ABA, LEA IN SEED MATURATION.pptx. Not now

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1. PLANT DEVELOPMENT The plant development starts with the seed, followed by the seedling , the vegetative phase, and end with the reproductive phase

2. Seed Development Delouche (1971) defined seed development and maturation as a process comprising of a series of morphological, physical, physiological and biochemical changes that occur from ovule fertilization to the time when seeds become physiologically independent of the parent plant.

3. Phases of seed development The phases of seed development can be broadly categorized into two phases, A morphogenesis phase constituting phases from endosperm development, cell divisions, and embryo and cotyledon differentiation and A maturation phase involving embryo growth at the expense of endosperm, desiccation of the seed and accumulation of seed storage materials Seed development programs in major dicot seed crops such as soybean and canola are similar to that in Arabidopsis Further, early stages of seed development in monocots are broadly similar to dicots. However, later stages of seed development vary in monocots

4. A morphogenesis phase Seed development begins with fertilization of both egg cell and central cell to form the diploid embryo and triploid endosperm Followed by syncytial phase in which endosperm nuclei divide rapidly without cell division and create seed cavity . In cellularization phase, rapidly divided endosperm nuclei are cellularized and finally in embryo growth phase embryo grows at the expense of endosperm to fill the seed cavity. A mature seed contains a single layer of endosperm and integuments become seed coat.

5. After completion of series of cell division and cell differentiation, seed development shifts to the maturation phase that can be divided into early (reserve accumulation) and late maturation (maturation drying) Maturation phase

6. Maturation Phase Seed moisture content increases during the initial part of development after fertilization and later begins to decline until equilibrium is established with environmental factors During this early seed development period , seed increased in size and gained fresh weight rapidly During this mid maturation period, seed continued to increase in size and fresh weight but more slowly than before. Eventually, seed reached a maximum size, fresh weight at Stage V . The seed dry weight also increased steadily and reached a plateau at Stage V Despite the gain in seed weight, the water content declined from Stage III to Stage V Seed were nearly spherical at Stage IV became flattened at Stage V Seed color remained green 

7. During this late-maturation/desiccation period , seed had constant dry weight but water content dropped dramatically from Stage V to Stage VII They were reduced in size and remained flattened. The color of seed was orange–brown at Stage VI and dark orange–brown at Stage VII. During early maturation, the seed can acquire desiccation tolerance . Desiccation tolerance is defined as the ability of a living entity to deal with extreme moisture loss to levels below 0.1 g water per gram dry weight, or drying to relative humidity below 50%, and subsequent re-hydration without accumulation of lethal damage

8. Physiological maturity when the dry weight reaches its maximum and that after this stage, the flow of nutrients to seeds from the mother plant generally ceases . The late maturation phase is also often called the maturation drying phase . A significant decline in seed moisture content occurs at the end of maturation, whereas the acquisition of desiccation tolerance begins during mid and late maturation At the same time, massive structural and physiological changes occur within the seed, Structural changes: accumulation of reserve food, seed coat development structural dormancy ( impermiable seed coat) longevity & viability. A strong reduction in metabolic activity and a transition to a quiescent (temporary inactivity & reversible) and frequently dormant state (temporary suspension due to avoid stresses) at the end of late maturation. Maximum dry weight

9. Based on the desiccation tolerance, seeds can be classified into orthodox and recalcitrant types Recalcitrant types Recalcitrant seeds usually do not show this quiescent state .( cant withstand dessication ) – no maturation drying phase, storage limitations, short term plantings Recalcitrant seeds are sensitive to dehydration and desiccation leads to damage and loss of viability Eg : rubber, avacado , Cacao

10. Orthodox Types The maturation in the orthodox seeds is accompanied with a water loss up to 5–10 % w/w, which allows them to sustain unfavourable environmental conditions , such as extremely high and low temperatures and drought. In orthodox seeds , the mechanisms behind the onset of desiccation tolerance are activated at the early stages of maturation Later on, desiccation tolerance is lost during germination, at the moment of radicle emergence

11. How dessication tolerance is acquired? Desiccation tolerance is acquired by seeds through accumulation of an array of small molecules and proteins maintain the structural integrity of critical cellular organelles, membranes and proteins They can persist during the dry state and resume their biological functions upon hydration.

