Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds II. Scutellum as the Site of Sucrose Synthesis

In a close parallel to the developmental pattern of α-amylase activity, a rapid increase of maltase activity occurred in the endosperm tissue of germinating rice seeds after about 4 days of the seed imbibition. The overall pattern of the 2 hydrolytic enzyme activities strongly suggest that amylolytic breakdown is the major metabolic route of starch utilization in the germinating rice seeds. Results of the chemical analyses of sugar constituents as well as the measurements of sucrose synthetase activity show that the scutellum is the site of sucrose synthesis in the germinating rice seeds. It is thus supported that glucose derived from the reserve starch in endosperm is transported to scutellum, where it is converted to sucrose. Sucrose is further mobilized to the growing tissues, shoots and roots.     

The relationship between carbohydrate composition of some tree seeds and their longevity

Fresh mature tree seeds of 16 species plus soybean were usedfor the analysis of soluble neutral sugar content, as well asfor the determination of longevity in terms of the time requiredfor the seeds to decrease to 50% of the original germinationunder 5C and 80–91% relative humidity. Oligosaccharideswere sometimes found in species of recalcitrant; although themass ratio of oligosaccharide/total sugar or mole ratio of stachyoseand raffinose/sucrose was less than 0.05 or 0.044, respectively.The time required for seeds to decrease to 50% germination variedfrom a few days to 8.3 months for the seeds of desiccation-sensitivespecies. The low ratio of oligosaccharide to sucrose is, however,most unlikely to be a cause of short life-span in recalcitrantseeds. It is suggested that 0.05 mole of oligosaccharide needs to beassociated with one mole sucrose to confer the seeds with desiccationtolerance. Orthodox seeds which have a high ratio of oligosaccharide/totalsugars happened to have a low ratio of disaccharide/total sugarindicating active biosynthetic activity of oligosaccharide.There was a positive correlation between the longevity and themass ratio of oligosaccharide/total sugar or oligo-/disaccharidefor those desiccation-tolerant seeds tested. These results supportthe conclusion that the ratio of oligo-/disaccharide plays arole in the desiccation tolerance and, consequently, the longevityof orthodox seeds. Key words: Tree seed, storage behaviour, recalcitrant, soluble carbohydrate, oligosaccharide, seed longevity     

Sucrose non‐fermenting kinase 1 (SnRK1) coordinates metabolic and hormonal signals during pea cotyledon growth and differentiation

Seed development passes through developmental phases such as cell division, differentiation and maturation: each have specific metabolic demands. The ubiquitous sucrose non‐fermenting‐like kinase (SnRK1) coordinates and adjusts physiological and metabolic demands with growth. In protoplast assays sucrose deprivation and hormone supplementation, such as with auxin and abscisic acid (ABA), stimulate SnRK1‐promoter activity. This indicates regulation by nutrients: hormonal crosstalk under conditions of nutrient demand and cell proliferation. SnRK1‐repressed pea (Pisum sativum) embryos show lower cytokinin levels and deregulation of cotyledonary establishment and growth, together with downregulated gene expression related to cell proliferation, meristem maintenance and differentiation, leaf formation, and polarity. This suggests that at early stages of seed development SnRK1 regulates coordinated cotyledon emergence and growth via cytokinin‐mediated auxin transport and/or distribution. Decreased ABA levels and reduced gene expression, involved in ABA‐mediated seed maturation and response to sugars, indicate that SnRK1 is required for ABA synthesis and/or signal transduction at an early stage. Metabolic profiling of SnRK1‐repressed embryos revealed lower levels of most organic and amino acids. In contrast, levels of sugars and glycolytic intermediates were higher or unchanged, indicating decreased carbon partitioning into subsequent pathways such as the tricarbonic acid cycle and amino acid biosynthesis. It is hypothesized that SnRK1 mediates the responses to sugar signals required for early cotyledon establishment and patterning. As a result, later maturation and storage activity are strongly impaired. Changes observed in SnRK1‐repressed pea seeds provide a framework for how SnRK1 communicates nutrient and hormonal signals from auxins, cytokinins and ABA to control metabolism and development.     

Differential Changes in Levels of mRNAs during Maturation of Wheat Seeds That Are Susceptible and Resistant to Preharvest-Sprouting

