J. Derek Bewley

Physiology and biochemistry of seeds in relation to germination

Physiology and biochemistry of seeds in relation to germination , Physiology and biochemistry of seeds in relation to germination , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Structure and Composition

Seeds are very diverse in their shape and size. In the mature state they contain an embryo, the next generation of plant, surrounded by a protective structure (the seed and/or fruit coat) and, in species in which the nutritive reserves are not stored within the cotyledons, by an alternative storage tissue (endosperm, perisperm, or megagametophyte). Most seeds contain large and characteristic quantities of polymeric reserves. The major ones are carbohydrates, oils, and proteins, with minor amounts of phosphate-rich phytin. Starch, a polymer of glucose, is the most common form of stored carbohydrate, contained within cytoplasmic starch granules; less common are the hemicelluloses, stored in secondary cell walls, usually as mannan polymers. Oils are triacylglycerols, each composed of glycerol and three fatty acids that are specific to the oil; these are present within oil bodies. Storage proteins, of which there are three types, albumins, globulins, and prolamins, are sequestered in protein storage vacuoles. These reserves are vital components of human and animal diets, and their production in crops is a basis of agriculture.

Breaking down the walls — a role for endo-β-mannanase in release from seed dormancy?

Dormancy is imposed in some seeds by the tissues that surround the embryo, which act as a mechanical barrier to the emergence of the radicle. Thus it has been proposed that germination is completed following the production of hydrolases within the surrounding tissues: these are secreted into the cell walls, causing weakening and so allowing the radicle to break out. Mannose is a prominent component of the endosperm cell walls of dormant seeds of several species and, in these, production of endo-β-mannanase has been suggested to be essential for germination. It is possible that this is the case in Datura ferox seeds, but evidence for a causal link between the enzyme and germination in seeds of tomato and lettuce is more equivocal.

Abscisic acid and osmoticum prevent germination of developing alfalfa embryos, but only osmoticum maintains the synthesis of developmental proteins.

Developing seeds of alfalfa (Medicago sativa L.) acquire the ability to germinate during the latter stages of development, the maturation drying phase. Isolated embryos placed on Murashige and Skoog medium germinate well during early and late development, but poorly during mid-development; however, when placed on water they germinate well only during the latter stage of development. Germination of isolated embryos is very slow and poor when they are incubated in the presence of surrounding seed structures (the endosperm or seed coat) taken from the mid-development stages. This inhibitory effect is also achieved by incubating embryos in 10−5 M abscisic acid (ABA). Endogenous ABA attains a high level during mid-development, especially in the endosperm. Seeds developing in pods treated with fluridone (1-methyl-3-phenyl-5[3-(trifluoromethyl)-phenyl]-4(1H)-pyridinone) contain low levels of ABA during mid-development, and the endosperm and seed coat only weakly inhibit the germination of isolated embryos. However, intact seeds from fluridone-treated pods do not germinate viviparously, which is indicative that ABA alone is not responsible for maintaining seeds in a developing state. Application of osmoticum (e.g. 0.35 M sucrose) to isolated developing embryos prevents their germination. Also, in the developing seed in situ the osmotic potential is high. Thus internal levels of osmoticum may play a role in preventing germination of the embryo and maintaining development. Abscisic acid and osmoticum impart distinctly different metabolic responses on developing embryos, as demonstrated by their protein-synthetic capacity. Only in the presence of osmoticum do embryos synthesize proteins which are distinctly recognizable as those synthesized by developing embryos in situ, i.e. when inside the pod. Abscisic acid induces the synthesis of a few unique proteins, but these arise even in mature embryos treated with ABA. Thus while both osmoticum and ABA prevent precocious germination, their effects on the synthetic capacity of the developing embryo are quite distinct. Since seeds with low endogenous ABA do not germinate, osmotic regulation may be the more important of these two factors in controlling seed development.