12. The embryo accumulates specific molecules that are associated with the cells’ ability to tolerate extreme water stress viz. low molecular weight antioxidants, oligosaccharides such as raffinose, stachyose, late embryogenesis abundant proteins (LEAs) and heat shock proteins (HSPs).

13. Further, structural changes occur at the cellular level such as folding of cell walls, condensation of chromatin and dismantling of thylakoids in chloroplasts These physiological and structural changes reduce metabolic activity while mitigating the mechanical stress of cell shrinkage during dehydration ( maturation drying process). Changes at this stage correspond with a gradual increase in seed longevity

14. late embryogenesis abundant proteins (LEAs) LEA proteins have relative high content of glycine, alanine, glutamate, lysine, arginine and threonine, while low amounts of cysteine and tryptophan residues. Due to this primary nature, LEA proteins are stable in a broad temperature range. During cell dehydration, LEA proteins act as chaperons , i.e., involved in structural stabilization of denatured proteins and promote their refolding through intensive hydrogen bond formation. LEA proteins are also responsible for sequestration of ionic compounds , accumulating during cell dehydration , and protection of membrane proteins and enzymes from the deleterious effects

15. Stresses usually cause protein dysfunction . Maintaining proteins in their functional conformations and preventing the aggregation of non-native proteins are particularly important for cell survival under stress. Chaperones are responsible for protein folding, assembly, translocation and degradation in many normal cellular processes, stabilize proteins and membranes, and can assist in protein refolding under stress conditions. They can play a crucial role in protecting plants against stress by re-establishing normal protein conformation and thus cellular homeostasis

16. Other structural adaptations that occur during this stage are chromatin compaction and nuclear size reduction , which are reversed during germination. Furthermore, metabolic activity is reduced and chlorophyll is degraded towards the end of seed maturation thereby minimizing the production of reactive oxygen species (ROS). Non-reducing sugars fill the free volume between large molecules, created during dehydration and the dehydrated cytoplasm forms a glassy matrix with very low molecular mobility . To protect the seed against oxidative damage, which cannot be repaired by enzyme activity under dry conditions. Seeds accumulate during seed maturation many antioxidants, such as ascorbate, glutathione, polyols, tocopherols, quinones, flavonoids and phenolics

17. Group II LEA proteins Group II LEA proteins accumulate highly in plant embryos during the late stages of seed development as an aid to embryo maturation under desiccation. In plant vegetative tissues , group II LEA proteins are rarely detected and are limited to young parts of plants, especially those that exhibit excessive cell division and cell elongation, for example, at the root tips, in expanding stems, and in petioles . Some group II LEA proteins are found in mature seeds. Localized: all the segments of the embryo and endosperm of mature seeds. accumulates in developing cotyledons during mid-to-late embryogenesis and in seedlings during dehydration stress

18. It comprises about 2% of the proteins in mature cotyledons. The carrot group II LEA gene, ECP40, is distributed in the zygotic embryos and endosperm of mature seeds In seed physiology, DHNs or group II LEA proteins are considered to be responsible for the persistence and longevity of seeds . Plant seeds are of special interest for investigating the proteins from the group II LEA family, since they are relatively abundant during seed maturation stages and in response to any external stimulus causing dehydration to the seeds . Seeds are classified as recalcitrant or orthodox based on their storage behaviors

19. Recalcitrant seeds do not go through maturation drying and drop with a relatively high content of moisture Seed recalcitrance is a major issue for the natural production of plant species that causes a serious problem in seed conservation and storage In recalcitrant seeds, a positive correlation was found between the seed moisture content and the germination rate These seeds cannot be maintained and stored in conventional freezers due to their low survivability after drying and freezing at −20 ◦C. The absence of resistance in recalcitrant seed drying was attributed to the lack of DHNs

20. Orthodox seeds, on the other hand, go through maturation drying and are dropped from plants at a low content of moisture These seeds have the potential to be dried to an internal seed water content of less than 12% and can be maintained, stored, and survived at freezing temperatures DHNs are synthesized in orthodox seeds , which are accumulated during the final stages of maturation and during seed desiccation It has been suggested that, in orthodox seeds, DHNs favor their tolerance towards moisture los s and osmotic stress during the stage of seed maturation

21. In orthodox seeds, as a response to the maturation drying, DHNs are synthesized, whereas recalcitrant seeds do not synthesize DHNs because of the absence of any maturation drying It is because of DHNs that orthodox seeds retain their viability during storage whereas recalcitrant seeds become unviable because of the absence of DHNs during maturation drying and storage conditions.

