Moreover, auxin also controls seed dormancy via activation of ABI3 expression by two auxin-responsive TFs, ARF10 and ARF16.
- A feed back loop has been observed where ABI3 represses the expression of miR160, leading to increased transcripts of ARF10 and ARF16 that upregulate, ABI3, resulting in seed dormancy
Verma 2022, Planta
: Transcriptional control of Arabidopsis seed development
In most angiospemrs, the seed is an outcome of a double fertilization process, in which one of the two sperm nuclei fuses with the egg cell to produce a diploid zygote, and the second sperm nucleus fuses with the binucleate central cell to generate the triploid endosperm
-Subsequently, the single cellular zygote undergoes highly coordinated cell divisions and cellular differentiation to develop into a multicellular embryo in a process termed embryogenesis
- In many dicots, including Arabidopsis, the endosperm develops as a coenocyte (series of mitosis without cytokinesis) followed by endosperm cellularization
- Later, in Arabidopsis, the endosperm is absorbed in part by the growing embryo.
- The seed coat is developed from the ovule’s integuments and consists of five cell layers.
- Two cell layers are derived from the outer integuments (OI): OI2 and OI1, and three cell layers are derived from the innter integuments (II): II2, II1’ and II1.
- During the development of the seed, cells of the outermost layer, i.e., OI2, produce mucilage which accumulates specifically at the outer conrners of the cell
- Besides, both outer integument cell layers (OI1 and OI2) produce starch granules, falvanols, and suberin (a lipophilic polymer).
- In contrast, cells of the innermost layer accumulate proanthocyanidins (PAs) which later oxidize and give the characteristic brown colour to the seed coat.
- In the end, the mature seed contains a filial embryo, a single-cell endosperm layer, and a protective covering, i.e., a seed coat developed from the ovule’s integuments
- Overall, embryo development can be classified into two distinct phases, (i) morphogenesis and (ii) maturation
- The morphogenesis phase begins immediately after fertilization and lasts until the late-heart stage.
- This phase is characterized by highly coordianted cell divisions and differentation, during which the basic body organization of the embryo is established
- By contrast, cell division and proliferation are ceased during embryo maturation.
- However, cell expansion occurs along with the accumulation of seed storage reserves such as carbohydrates, proteins, and fatty acids.
- Embyro maturation partially overlaps with the morphogeneiss phase.
- It begins around the early-heart stage and lasts until the seed is fille dwith nutrients, becomes dry, and acquires a dormant state (e.g., seed maturation).
- It has been observed that the developmental stage, not the time elpased since fertilization, determines the onset of maturation
- Seed devleopment depends on the spatiotemporal expression of various genes invovled in different processes occurring during this period, including cell division, differentaition, seed filling, desiccation, and dormancy
- Therefore, control of spatiotemporal patttern and expression levels of such genes is crucial, which results from the regulation of their transcription by different TFs.
- TFs are critical regulatory proteins that act in a combinaotorial manner together with other proteins to orchestrate this transcriptional regulation
- They work through binding to specific DNA sequences (cis-regulatory elements) present over the promoters of their target genes.
- These regulatory proteins are themselves regulated by otehr TFs, hormones, thereby creating multiple layers and networks of regulation to control a particular developmental aspect
Transcriptional control of early embryogenesis (morphogenesis)
Embryogenesis begins when the zygote undergoes an assymetric division that generates a small apical cell and a large basal cell
- The apical cell gives rise to the spherical embryo proper (proembryo) that will create most of the mature embryo
- Two rounds of longitutinal and one round of transverse divisions convert the apical cell into an eight-celled embryo.
- In this stage, two domains, termed the upper tier and lower tier, are distinguished
- The basal cell divides transversely and gives rise to a 7 - 9 celled filamentous suspensor
- Later, the uppermost suspensor cell is specificed as hypophysis, which ultimately protrudes into the embryo.
- The upper tier gives rise to the cotyledons and shoot apical meristem (SAM)
- The lower tier and hypolysis generate the cotyledon’s abaxial part, hypocotyl, root apical meristem (RAM), and embryonic root.
- After a periclinal division, a 16-celled embryo results from the first visiable cell differentation whehn the outermost layer is specified as protoderm
- Subsequent divisions give the embryo a globular appearance
- At this stage, precursor cells of ground tissue and vascular tissue are specified.
