1. Arabidopsis seed, development, germination Flashcards

Question

Ohto 2009, Sex Plant Repro : Effects of APETALA2 on embryo, endosperm, and seed coat development determine seed size in *Arabidopsis*

Answer 1

- Arabidopsis APETALA2 (AP2) controls seed mass maternally, with *ap2* mutants producing larger seeds than wild type. - AP2 appears to have a significant effect on endosperm development. -* ap2* mutant seeds undergo an extended period of rapid endosperm growth early in developement relative to wild type. - this early expanded grwoth period in* ap2 *seeds is associated with delayed endosperm cellularization and overgrowth of the endosperm central vacuole - Seed coat development is affected; integument cells of *ap2* mutants are more elongated than wild type. - Endosperm overgrowth and/or integument cell elongation create a larger postfertilization embryo sac into which the *ap2* embryo can grow - Morphological development of the embryo is initially delayed in *ap2* compared with wild-type seeds, but *ap2* embryos become larger than wild type after the bent-cotyledon stage of development - *ap2* embryos are able to fill the enlarged postfertilziation embryo sac, because they undergo extended periods of cell proliferation and seed filling - Maternally actomg AP2 influences development of the zygotic embryo and endoserpm to repress seed size - During the early morphogenesis phase, the embyro undergoes a series of differentiation events in which the plant body plan is established with formation of the embryonic tissue and organ systems - It is also during this phase that the endosperm mother cell initially undergoes nuclear divisions without cytokinesis to form a syncytium - Syncytial nuclei are sequestered into individual endosperm cells thorugh the process of cellularization, and the endosperm continues to grow throhgh periclinal cell divisions at the periphery of the endosperm. - Later in embryogenesis during the maturation phase, the embryo and endosperm accumulate reserves such as storage lipids and proteins, and the embryo acquires the ability to withstand desiccation - In Arabidopsis, the endosperm is largely consumed by the developing embryo such that only a few endosperm cell laryers remain in the mature seed - 1st pathway: Parent-of-origin effects on seed size: Interploidy crosses between diploid and polyploid plants produce offspring with an excess of either maternal or paternal genomes, which cause an under-or over-proliferation of endosperm nuclei, an acceleration or delay in the onset of endosperm cellularization, and the production of smaller or larger seeds, respectively. - Reciprocal crosses of wild-type plants with hypomethylated plants defective in the expression of *MET1*, encoding a DNA methyltransferase, phenocopies interploidy crosses, suggesting that parent-of-origin effects involve DNA methylation. - 2nd pathway genes: *IKU2 *and *MINI3*, expressed int he endosperm that act to control endosperm growth and seed size - 3rd pathway: genes that in the maternal sporophyte to influence seed size, including *AP2, TTG2,* and* ARF2.* Loss-of-function mutations in *TTG2* and *ARF2 *affects the lengths and number of integument cells of the ovule, respectively, and the inlfuence of these genes on seed size has been primarily attributed to their effect on the size of the postfertilizastion embryo sac - AP2 (APETALA2), the defining member of the AP2 DNA binding domain class of transcription factors, is invovled in controlling seed size - *AP2 *was identified originally as a gene requried to specify floral organ and floral meristem identity and to control ovule and seed coat development - Although its most conspicuous functions are in flower development, *AP2* RNA is detected in seedlings, leaves, and stems in addition to flowers, suggesting its function in other processes. - Loss-of-function mutations in *ap2 *affect seed mass maternally, although it has been suggested that *AP2* acts in the endosperm to influence seed size - The *ap2* mutation causes an increase in both the number and size of cell sin the mature embryo and int he accumulation of storage proteins and lipids in mutant seeds when comapred with wild type