Germination of Arabidopsis thaliana seeds is not completed as a result of elongation of the radicle but of the adjacent transition zone and lower hypocotyl

The completion of germination of seeds of Arabidopsis thaliana is marked by the appearance of the radicle through the surrounding endosperm and testa. Using confocal microscopy and green fluorescent protein (GFP)-transformed embryos to highlight the epidermal cell walls it has been possible to conduct time-lapse photography of individual embryos during their germination. This reveals that the elongation of embryo cells to effect completion of germination does not occur within the radicle itself, but rather within a discrete region that is immediately proximal to the radicle. This region, identifiable as the lower hypocotyl and hypocotyl-radicle transition zone, is also definable by accumulation of carbohydrate-containing bodies during germination, and distinct GFP expression of GAL4-GFP in enhancer trap lines. Flow cytometric studies show that there is an increase in the proportion of 4C nuclei in the axis which coincides with a considerable increase in length of the hypocotyl, and the occurrence of endopolyploid (8C and 16C) nuclei accompanies the 2-fold increase in mean cell size in the region of elongation, the lower hypocotyl, and hypocotyl-radicle transition zone. Thus the observed cell elongation during germination is accompanied by an increase in nuclear DNA content, and the resultant elongation of the axis to effect radicle emergence is due to cell expansion, not to cell division. When studying the molecular events involved in the completion of germination, therefore, it may be prudent to focus on this region of elongation.

procera is a putative DELLA mutant in tomato (Solanum lycopersicum): effects on the seed and vegetative plant

The procera (pro) mutant of tomato exhibits a well-characterized constitutive gibberellic acid (GA) response phenotype. The tomato DELLA gene LeGAI in the pro mutant background contains a point mutation that results in an amino acid change in the conserved VHVID putative DNA-binding domain in LeGAI to VHEID. This same point mutation is in four different genetic backgrounds exhibiting the pro phenotype, suggesting that this mutation co-segregates with the pro phenotype. Complementation of the mutant with a constitutively expressed wild-type LeGAI gene sequence was not conclusive due to the infertility of transgenic plants. The pro mutation alters tomato branching architecture through differential suppression of axillary bud development, indicating a role for DELLA proteins in the regulation of plant structure. Isolated gib-1 pro double mutant embryo axes, which are unable to synthesize GA, germinate faster than their wild-type counterparts, and exert greater embryo growth potential. The pro mutation is therefore regulating GA responses within the tomato embryo. Transient expression of a LeGAI-GFP (green fluorescent protein) fusion protein in onion epidermis results in its location to the nucleus, and this protein is rapidly degraded by the proteasome in the presence of GA.

A dual rôle for the endosperm and its galactomannan reserves in the germinative physiology of fenugreek (Trigonella foenum-graecum L.), an endospermic leguminous seed.

Some 30% of the reserve material in the fenugreek seed is galactomannan localised in the endosperm; the remainder is mainly protein and lipid in the cotyledons of the embryo. The importance of galactomannan to the germinative physiology of fenugreek has been investigated by comparing intact and endosperm-free seeds. From a purely nutritional point of view the galactomannans rôle is not qualitatively different from that of the food reserves in the embryo. Nevertheless, due to its spatial location and its hydrophilic properties, the galactomannan is the molecular basis of a mechanism whereby the endosperm imbibes a large quantity of water during seed hydration and is able to “buffer” the germinating embryo against desiccation during subsequent periods of drought-stress. The galactomannan is clearly a dual-purpose polysaccharide, regulating water-balance during germination and serving as a substrate reserve for the developing seedling following germination. The relative importance of these two rôles is discussed.Some 30% of the reserve material in the fenugreek seed is galactomannan localised in the endosperm; the remainder is mainly protein and lipid in the cotyledons of the embryo. The importance of galactomannan to the germinative physiology of fenugreek has been investigated by comparing intact and endosperm-free seeds. From a purely nutritional point of view the galactomannans rôle is not qualitatively different from that of the food reserves in the embryo. Nevertheless, due to its spatial location and its hydrophilic properties, the galactomannan is the molecular basis of a mechanism whereby the endosperm imbibes a large quantity of water during seed hydration and is able to “buffer” the germinating embryo against desiccation during subsequent periods of drought-stress. The galactomannan is clearly a dual-purpose polysaccharide, regulating water-balance during germination and serving as a substrate reserve for the developing seedling following germination. The relative importance of these two rôles is discussed.

