Proteolytic activity in the maize pollen wall

A new protease from maize ( Zea mays L.) pollen is described. It was purified using gel filtration, ion exchange and high performance liquid chromatography. SDS-PAGE and HPLC showed that the enzyme has a dimeric structure of M, ca 60,000. Inhibitor investigations indicated an aspartic acid residue in its active site. The optimum pH for maize pollen aspartic proteinase activity was 5.6, and the optimum temperature was 45°C. The enzyme is easily eluted from the pollen grains and, as confirmed by enzymoblotting after isoelectric focusing, it is located in the pollen wall. Similar to metallo-proteinases, its activity is inhibited by Zn₂₊. The pL value for purified aspartic proteinase, as estimated after IEF, was 5.0. Two-dimensional electrophoresis analysis of proteins eluted from maize pistils suggests that the enzyme digests the proteins and may be involved in pollen-tube germination. The properties of serine and aspartic proteinases from maize pollen are compared.     

Content of low-molecular-weight thiols during the imbibition of Pea seeds

The metabolism of low-molecular-weight thiols was investigated in seeds of Pisum sativum L. cv. Kleine Rheinländerin during imbibition in water for 14 h. The amount of oxidized glutathione (GSSG) decreased from 319 nmol (g dry weight)⁻¹ in dry seeds to 38 nmol (g dry weight)⁻¹ within the first 14 h of imbibition. The decrease may have been due to the reduction of GSSG to reduced glutathione (GSH), catalyzed by the enzyme glutathione reductase (GR; EC 1.6.4.2). The enzyme activity was high in dry seeds [25 nkat (g dry weight)⁻¹] and decreased to 20 nkat (g dry weight)⁻¹ within 14 h of imbibition. The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) decreased from 100 nkat (g dry weight)⁻¹ in dry seeds to 67 nkat (g dry weight)⁻¹ after 14 h of imbibition. Within 14 h the amount of γ-glutamyl-cysteine (γ-GC) decreased from 135 to 38 nmol (g dry weight)⁻¹, whereas the cysteine content rose from 81 nmol (g dry weight)⁻¹ in dry seeds to a maximum of 170 nmol (g dry weight)⁻¹ after 12 h of imbibition, which may be due to the degradation of γ-GC into cysteine.     

Energy charge during aerobic and anaerobic imbibition of Kalanchoë blossfeldiana seeds

Abstract Adenosine phosphate levels were measured in Kalanchoë blossfeldiana cv. feuerblüte seeds during dark imbibition in aerobic and anaerobic conditions. A correlation between the depth of primary dormancy (light requirement) and the increase of ATP-content during the first hours of aerobic imbibition was found, but the rise of energy charge from about 0.2 in dry seeds to 0.8 after 18 h is not related to the breaking of dormancy and consequently to germination. In an anaerobic environment, the increase in ATP-content is dramatically lowered and the energy charge value stabilizes at about 0.4, most probably as the result of fermentation activity.     

Role of the testa in preventing cellular rupture during imbibition of legume seeds

Studies with the seeds of soybean, navy bean, pea, and peanut were made to determine the extent of leakage of intracellular enzymes during imbition. Embryos with intact testae from all four species were found to leak detectable activities of either intracellular enzymes of the cytosol (glucose-6-phosphate dehydrogenase) or enzymes found in both the cytosol and organelles (malate dehydrogenase, glutamate dehydrogenase, glutamate oxaloacetate transaminase, and NADP-isocitrate dehydrogenase) after 6 hours imbition at 25 C. Pea and peanut embryos with testae leaked considerably lower levels of activity for these enzymes than did those of soybean and bean. Leakage of mitochondrial marker enzymes (fumarase, cytochrome c oxidase, and adenylate kinase) was not detected from embryos with testae, suggesting that a differential diffusion of intracellular components out of cells occurred. Soybean and bean embryos without testae leaked high, and proportionally (per cent dry seed basis) similar, levels of all cytosol, cytosol-organelle, and mitochondrial marker enzymes and protein during imbibition, indicating that cell membranes were not differential to leakage and that they had ruptured. Pea and peanut embryos without testae leaked detectable activities of all cytosol and cytosol-organelle enzymes, although fumarase was the only detectable mitochondrial marker enzyme leaked, suggesting that some degree of differential leakage may have occurred in these species. The outermost layers of embryo cells of seeds without testae of all four species absorbed and sequestered the nonpermeating pigment Evan's blue after 5 to 15 minutes imbibition, indicating that membranes had ruptured. This occurred to a much lesser extent in seeds with intact testae. Both soybean and bean embryos without testae were observed to disintegrate during imbibition, whereas those of pea and peanut did not. These data indicate that seeds of certain legumes are susceptible to cellular rupture during imbibition when seed coats are damaged or missing.     