- Later, with the development of cotyledon primordia, the embryo takes the shape of a heart
- At this stage, SAm is established, cotyledons are formed, and they begin to elongate, marking the beginning of the maturation phase
**Transcriptional regulation of seed maturation **
- In oilseeds, like Arabidopsis, embryo greening appears necessary for photosynthesis and eventually accumulation of storage lipids.
- In Arabidopsis seeds, lipids as triacylglycerols (TAGs) and seed storage proteins (12S globulins and 2S albumins) are the major constitutients of seed storage reserves
- ABI3, FUS3, and LEC2 (B3-AFL) are the members of the B3 domain-containing TFs, whereas LEC1 encodes the member of the NF-YB protein family(NF-YB9, HAP3 subunit of the CCAAT box-binding factors)
- LEC1 and LEC2 have a distinct role in initiating and maintaining embryonic fate
- Their ectopic expression is sufficient to induce somatic embryogenesis in vegetative tissues
- And their loss-of-function mutants exhibit trichomes and anthocyanin accumulation on cotyledon’s surface
- Recently, it has been observed that the expression of LEC1 in the embryo is not sufficient to initiate the maturation process.
- The endosperm–produced LEC1 protein, trafficked to the embryo through the suspensor, performs this function
- ABI3 and FUS3 reuglate each other’s expresison and can autoactivate themselves
- Furthermore, their expression is positively regulated by LEC1 and LEc2
- B3-AFL AFL directly regulate maturation genes by binidng to the RY element (CATGCA)
- LEC1 does not exhibit specifci DNA-binding activity
- It interacts with NF-YA and NF-YC subunits to form the NF- complex, recognizing the CCAAT motif to regulate the transcription of its target genes
- ABI3 cooperates with the members of bZIP TFs such as bZIP10, 25 in regulating SSP (At2S) gene expression
- Likewise, LEC1 and its paralogs LEC1-like (LIL) inteact with an NF-YC subunit and bZIP67
- This trimeric complex (LEC1/LIL-NFYC2-bZIP67) binds to ABRe/G-box elements through bZIP67 to activate the expression of genes invovled in storage, such as CRUCIFERIN and FATTY ACID DESATURASE3 (FAD3)
- WRI1 acts downstream to LEC1 and LEC2
- Previously, FUS3 has been shown to affect the expressino of fatty acid biosynthetic genes, probably by acitvating WRI1 expression
- WRI has appeared as the direct target of LEC1 and FUS3
- ABI3 directly induces WRI and other fatty acid biosynthesis-related genes such as FAD3 and SSI2
- The plant hormone abscisic acid (ABA) plays a central role in inducing and maintaining seed dormancy
- Moreover, auxin also controls seed dormancy via activation of ABI3 expression by two auxin-responsive TFs, ARF10 and ARF16.
- A feed back loop has been observed where ABI3 represses the expression of miR160, leading to increased transcripts of ARF10 and ARF16 that upregulate, ABI3, resulting in seed dormancy
Weigel 2008, Genetic Analysis of Arabidopsis Mutants
-Newly identified mutations can be examined intially using segregation analysis, followed by backcrossing and cosegregation of removal of extraneous mutations and assessment of pleiotrophy
SEGREGATION ANALYSIS
Segregation analysis is used to determine whether a mutant phenotype is caused by dominant or recessive muations, how many mutations are required for the pehnotype, and whether the muations are in cytoplasmic or nuclear genes
- With a newly identified mutation, it is advisable to examine the segregation pattern of the phenotype first in the self-progeny, then in teh F1 of a cross to wild type and in teh F2 progeny, and finally in several backcrosses to wild type to remove extraneous mutations
- Self-progeny:
- Dominant mutations can be identified as heterozygotes or homozygotes.
- If heterozygous, one quarter of the self progeny will exhibit a wild type phenotype (Aa x Aa -> 1/4 AA: 1/2 Aa: 1/4 aa).
- In this situation, homozygous mutants can be identified as plants that are true breeding for the mutation (do not yield wild-type offspring).
- F1 progeny:
- To determine whether the mutation can be transmitted through both male and female gametes, it is importnat to carry out reciprocal crosses, pollinating wild-type carpels with mutant pollen and mutant carpels with wild-type pollen.
- If the resulting F1 plants are wild type, then the mutation is nuclear and recessive; If mutant, then the mutation is nuclear and domiannt
- F2 progeny:
- Grow 100 F2 plants and determine whwat fraction of the plants exhibit the mutant phenotype.
- If the mutant phenotype is caused by a single, recessive, nuclear mutation, one-quarter of the paltns will display the mutant phenotype.