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23. Functional mechanism As chaperones and bind to DNA and other protein biomolecules by shielding them and thereby preserve the functions of these proteins during the stress. The ability to bind to DNA and protect it from immoderate ROS such as H2O. DHNs preserve the activities of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) under freezing and thawing stress damage. Such stabilization of cell structures and organelles was evident through the overexpression of DHN genes in transgenic tomatoes , which improved the relative water content (RWC) and lowered the rate of water loss in the tomato

24. Under the dessication , the plant senses the stress and signals its organelles for the presence of stress through the release of free metal ions or through the accumulation of ROS. After the signal transduction, DHN genes are upregulated within the nucleus, and DHNs are synthesized for the stress tolerance mechanism. Both phenomena can occur within plant cells based on the signal transduction pathway. (A) DHNs act as antioxidants and scavenge ROS that accumulate within the plant cells. (B) DHNs, through their property of metal-ion binding , also scavenge the free metal-ion radicals that arise within plant cells under abiotic stress.

25. Functional role of DHNs under the dessication A—ROS scavenging as antioxidant, B—Metal-ion binding

26. Binding of DHNs to membrane phospholipids. The unstructured DHNs that are synthesized during an abiotic stress in the cytoplasm move close to the cell membranes. Through their phospholipid binding property, the unstructured DHNs bind to the membrane’s anionic phospholipids, attain a helical structure, and generate stress responses. The stress responses include structured DHNs that bind to other stress-sensitive protein molecules and protect them from the damage caused by the stress

27. Metal-Ion-Binding Protein DHNs function through their metal-ion-binding properties under certain environmental stresses The catalytic metal ions, copper and zinc, mainly occur as complexes of metal and protein molecules in plants growing under favorable habitats However, as plants move under stress conditions, these metal ions can be released as free ions. These ions are involved in ROS production through the Haber–Weiss reaction

28. Metal ions are a common target for a number of DHNs . Abiotic stresses, such as water stress, result in the release of metal ions from the membranes and organelles and increase the concentration of free metals in the intracellular spaces It has been hypothesized that DHNs bind to these free metal ions and decrease the damage they cause The binding of DHNs to metal ions has been reported in Arabidopsis thaliana and citrus DHNs, AtHIRD11 and CuCOR15 , which are able to bind to iron and cobalt over magnesium and calcium and prevent the release of free ions It has also been found that CuCOR15 acts as a radical scavenger that reduces the metal toxicity in plants under drought stress Moreover, an ion transport protein (ITP), KS-DHN , from Ricinus communis was indicated as an active transporter of metal ions within plants.

29. Phospholipid-Binding Protein DHNs tend to bind to phospholipids because of their rich K-segments and histidine motifs Their binding to phospholipids triggers the accumulation of a crucial stress signaling phospholipid, phosphatidic acid The concentration of PA in an inflated plasma membrane is very low, about 1% , but increases under drought stress The increase in PA concentration is due to low water content within cells or release of ABA The presence of basic amino acids such as arginine and lysine in DHNs enables them to bind to anionic phospholipids The interaction between dehydrins and membranes changes certain membrane properties, such as water content and temperature within cells DHNs bind to charged lipids by the occurrence of electrostatic interactions . Some DHNs gain their helicity structure through binding with acidic phospholipids This enables them to bind to other biomolecules within the cytoplasm and protect them from stress As DHNs bind particularly to acidic phospholipids, it can be postulated that DHNs may interact with membranes of the cell at specific regions