Seed dormancy is strongly related to the physiological conditions,especially as they relate to responsiveness to ABA, of embryocells during maturation of seeds. In this study, seeds of Triticumaestivum L. cv. Chihoku, which showed nondormancy at harvest,and line Kitakei-l354 (referred to as Kitakei), which showedpost-harvest dormancy, were collected 30 days after anthesis(DPA 30) and at the mature stage (DPA 60). Poly(A)⁺RNA was extractedfrom the embryos of the seeds and translated in a wheat germsystem. The majority of products of translation from the twogenotypes migrated to the same positions in two-dimensionalgels. Levels of six (for polypeptides h, i, k, m, n, and o)out of 14 Chihoku-specific mRNAs decreased dramatically duringseed maturation, concurrently with the loss of dormancy. Bycontrast, levels of 3 (for polypeptides c, e and f) out of 6Kitakei specific mRNAs were maintained during maturation andduring a 48-h imbibition of the dormant seeds but decreasedat germination. Polypeptides n of Chihoku and e of Kitakei hadthe same molecular size and slightly different pI values. Thesetwo polypeptides may be encoded by the same gene and may playsome role in the maintenance of seed dormancy. Levels of mRNAsfor 10 polypeptides, found in both Chihoku and Kitakei embryosat DPA 30, changed to different extents during maturation. Outof the 10 mRNAs, the relative abundance of 4 mRNAs of Kitakeidid not change dramatically during seed maturation, while inChihoku these mRNAs decreased in level or disappeared duringthe same maturation period. In addition, levels of 2 of these4 mRNAs did not decrease significantly during imbibition ofthe dormant Kitakei seeds but disappeared upon treatment forbreaking of dormancy. The maintenance of these mRNAs in thedormant seeds during maturation and imbibition suggests thatthe respective gene products are involved in the maintenanceof dormancy in wheat seeds. (Received December 5, 1991; Accepted April 1, 1992)     

Changes in Seed Constituents During Germination and Seedling Growth of Precociously Matured Soybean Seeds(Glycine max)

During 7 d of precocious maturation of soybean seed (Glycinemax), the starch content declined and soluble sugar levels increasedin patterns similar to natural seed dehydration and maturation.Total seed protein content and total seed dry weight increasedwhereas oil content remained relatively unchanged. Overall,the proportions of the constituents in precociously maturedseeds were comparable to naturally mature seeds. Precociouslymatured soybean seeds showed much the same germination and seedlinggrowth frequency patterns as naturally matured seeds. Duringgermination and seedling growth of precociously matured seeds,starch, soluble sugar, protein and oil levels followed patternssimilar to naturally mature, germinating seeds and seedlings.Therefore, precocious maturation may be used as a model systemto investigate the control of the physiological and biochemicalevents occurring during seed maturation which lead to germinationand subsequently, seedling growth. Glycine max (L.) Merr., soybean, cotyledons, maturation, germination/seedling growth     

Changes in starch,oligosaccharides and soluble sugars in developing pod wall and seed of chickpea

The accumulation of starch in the seed of chickpea accompanied by a decline in the pod wall during the early stages of development probably indicates that seed and pod wall did not compete with each other for starch accumulation. During the early stages of maturation, the reducing and non-reducing sugars showed a decline in seeds whereas non-reducing sugars decreased in the pod wall. The accumulation of the oligosaccharides raffinose, stachyose, and some other unidentified oligosaccharides was accompanied by a decline in the mono- and disaccharides in the developing seeds.     

Temporal profiling of the heat-stable proteome during late maturation of Medicago truncatula seeds identifies a restricted subset of late embryogenesis abundant proteins associated with longevity

Developing seeds accumulate late embryogenesis abundant (LEA) proteins, a family of intrinsically disordered and hydrophilic proteins that confer cellular protection upon stress. Many different LEA proteins exist in seeds, but their relative contribution to seed desiccation tolerance or longevity (duration of survival) is not yet investigated. To address this, a reference map of LEA proteins was established by proteomics on a hydrophilic protein fraction from mature Medicago truncatula seeds and identified 35 polypeptides encoded by 16 LEA genes. Spatial and temporal expression profiles of the LEA polypeptides were obtained during the long maturation phase during which desiccation tolerance and longevity are sequentially acquired until pod abscission and final maturation drying occurs. Five LEA polypeptides, representing 6% of the total LEA intensity, accumulated upon acquisition of desiccation tolerance. The gradual 30-fold increase in longevity correlated with the accumulation of four LEA polypeptides, representing 35% of LEA in mature seeds, and with two chaperone-related polypeptides. The majority of LEA polypeptides increased around pod abscission during final maturation drying. The differential accumulation profiles of the LEA polypeptides suggest different roles in seed physiology, with a small subset of LEA and other proteins with chaperone-like functions correlating with desiccation tolerance and longevity.     

Repressing the expression of the SUCROSE NONFERMENTING-1-RELATED PROTEIN KINASE gene in pea embryo causes pleiotropic defects of maturation similar to an abscisic acid-insensitive phenotype

The classic role of SUCROSE NONFERMENTING-1 (Snf1)-like kinases in eukaryotes is to adapt metabolism to environmental conditions such as nutrition, energy, and stress. During pea (Pisum sativum) seed maturation, developmental programs of growing embryos are adjusted to changing physiological and metabolic conditions. To understand regulation of the switch from cell proliferation to differentiation, SUCROSE NONFERMENTING-1-RELATED PROTEIN KINASE (SnRK1) was antisense repressed in pea seeds. Transgenic seeds show maturation defects, reduced conversion of sucrose into storage products, lower globulin content, frequently altered cotyledon surface, shape, and symmetry, as well as occasional precocious germination. Gene expression analysis of embryos using macroarrays of 5,548 seed-specific genes revealed 183 differentially expressed genes in two clusters, either delayed down-regulated or delayed up-regulated during transition. Delayed down-regulated genes are related to mitotic activity, gibberellic acid/brassinosteroid synthesis, stress response, and Ca2+ signal transduction. This specifies a developmentally younger status and conditional stress. Higher gene expression related to respiration/gluconeogenesis/fermentation is consistent with a role of SnRK1 in repressing energy-consuming processes in maturing cotyledons under low oxygen/energy availability. Delayed up-regulated genes are mainly related to storage protein synthesis and stress tolerance. Most of the phenotype resembles abscisic acid (ABA) insensitivity and may be explained by reduced Abi-3 expression. This may cause a reduction in ABA functions and/or a disconnection between metabolic and ABA signals, suggesting that SnRK1 is a mediator of ABA functions during pea seed maturation. SnRK1 repression also impairs gene expression associated with differentiation, independent from ABA functions, like regulation and signaling of developmental events, chromatin reorganization, cell wall synthesis, biosynthetic activity of plastids, and regulated proteolysis.     