- If it is caused by a sinlge, dominant, nuclear muation, three-quarters of the plants will show the mutant phenotype.
- heterzygous plants -> 1:2:1 progeny
- two unlinked recessive mutations (1/16 mutant phenotype); 1 recessive, 1 unlinked dominant mutation (3/16 mutant phenotype); two unlinked dominant mutations (9/16 mutant); Either 1 of 2 unlinked recessive mutations (7/16 mutant).
- iie., F2 population consists of 21 phenotypically mutant plants and 79 phenotypically wild-type plants. Is the phenotype casused by a single recessive muation?
- 0.85 < chi-squared of 0.95 = 3.841 -> Data fall within the range that is expected for 95 % of samples ets occuring from segregation of single recessive gene. Accept hypothesis taht results are reasonable for 3:1 segregation.
- The first backgcross of a newly identified mutant yields few mutant plants in the F2 generation than expected. Skewed ratio can be from segregation of multiple, unrelated mutations in a mutagenized background.
- For example, the original isolate may carry a second, linked mutation, with deleterious effects on survival or growth, casuign the mutant phenotype to be underrepresented in the F2 popualtions.
BackCROSSES and COSEGREGATION
- plants from mutagenized popualtions frequently carry multiple mutations.
- Some of these may affect the phenotype of interest.
- It is advisable to backgross mutant plants to their wild-type parents several times to remove extaneous mutations
- Each time a mutatnt is crossed to wild type, one-half of the mutant genome is replaced with wild-type genes.
- Subsequent analysis of the F2 popualtion allows the plants that are homozygous for the recessive mutation of interest to be recovered, but does not increase the likelihood of removing unlinked secondary mutations becasue only 1/4 of the plants will have lost such a muation (an “improvement”), whereas 1/2 of the plants will be heterozygous, and 1/4 of the plants will be homozygous for the secondary mutation
- Consequently, each backcross removes half of the unlinked secondary mutations, and the probability that a particular unlinked mutation remains after n rounds of backcrossing is (1/2)^n.
- The chance of such a mutation remaining after four rounds of backcrossing is 0.5^4 = 0.062.
- Of course, if a secondary mutation is linked to the mutation of interest, the probability of removing it in a backcross is much less than one-half and approaches zero as the distance between the primary and secondary mutations decreases
Confirmation that a phenotype of interest is due to a particular mutation can be obtained by identifying the corresponding wild-type gene, introducing that gene into mutant plants, and ecmonstrating that the mutant pehnotype is rescued (complementation).
In the case of dominant mutations, it is necessary to demonstrate that a transgene construct carrying the dominant allele transofmrs wild-type plants to the mutant phenotype.
if a mutant exhibits two or more phenotypes, it is usually necessary to determine whether both phenotypes result from the same mutation (pleiotropy) or from two or more different mutations. These possbilites can usually be distinguished by multiple rounds of backcrossing or by testing for cosegregation of the phenotypes, which is generally much faster
- The probability that they are not caused by two unlinked, recessive mutations is 1 - p.
- Among the F2 progeny of a backcross to WT, if two mutations are recessive and unlinked, the probability of a plant that is homozygous for one mutation (genotype aa showing phenotype “a”) also being homozygous for the second mutation (genotype bb, phenotype ‘b”) is 1/4.
- For example, the probabiltiy taht two recessive phenotypes, which cosegregate among four plants, are caused by two unlinked mutation is 0.004 i.e., 0.25^4.
- The probability that this is not ture is 0.996 (i.e., 1 - 0.004).
- The phenotypes are almost certainly caused by the same mutation or by linked mutations.
-
Weigel 2008, Genetic Analysis of Arabidopsis Mutants
-Newly identified mutations can be examined intially using segregation analysis, followed by backcrossing and cosegregation of removal of extraneous mutations and assessment of pleiotrophy
SEGREGATION ANALYSIS
Segregation analysis is used to determine whether a mutant phenotype is caused by dominant or recessive muations, how many mutations are required for the pehnotype, and whether the muations are in cytoplasmic or nuclear genes
- With a newly identified mutation, it is advisable to examine the segregation pattern of the phenotype first in the self-progeny, then in teh F1 of a cross to wild type and in teh F2 progeny, and finally in several backcrosses to wild type to remove extraneous mutations
- Self-progeny:
- Dominant mutations can be identified as heterozygotes or homozygotes.
- If heterozygous, one quarter of the self progeny will exhibit a wild type phenotype (Aa x Aa -> 1/4 AA: 1/2 Aa: 1/4 aa).