Answer 2

-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. -**

Answer 3

- 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. -

Answer 4

- **Germination vigor** is driven by the ability of the plant embryo, embeeded within the seed, to resume its metabolic activity in a coordinated and sequential manner. - The seed results from double fertilizastion of the ovule by th epollen grain . - It houses both a zygotic embryo that will form the new plant as well as storage tissue to supply nutrients that support seedling gfowth following germination - This latter storage tissue is usually triploid (e.g., the endosperms of cereal grains); however, in some species, the storage tissue may derive only from the maternal nucellus, such as the perisperm in sugar beet - In other angiosperm speices, the embryo absorbs the endosperm as the latter grows within the devloping seed, and the cotyledons of the embryo become filled with these storage compounds; at maturity, seeds of these species (i.e., pea, sun flower, Arabidopsis) have only residual endosperm and are termed exalbuminous seeds - Seeds fall into two broad categories (a) orthodox seeds, which sustain intense deseiccation by the end of their maturation on the mother plant and retain their germination potential over long periods of dry storage, and b) the recalcitrant seeds, which do not survive drying during ex situ conservation. - From an evolutionary perspective, seed desiccation tolerance seems associated with plants grown in drier environments, whereas desiccation sensitivity is most common in moist envrironments. - Because of this unique property of survival during dry storage, orthodox seeds are the most commonly used in agriculture. - However, economically important species such as avocado, mango, lychee, cocoa, coffee, citrus, and rubber produce recalcitrant seeds - Here, we focus on the orthodox seeds for which desiccation during maturation allows metabolic activity to be interrupted and restarted during germination following a simple imbibition - In such seeds, preservation of embryonic cell viability in the dry state can extend over centuries - This implies the existence of specific mechanisms to maintain the state of metabolic quiescence in mature dry seeds while preserving their integrity to ensure that cell metabolsim is activated and restarted during germination - The mature dry seeds of most species require a period of dry storage known as after-ripening to release them from dormancy, a physiological state in which seeds will not germinate even under optimal conditions that are otherwise favorable to theri germination when they become nondormant. - The phytohormone ABA, a sesquiterpene compound resulting from the cleavage of carotenoids, controls stroage reserve accumulation and desiccation tolerance of orthodox seeds - This hormone induces the expression of late embryogenesis abundant (LEA) proteins, which become abundant during late maturation and are thought to act as chaperones to protect macromolecular structures against desiccation injury - Importantly, during seed germination, ABA also exerts an inhibitory effect on mechanisms triggering precocious and deleterious germination of devloping seeds on the mother plant (pre-harvest sprouting), thereby allowing the maturation process to be sustained and seeds to be formed that are endowed with appropriate reserves required for establishment of vigorous plantlets following germinatino - To succesfully germinate following imbibtion, a nondormant seed must (a) establish a specific catabolsim, decrease in sensitivity, and biosynthesis inhibition to reduce the active level of this hormone, and (b) synthesize another class of hormones represented by a large family of tetracylclic diterpenes, the gibberellins (GAs), which are essential germination activators - GAs negatively regulate proteins behaving as repressors of germination - Consistent with the idea that the ABA/GA ratio regulates the metabolic transition required for germination, imbibed dormant seeds retain high levels of bioactive ABA

Answer 5

Seed development comprises two major phases: embryo development and seed maturation - Embryogenesis starts with a morphogenesis phase and ends at the heart stage when all embryo structures have been formed - It is 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 - Later during developemnt the seed, containing a full size embryo, undergoes maturation during which food reserves accumulate and dormancy and desiccation tolerance develop -

Answer 6

- In angiosperms, seed development begins with double fertilization - This process involves the joining of a female gametophyte with two male gametes, leading to the formation of a diploid embryo and a triploid endosperm - One of the sperm cells fuses with the egg cell to form the zygote - The zygote undergoes elongation and a gradual transition to establish the basic embryo pattern, which contains a shoot meristem, cotyledon(s), hypocotyl, root and root meristem - The endoserpm arises from the central cell, which ctonatins two identical haploid genomes and combines with other sperm cells - Endosperm development can be divided into four phases: synctial, cellularization, differentiation, death - In monocots and some dicots, the endosperm constitutes the primary volume of the mature seed - In some dicots, such as Arabidopsis, the embryo grows to fill the seed and consumes most of the endosperm when the seed matures - The maternal integument surrounding the developing embryo and endosperm forms the seed coat after fertilization - Therefore, the seed size is determined by the coordinated growth of the triploid endosperm, the diploid maternal integument, and the diploid embryo, that is, the endosperm, maternal sporophytic tissues, and zygotic tissues, respecitvely - In the IKU pathway, downregualtion of *HAIKU1 (IKU1), HAIKU2 (IKU2), and MINISEED3 (MINI3)* reduces seed size a s result of abnormal development of the endosperm - TF TRANSPARENT TESTA GLABRA2 (TTG2) and APETALA2 (AP2) regulate cell expansion in the maternal integument and influence seed size - AUXIN RESPONSE FACTOR2 (ARF2) control seed dize by regulating cell proliferation in the maternal integument - Morever, ubiquitin receptor DA1 and DA1-related protein (DAR1) act redundantly to restrict cell proliferation in integuments and reduce seed size - The E3 ubiquitin ligases DA2 and BIG BROTHER (BB) function synergistically with DA1 to restrict the size of the seed and other orangs.