The outermost cuticle of soybean seeds: chemical composition and function during imbibition

Seeds of different cultivars of Glycine max (L.) Merr. (soybean) have strikingly different rates of water imbibition. Seeds that readily imbibe water are termed 'soft', while those that remain non-permeable, even after several days in water, are referred to as 'hard', 'stone', or 'impermeable' seeds. What prevents soybean hard seeds from taking up water? Previous work established that the initial imbibition of soft soybean seeds correlates with the presence of small cracks in the outermost cuticle that covers the seed coat, prompting a detailed analysis of soybean seed coat cutin. In this paper, it is shown that the outermost cuticle of the seed coat has an unusual chemical composition, lacking typical mid-chain-hydroxylated fatty acids but being relatively rich in other types of hydroxylated fatty acids. The cuticle of the impermeable cultivar studied contained a disproportionately high amount of hydroxylated fatty acids relative to that of the permeable ones. Moreover, a brief treatment with hot alkali released the omega-hydroxy fatty acid component of the outermost cuticle and created holes in it that caused the seeds to become permeable. This demonstrates that the outermost cuticle of the seed is the critical structure that prevents water uptake by hard seeds.     

Effect of some nitrogenous salts on nitrogen transfer and protease activity in germinatingZea mays L. seeds

Maize seeds were allowed to germinate in the presence of different nitrogenous salts for 72 h. Changes in the ethanol soluble and insoluble nitrogen were studied in the embryo and in the endosperm. Supply of Ca(NC₃)₂ enhanced germination and protease activity in the endosperm resulting in greater solubilisation of protein to soluble nitrogen in the seeds. NH₄NO₃ and (NH₄)₂SO₄ were less effective as compared to Ca(NO₃)₂. Cycloheximide inhibited germination and protease activity. Pretreatment also resulted in increase in growth, soluble and insoluble nitrogen, and nitrate reductase activity in the primary leaves. Ca(NO₃)₂ was more effective than NH₄NO₃ and (NH₄)₂SO₄.     

Endomycorrhizal fungi in nitrogen transfer from soybean to maize

Using ¹⁵N as a tracer, interspecific N-transfer was studied during the course of plant development. The use of barriers of differing permeabilities between donor and receiver plants allowed separation of the effect of mycorrhizal colonization, root or hyphal contact and interplant hyphal bridging, on ¹⁵N-transfer from soybean (Glycine max (L.) Merrill) to maize (Zea mays L.). More transfer was measured between mycorrhizal plants, but transport of ¹⁵N from the labelled host plant to Glomus versiforme (Karsten) Berch did not seem to occur at the symbiotic interface, suggesting that the fungus is independent of its host for its N-nutrition, and that the role of hyphal bridges in N-transfer between plants, is not significant. Uptake by the receiver plant of the N excreted by the donor plant root system appears to be the mechanism of N-transfer between plants. The factor most affecting ¹⁵N-transfer between plants was found to be the extent of the contact between plant root systems. The presence of the endomycorrhizal fungus in plant roots reduced ¹⁵N-loss from soybean, but at the same time, its extensive hyphal network improved the efficiency of the maize root system for the recovery of the ¹⁵N excreted by soybeans. The net result was a better conservation of the N resource within the plant system. The transfer of N between mycorrhizal plants was particularly enhanced by the death of the soybean.     