30. Hormonal Regulation of Seed Development and Maturation Plant hormones are signal molecules that are produced in the plant and are active at very low concentrations. The hormones abscisic acid (ABA), gibberellins (GAs), auxin (IAA), cytokinins , ethylene and brassinosteroids regulate cellular processes in targeted cells, which may or may not be the cells in which they are synthesize. Measurements of endogenous hormone concentration have suggested the high transient expression of cytokinins , GAs and IAA during the early phase of seed development In studies with tomato, GAs were found essential to produce fertile pollen , but pollination of a GA-deficient (female) mutant with mutant pollen, obtained upon spraying the male plant with GAs, resulted in the development of normal-looking healthy seeds that only needed GAs for germination

31. Cytokinins have also been implicated in promoting suspensor function, but may be even more significant in promoting endosperm growth and grain filling via promotion of cell division (Bewley et al. 2013 ). In contrast, during early embryogenesis, auxins play a major role in establishing the embryonic body plan via effects on apical-basal polarity or pattern formation (transition of embryo from globular to heart shape and cotyledon separation at later stages) and vascular development

32. During maturation , seeds of most species acquire the capability to endure desiccation. The maturation phase begins when the embryo and endosperm have accomplished the morphogenesis and patterning stages ( Wobus and Weber 1999). This phase is categorized by a growth arrest , followed by the synthesis and accumulation of reserves, whose degradation upon germination will provide nutrients to the growing seedling before the photosynthetic capacity is fully acquired (Baud et al. 2002 ) . Early and mid-phases of maturation are controlled by the action of ABA, initially synthesized in the maternal tissues and later on, although to a lower extent, in the embryo and endosperm ( Nambara and Marion-Poll 2003). Seed maturation coincides with an increase in seed ABA content ; consistent with the fact that ABA induces expression of a cyclin-dependent kinase inhibitor (ICK1) that could lead to cell cycle arres

33. In the later stage, a decline in ABA level occurs and synthesis of LEA proteins follows, which is characteristic to the late maturation phase . Maturation is not always an obligatory process, if ABA effects are eliminated by removing the embryo from the seed would lead to development of seedlings (Berger 2003 ). But due to their low vigour , planting these immature seeds in the field will not result in the development of a healthy seedling.

34. Role of ABA biosynthesis genes in seed development Maternal ABA plays a significant role in embryo development and seed maturation. ABA is also de novo synthesized in embryo and testa during embryo development, as well as accumulates during seed maturation, F acilitates late seed maturation processes, Synthesis of storage proteins to prevent seed abortion, I nduces primary dormancy and allows successful germination as well as a successive seedling enterprise So , de novo synthesis of active ABA plays a more important role in seed development and later germination

35. Active ABA is synthesized through an indirect pathway from xanthophylls (e.g., zeaxanthin, violaxanthin, and neoxanthin). Three types of genes are responsible for the successive steps of ABA biosynthesis such as ZEAXANTHIN EPOXIDATION (ZEP), OXIDATIVE CLEAVAGE OF 9-CIS-EPOXYCAROTENOIDS (NCED), and ABSCISIC ALDEHYDE OXIDATION (AAO). The ZEP/ABA gene was firstly identified in Arabidopsis thaliana and Nicotiana plumbaginifolia .

36. Regulation of seed development and dormancy by ABA biosynthesis through the carotenoid pathway started from b-carotene (C40 ). The complete ABA synthesis process takes place in plastids and cytoplasm where ZEAXANTHIN EPOXIDASE (VPs, ZEP, ABA1/2) converts zeaxanthin into antheraxanthin and all trans- violaxanthin . ABA4 catalyzes the conversion from all-trans- violoxanthin to the all-trans- neoxanthin . The conversion of xanthoxin from 90 - cis-neoxanthin and 90 - cis-violaxanthin is exerted by VP14 and NCEDs (NINE-CIS-EPOXYCAROTENOID DIOXYGENASE), among which the NCEDs display different subcellular localization of plastid or cytoplasm . The oxidation of abscisic aldehyde by AAO3 (ABSCISIC ALDEHYDE OXIDASE3) is responsible for the conversion from abscisic aldehyde into ABA, which in turn induces and maintains seed dormancy ..