Answer 7

- 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

Answer 8

- 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 A*GL62* 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

Answer 9

- The life cycle of platns invovles an alternation of generations between the haploid gametophyte and the diploid sporophyte. - In angiosperms (flowering plants), seed devleopment begins with double fertilziation - Pollen grains (male gametophytes) carry two haploid sperm cells, which fertilize the egg cell and the central cell fo the haploid embyro sac (female gametophyte) contained within the maternal tissues of the ovule - This event results in the formation of the diplid embryo and the triploid endosperm, respetively, the latter arising from the central cell that contains two identical haploid sets of chromosomes - Seed development is marked by the rapid growth of the endosperm and the embryo, until seed maturation, which is accompanied by desiccation - Simultaneously, the maternal ovule also undergoes regulated growth to accomodate the growing embryo and endosperm, and the integuments of the ovule ultimately constitute the coat of the mature seed - The endosperm grows much more rapidly than the embryo, growing initially through nuclear divisions as a syncitium for several mitotic cycles and subsequentaly cellularizing followed by decreased rate of growth - The growth of the seed is coupled with the growth of the endosperm, with the major increase in seed volume occuring in concordance with the rpaid growth of the endosperm - In monocots and some dicots, the endosperm contitutes the major contribution to the volume of the mature seed - In Arabidopsis and many other dicots, the endosperm is eventually consumed, being replaced by the growing, which then constitutes most of the mature seed. - However, in all cases the growth of the seed is primarily associated with the initial growth of the endosperm, and not with the later growth of the embryo. - Thus, the size of the seed is the result of three different growth programs: those of the diploid embryo, the triploid endosperm, and the diploid maternal ovule -

Answer 10

- 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 -

Answer 11

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

Answer 12

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

Answer 13

-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. -

Answer 14

-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. -

Answer 15

- The ABA signaling pathway consists of receptor RCAR/PYR/PYLs, protein phosphatase PP2C, kinase SnRK2s and the targeting substrates - ONce bound and activated by ABA, PYR/PYL/RCARs form a trimeric complex with PP2Cs, which inhibits the phosphatase activity of PP2Cs. - SnRKs are then released from the associated with PP2Cs. - Released SnRK2s can be activated by autophosphorylation, and, in turn, phosphorylate the downstream TFs and ion channel proteins - Among SnRK2 targets, AREBs/ABFs are the downstream TFs in the ABA signaling pathway. - Some other TF families, such as WRKY, MYB, and NF-Ys, are also involved in drought response and adaptation

Answer 16

- The ABA signaling pathway consists of receptor RCAR/PYR/PYLs, protein phosphatase PP2C, kinase SnRK2s and the targeting substrates - ONce bound and activated by ABA, PYR/PYL/RCARs form a trimeric complex with PP2Cs, which inhibits the phosphatase activity of PP2Cs. - SnRKs are then released from the associated with PP2Cs. - Released SnRK2s can be activated by autophosphorylation, and, in turn, phosphorylate the downstream TFs and ion channel proteins - Among SnRK2 targets, AREBs/ABFs are the downstream TFs in the ABA signaling pathway. - Some other TF families, such as WRKY, MYB, and NF-Ys, are also involved in drought response and adaptation

Answer 17

- Seed development determines plant evolutionary fitness and crop yield in flowering plants - Among various growth parameters of seed develpment, seed size is closely associated with nutrient levels for seed germination and the capacity of seed tolerance to abiotic stress during seedling establishment - Thus, seed size is always an essential agronomic trait during domestication and breeding in many crops - By now, understanding of seed size control in the model plant *Arabidopsis thaliana* has provided important insights into molecular breeding for crop improvement - Seed development in Arabidopsis starts with rapid growth of the endosperm and integument to form a large seed cavity, followed by replacement of the endosperm by the embryo - The volume of seed cavity is an essnetial factor that influences the final seed size by spatially confining the embryo growth - Integument elongation and endosperm expansion usually terminate after endosperm cellularization, which defines the available cavity space so that the seed maintains almost hte same size in the course of subsequent embryo enlargement. - Thus, the final seed size is tightly controlled by the timing of endosperm cellularizaiotn, and disruption of endosperm cellularization is usually associated with chances in seed size as exemplified by mutations in the regulators in the IKU pathway. - In this pathway, LOF of *HAIKU1 (IKU1), HAIKU2 (IKU2)* and *MINISEED3 (MINI3)* shows precocious endosperm cellularization and consequently results in a small seed phenotype, whereas gain of function of SHORT *HYPOCOTYL UNDER BLUE1 (SHB1)* causes delayed endosperm cellulariation and big seeds - Folllowing the fertiliztion of a sperm nucleus with the binucleate central cell, the resulting primary endosperm nucleus undergoes mitoses wihtout cytokinesis, generating the synctial endosperm that is spatially divided into three domains; micropylar, chalazal, and peripheral endosperm. - The **micropylar endosperm** surrounds the emryo and suspensor, while the chalazal endosperm is at the chalazal pole located opposite the embryo. - The peripheral endosperm spreads as a peripheral layer due to the force of a large central vacuole at the initial stage of seed development. - After eight rounds of syncytial mitoses, the micropylar and peripheral endosperm subsequently undergo cellularization, whereas the chalazal endosperm always keep its syncytial state - Ultrastructural studies have indicated that the chalazal endosperm, which is close to the maternal vascular tissues, serves as an endospermic haustorium in the uptake, reprocessing and transport of nutrients or signals to developing seeds

1. Arabidopsis seed, development, germination Flashcards

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