Endo-β-mannanase isoforms are present in the endosperm and embryo of tomato seeds, but are not essentially linked to the completion of germination

A current hypothesis is that endo-β-mannanase activity in the endosperm cap of tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds is induced by gibberellin (GA) and weakens the endosperm cap thus permitting radicle protrusion. We have tested this hypothesis. In isolated parts, the expression of endo-β-mannanase in the endosperm after germination is induced by GAs, but the expression of endo-β-mannanase in the endosperm cap prior to radicle protrusion is not induced by GAs. Also, abscisic acid (ABA) is incapable of inhibiting endo-β-mannanase activity in the endosperm cap, even though it strongly inhibits germination. However, ABA does inhibit enzyme activity in the endosperm and embryo after germination. There are several isoforms in the endosperm cap and embryo prior to radicle protrusion that are tissue-specific. Tissue prints showed that enzyme activity in the embryo spreads from the radicle tip to the cotyledons with time after the start of imbibition. The isoform and developmental patterns of enzyme activity on tissueprints are unaffected when seeds are incubated in ABA, even though germination is inhibited. We conclude that the presence of endo-β-mannanase activity in the endosperm cap is not in itself sufficient to permit tomato seeds to complete germination.

Down-Regulation of DELLA Genes Is Not Essential for Germination of Tomato, Soybean, and Arabidopsis Seeds

The relationship between expression of a negative regulator of GA signal transduction (RGL2) belonging to the DELLA gene family and repression of Arabidopsis seed germination has been studied (Lee S, Cheng H, King KE, Wang W, He Y, Hussain A, Lo J, Harberd NP, Peng J [2002] Genes and Development 16: 646–658). There is one DELLA gene (LeGAI) present in tomato (Lycopersicon esculentum Mill.), which is expressed in both vegetative and reproductive tissues. During germination of wild-type tomato seed, there was no decline in the expression of LeGAI in either the embryo or the endosperm. Rather, LeGAI transcripts increased in these tissues following imbibition and remained high during and following germination. A similar increase in LeGAI transcripts occurred in the endosperm and embryo of GA-treated gib-1 mutant seed during and following germination. Likewise in soybean (Glycine max) seed, there was no decline in the expression of two DELLA genes in the radicle before or after germination. Upon reexamination of RGL2 in Arabidopsis seeds, a decline in its expression was noted but only after radicle emergence, i.e. after germination had been completed. Taken together, these data are consistent with GA-induced down-regulation of DELLA genes not being a prerequisite for germination of tomato, soybean, and Arabidopsis seeds.

Molecular cloning of a cDNA encoding a (1-4)-β-mannan endohydrolase from the seeds of germinated tomato (Lycopersicon esculentum)

Abstract. Mannose-containing polysaccharides are widely distributed in cell walls of higher plants. During endosperm mobilization in germinated tomato seeds (1→4)-β-mannan endohydrolases (EC 3.2.1.78) participate in the enzymic depolymerization of these cell wall polysaccharides. A cDNA encoding a (1→4)-β-mannanase from the endosperm of germinated tomato (Lycopersicon esculentum Mill.) seeds has been isolated and characterized. The amino acid sequence deduced from the 5′-region of the cDNA exactly matches the sequence of the 65 NH2-terminal amino acids determined directly from the purified enzyme. The mature enzyme consists of 346 amino acid residues, it has a calculated Mr of 38 950 and an isoelectric point of 5.3. Overall, the enzyme exhibits only 28–30% sequence identity with fungal (1→4)-β-mannanases, but more highly conserved regions, which may represent catalytic and substrate-binding domains, can be identified. Based on classification of the tomato (1→4)-β-mannanase as a member of the family 5 group of glycosyl hydrolases, Glu-148 and Glu-265 would be expected to be the catalytic acid and the catalytic nucleophile, respectively. Southern hybridization analyses indicate that the enzyme is derived from a family of about four genes. Expression of the genes, as determined by the presence of mRNA transcripts in Northern hybridization analyses, occurs in the endosperm of germinated seeds; no transcripts are detected in hypocotyls, cotyledons, roots or leaves.