37. Role of ABA signaling components in different seed developmental stages The ABA signaling pathway is involved in seed development . In the absence of ABA: Receptors PYLs release and activate protein phosphatase 2C (PP2C) such as ABI1/2 and AGH1/3. Downstream SNF1-RELATED PROTEIN KINASE subfamily (SnRK2s) genes are inactivated by active PP2C which leads to premature germination and the nondormant seed through repression of lots of transcription factors such as ABI1/2/3/4/5 and bZIP67. presence of ABA: Receptors PYR/PYL/RCAR bind ABA and PP2C together to inhibit the activity of PP2C, which release the activity of SnRK2s downstream transcription factors such as ABI3 by protein phosphorylation, then regulate downstream genes SGR1/2 function to mediate seed de-greening process.

38. Additionally, the active LAFL (ABI3, FUS3, LEC1, and LEC2) network by ABA along with WRI1 regulates the At2S3 gene; an active bZIP22 function downstream of SnRK2s to promote gene transcription of 27-kD c- zein for protein reserve accumulation in the seed. Along with seed de-greening and storage product accumulation, SnRK2s function upstream of ABI3/5 and ABFs to regulate LEAs and HSPs that are pivotal for desiccation tolerance. In other branches, DOG1 also plays a role upstream of ABI3/5/ABFs as well as functions as a repressor of PP2Cs (AHG1/3) to involve seed desiccation tolerance acquirement. ABA signaling components (SnRK2s, ABI3, ABI4, ABI5, ZmbZIP22, bZIP67, and ABFs) are involved in storage product accumulation, de-greening, and desiccation tolerance with different function pathways to provide a mature and dormant seed. Letters ‘‘P” and ‘‘T” in the color circles indicate the two manners of protein phosphorylation and gene transcription regulation, respectively. Activated and repressive effects are shown by arrows and bars, respectively

39. The function of ABA in seed germination and seedling establishment . Seed completes germination successfully through degradation of active ABA into PA ( phaseic acid) and DPA ( dihydrophaseic acid)/DPAG with CYP707As regulated by REF6 and phaseic acid reductase (ABH2 and GT) respectively. During germination and seedling establishment, the core ABA signaling component SnRK2s and downstream ABI3/4/5 are activated or repressed by many factors directly or indirectly to promote seed germination and seedling establishment.

40. Ethylene The ethylene pathway studies in relation to seed development and maturation are extremely limited. I n plant tissues, ethylene affects chlorophyll metabolism ( Matilla 2000). Because chlorophyll loss is triggered during the final stages of embryogenesis (during acquisition of seed vigour ), this process may be affected by ethylene. Mustard and canola seeds produce significant amounts of ethylene during embryogenesis, specifically in the early pre-desiccation stages (Child et al. 1998). Hence the role of ethylene can be attributed as minor during seed development and maturation and may be associated with the embryo de-greening process.

41. Heat shock proteins Higher plants are unable to cope up with the extended exposure to temperatures above 45 ° C cellular homeostasis The loss of biological activity of proteins upon high temperature stress may be due to aggregation and/or protein misfolding The stress-induced accumulation of aggregated and mis -folded proteins is irreversible and deleterious to the cell functioning. To balance the homeostasis of cellular proteins under heat stress, plant cell upregulates several heat inducible genes, commonly referred as “heat shock genes” (HSGs ) chaperone activity

42. Heat Shock proteins Hsps are broadly divided into two major families i.e., low and large molecular weight Hsps sizes Hsp100/ Clp , Hsp90, Hsp70, Hsp60/ chaperonin and sHsps

43. Functions ( 1) folding or assisting folding of newly synthesized proteins (Hsp70, Hsp60), ( 2) guiding translocation of proteins across organellar membranes and between intercellular compartments (Hsp70) ( 3) preventing aggregation , desegregation of oligomeric proteins, and unfolding (Hsp70, Hsp100, Hsp90, small Hsps ) ( 4) facilitating proteolytic degradation of unstable proteins ( Hsp70, Hsp100), ( 5) maturation of signaling molecules , signal transduction and transcriptional activation of transcription factors (Hsp70, Hsp90