- In this situation, homozygous mutants can be identified as plants that are true breeding for the mutation (do not yield wild-type offspring).
- F1 progeny:
- To determine whether the mutation can be transmitted through both male and female gametes, it is importnat to carry out reciprocal crosses, pollinating wild-type carpels with mutant pollen and mutant carpels with wild-type pollen.
- If the resulting F1 plants are wild type, then the mutation is nuclear and recessive; If mutant, then the mutation is nuclear and domiannt
- F2 progeny:
- Grow 100 F2 plants and determine whwat fraction of the plants exhibit the mutant phenotype.
- If the mutant phenotype is caused by a single, recessive, nuclear mutation, one-quarter of the paltns will display the mutant phenotype.
- If it is caused by a sinlge, dominant, nuclear muation, three-quarters of the plants will show the mutant phenotype.
- heterzygous plants -> 1:2:1 progeny
- two unlinked recessive mutations (1/16 mutant phenotype); 1 recessive, 1 unlinked dominant mutation (3/16 mutant phenotype); two unlinked dominant mutations (9/16 mutant); Either 1 of 2 unlinked recessive mutations (7/16 mutant).
- iie., F2 population consists of 21 phenotypically mutant plants and 79 phenotypically wild-type plants. Is the phenotype casused by a single recessive muation?
- 0.85 < chi-squared of 0.95 = 3.841 -> Data fall within the range that is expected for 95 % of samples ets occuring from segregation of single recessive gene. Accept hypothesis taht results are reasonable for 3:1 segregation.
- The first backgcross of a newly identified mutant yields few mutant plants in the F2 generation than expected. Skewed ratio can be from segregation of multiple, unrelated mutations in a mutagenized background.
- For example, the original isolate may carry a second, linked mutation, with deleterious effects on survival or growth, casuign the mutant phenotype to be underrepresented in the F2 popualtions.
BackCROSSES and COSEGREGATION
- plants from mutagenized popualtions frequently carry multiple mutations.
- Some of these may affect the phenotype of interest.
- It is advisable to backgross mutant plants to their wild-type parents several times to remove extaneous mutations
- Each time a mutatnt is crossed to wild type, one-half of the mutant genome is replaced with wild-type genes.
- Subsequent analysis of the F2 popualtion allows the plants that are homozygous for the recessive mutation of interest to be recovered, but does not increase the likelihood of removing unlinked secondary mutations becasue only 1/4 of the plants will have lost such a muation (an “improvement”), whereas 1/2 of the plants will be heterozygous, and 1/4 of the plants will be homozygous for the secondary mutation
- Consequently, each backcross removes half of the unlinked secondary mutations, and the probability that a particular unlinked mutation remains after n rounds of backcrossing is (1/2)^n.
- The chance of such a mutation remaining after four rounds of backcrossing is 0.5^4 = 0.062.
- Of course, if a secondary mutation is linked to the mutation of interest, the probability of removing it in a backcross is much less than one-half and approaches zero as the distance between the primary and secondary mutations decreases
Confirmation that a phenotype of interest is due to a particular mutation can be obtained by identifying the corresponding wild-type gene, introducing that gene into mutant plants, and ecmonstrating that the mutant pehnotype is rescued (complementation).
In the case of dominant mutations, it is necessary to demonstrate that a transgene construct carrying the dominant allele transofmrs wild-type plants to the mutant phenotype.
if a mutant exhibits two or more phenotypes, it is usually necessary to determine whether both phenotypes result from the same mutation (pleiotropy) or from two or more different mutations. These possbilites can usually be distinguished by multiple rounds of backcrossing or by testing for cosegregation of the phenotypes, which is generally much faster
- The probability that they are not caused by two unlinked, recessive mutations is 1 - p.
- Among the F2 progeny of a backcross to WT, if two mutations are recessive and unlinked, the probability of a plant that is homozygous for one mutation (genotype aa showing phenotype “a”) also being homozygous for the second mutation (genotype bb, phenotype ‘b”) is 1/4.
- For example, the probabiltiy taht two recessive phenotypes, which cosegregate among four plants, are caused by two unlinked mutation is 0.004 i.e., 0.25^4.
- The probability that this is not ture is 0.996 (i.e., 1 - 0.004).
- The phenotypes are almost certainly caused by the same mutation or by linked mutations.
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Weitbrecht 2011, Journal of Experimental Botany
:First off the mark: early seed germination