Soluble carbohydrates of dry and developing seeds

Sucrose was present in seeds of 31 species at all ages and stages of their development. The raffinose family of oligosaccharides is present in most mature and dry seeds; tomato and tobacco seeds contain planteose, whereas sesame seeds contain this sugar and a higher member of the planteose series. Cotton seeds contain raffinose, stachyose, verbascose and an unidentified ketose. Free monosaccharides were not detected in any of the dry seeds; although free glucose and fructose were detected in some immature seeds, these sugars decreased in amount and eventually disappeared during seed maturation. Sucrose, stachyose, raffinose and verbascose accumulated, in developing soybeans, in that sequence. Maltose, a sugar rarely found in plant tissues, is present in immature soybean and honey locust seeds but does not occur in the other seeds examined. It increases to a maximum during development, subsequently decreases in amount during maturation and ripening and eventually disappears completely. The petioles of old leaves and stems of the soybean plant contain maltose, but the petioles of young soybean leaves, empty pods, leaf blades and roots do not.     

The use of an ELISA to quantitate the extent of 11S globulin mobilization in untreated and primed sugar beet seed lots

Seed priming (controlled imbibition) is a widely used technique for improving crop establishment, because it allows a reduction of the time to radicle emergence following seed imbibition and synchronization of individual seeds within seed lots with respect to germination timing. The major problem encountered in seed priming is the control of seed imbibition to a level permitting pre-germinative processes to proceed but that blocks radicle emergence. If not, the consequence of drying back the seeds to initial moisture content for storage purposes could be a total loss of the treated batch. This is because, as long as radicle growth has not begun, seeds may be re-dried without any permanent deleterious effects upon subsequent germination or growth. Recently, we reported the discovery of a molecular marker of sugar beet seed priming, corresponding to the basic B-subunit of the seed storage protein 11S globulin. An ELISA based upon this molecular marker has been used to analyse how different sugar beet seed lots respond to a priming treatment. The results demonstrate that this ELISA allows us to readily distinguish between the primed seeds and the corresponding untreated seeds.     

Enzymic mechanism of starch breakdown in germinating rice seeds I. An analytical study

Time-sequence analyses of carbohydrate breakdown in germinating rice seeds shows that a rapid breakdown of starch reserve in endosperm starts after about 4 days of germination. Although the major soluble carbohydrate in the dry seed is sucrose, a marked increase in the production of glucose and maltooligosaccharides accompanies the breakdown of starch. Maltotriose was found to constitute the greatest portion of the oligosaccharides throughout the germination stage. α-Amylase activities were found to parallel the pattern of starch breakdown. Assays for phosphorylase activity showed that this enzyme may account for much smaller amounts of starch breakdown per grain, as compared to the amounts hydrolyzed by α-amylase. There was a transient decline in the content of sucrose in the initial 4 days of seed germination, followed by the gradual increase in later germination stages. During the entire germination stage, sucrose synthetase activity was not detected in the endosperm, although appreciable enzyme activity was present in the growing shoot tissues as well as in the frozen rice seeds harvested at the mid-milky stage. We propose the predominant formation of glucose from starch reserves in the endosperm by the action of α-amylase and accompanying hydrolytic enzyme(s) and that this sugar is eventually mobilized to the growing tissues, shoots or roots.     

Expression of a GALACTINOL SYNTHASE gene in tomato seeds is up-regulated before maturation desiccation and again after imbibition whenever radicle protrusion is prevented

Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance.     

Temperature effects on seed imbibition and leakage mediated by viscosity and membranes

The possible involvement of membranes and water viscosity in the temperature effects on imbibition and solute leakage of radish (Raphanus sativa var. Early Scarlet Globe) seeds and excised sugar pine (Pinus lambertiana Dougl.) embryos was evaluated. In these two seed materials, the temperature effect on initial rates of imbibition and solute leakage could be accounted for primarily by changes in water viscosity, the relationship being approximately linear. It appears that membranes are involved both in water uptake and solute leakage. Heat-killed radish seeds and sugar pine embryos exhibited significantly higher rates of imbibition and solute leakage than did viable ones. In addition, sugar pine embryos exhibited an abrupt change in rates of imbibition and solute leakage between 15 and 20°C, resulting in abnormally high water uptake and solute leakage above this temperature.