44. sHsps are found to be most prevalent in plants and their expression can be increased up to 200 folds under stress sHsps range in size from 10 to 42 kDa localized in compartments like cytosol, endoplasmic reticulum (ER), mitochondria and chloroplast sHsps do not actively participate in refolding of non-native proteins They possess a high capacity of binding to nonnative proteins, through hydrophobic interaction The abundance of sHsps in plants and their functional characteristics of binding and stabilizing denatured proteins suggest that sHsps play an important role in plant-acquired stress tolerance

45. TaHSP26  is induced by heat stress in vegetative and floral tissues in both wheat and  Arabidopsis . The induction of  TaHSP26  at 37 °C is quite high and rapid (induced within 10 min) in 10-day-old shoots and root tissues. The induction of  sHSP26  is faster in wheat (present study) than in rice, where  reported 20 min of lag period in its induction upon high-temperature stress . Similar kinetics has also been reported for other plant small  HSPs   This may be indicative of the fact that wheat is a temperate crop while rice is a predominately sub-tropical crop. Additionally ,  TaHSP26  transcript also accumulated in early stages of seed development upon heat stress, followed by constitutive accumulation at advanced stages of seed maturation when it undergoes the desiccation phase. Transcript accumulation also takes place in  Arabidopsis  seedling tissues, inflorescence and developing siliques upon heat stress during early and later stages; however, unlike wheat, we did not find accumulation of  AtHSP26  transcript in  Arabidopsis   siliques under non-stressed conditions

46. Physiological Maturity, Mass Maturity and Harvest Maturity Two stages of maturity have been defined viz. physiological (or mass) maturity and harvest maturity . Physiological maturity is the end of the seed-filling period (Harrington 1972), whereas harvest maturity is the point of time that coincides with the end of maturation drying

47. Seed Development and Maturation in Relevance to Seed Quality Since protection mechanisms are mainly built during the late seed maturation phase, the stage of harvest becomes the most critical factor for seed quality and storability. Harvesting seeds too early when there is inadequate development of essential structures and protection mechanisms may result in poor quality. Similarly, harvesting too late may increase the risk of shattering and may decrease the quality of seed due to ageing. If harvesting is delayed, incidence of adverse environmental conditions such as rain and humidity may result in precocious germination

48. Function of Abscisic Acid   Plants have openings on the bottom side of their leaves, known as stomata. Stomata take in carbon dioxide and regulate water content. Abscisic acid has been found to  function in the closing of these stomata  during times when the plant does not require as much carbon dioxide or during times of drought when the plant cannot afford to lose much water through transpiration.  One of the crucial functions of abscisic acid is to  inhibit seed germination . ABA functions in many plant developmental processes, including seed and bud dormancy, the control of organ size and stomatal closure. It is especially important for plants in the response to environmental stresses, including drought, soil salinity, cold tolerance, freezing tolerance, heat stress and heavy metal ion tolerance. 

49. Abscisic acid (ABA) is one of the most important phytohormones that influence seed development and germination . At the molecular level, ABA biosynthesis, degradation, and signaling genes were identified to play important roles in seed development and germination Additionally, the crosstalk between ABA and other hormones such as gibberellins (GA), ethylene (ET), Brassinolide (BR), and auxin also play critical roles

50. Functional ABA biosynthesis genes show specific roles for ABA accumulation at different stages of seed development and seedling establishment. De novo ABA biosynthesis during embryogenesis is required for late seed development, maturation, and induction of primary dormancy. ABA plays multiple roles with the key LAFL hub to regulate various downstream signaling genes in seed and seedling development. Key ABA signaling genes ABI3, ABI4, and ABI5 play important multiple functions with various cofactors during seed development such as degreening , desiccation tolerance, maturation, dormancy, and seed vigor. The crosstalk between ABA and other phytohormones are complicated and important for seed development and seedling establishment.

51. There are two important phases of seed development which include zygotic embryogenesis, seed maturation

52. Physiological maturity is marked as the time when seeds attain maximum dry weight and thereby, maximum yield when it concerns crop production.