Expression profile of maize (Zea mays) scutellar epithelium during imbibition

The scutellum is a shield-shaped structure surrounding the embryo axis in grass species. The scutellar epithelium (Sep) is a monolayer of cells in contact with the endosperm. The Sep plays an important role during seed germination in the secretion of gibberellins and hydrolytic enzymes and in the transport of the hydrolized products to the growing embryo. We identified 30 genes predominantly expressed after imbibition in the Sep as compared to other parts of the scutellum. A high proportion of these genes is involved in metabolic processes. Some other identified genes are involved in the synthesis or modification of cell walls, which may be reflected in the changes of cell shape and cell wall composition that can be observed during imbibition. One of the genes encodes a proteinase that belongs to a proteinase family typical of carnivorous plants. Almost nothing is known about their role in other plants or organs, but the scutellar presence may point to a "digestive" function during germination. Genes involved in the production of energy and the transport of peptides were also identified.     

Enzyme activities during imbibition and germination of seeds of tamarack (Larix laricina)

Activities of six enzymes from extracts of separated embryos and gametophytes of tamarack [ Larix laricina (Du Roi) K. Koch] seeds were assayed at various stages of imbibition and germination. On a per seed part basis, activities of 6-phosphogluconate dehydrogenase (6-PGD, EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G-6-PD, EC 1.1.1.49), malate dehydrogenase (NAD⁺–MDH, EC 1.1.1.37), isocitrate dehydrogenase (NADP⁺–IDH, EC 1.1.1.42), soluble peroxidase (PER, EC 1.11.1.7), and acid phosphatase (ACP, EC 3.1.3.2) from both the embryo and gametophyte tissues generally increased slowly, following cold stratification for 30 days and imbibition under germinating conditions for 5 days, but then increased at a faster rate with emergence of the radicle and subsequent growth of the seedling. The rate of increase of enzyme activity was highest for PER. Soluble protein levels also increased with imbibition and germination, with about 3 times greater levels present in the gametophyte than in the embryo. Heat inactivation experiments showed that, except for G-6-PD, activities were stable up to 40°C. Inactivation occurred at lower temperatures for G-6-PD, while higher temperatures were required for PER. Incubation of extracts for 7 days at 4°C indicated that loss of enzyme activity was greatest for G-6-PD (3.9% remaining) and least for PER and ACP (94 and 95% remaining, respectively).     

Massive cellular disruption occurs during early imbibition of Cuphea seeds containing crystallized triacylglycerols

The transition from anhydrobiotic to hydrated state occurs during early imbibition of seeds and is lethal if lipid reserves in seeds are crystalline. Low temperatures crystallize lipids during seed storage. We examine the nature of cellular damage observed in seeds of Cuphea wrightii and C. lanceolata that differ in triacylglycerol composition and phase behavior. Intracellular structure, observed using transmission electron microscopy, is profoundly and irreversibly perturbed if seeds with crystalline triacylglycerols are imbibed briefly. A brief heat treatment that melts triacylglycerols before imbibition prevents the loss of cell integrity; however, residual effects of cold treatments in C. wrightii cells are reflected by the apparent coalescence of protein and oil bodies. The timing and temperature dependence of cellular changes suggest that damage arises via a physical mechanism, perhaps as a result of shifts in hydrophobic and hydrophilic interactions when triacylglycerols undergo phase changes. Stabilizers of oil body structure such as oleosins that rely on a balance of physical forces may become ineffective when triacylglycerols crystallize. Recent observations linking poor oil body stability and poor seed storage behavior are potentially explained by the phase behavior of the storage lipids. These findings directly impact the feasibility of preserving genetic resources from some tropical and subtropical species.This publication is dedicated in memory of Dr. Vincent Franceschi, a mentor and friend. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

Protein and nucleic acid synthesis during imbibition of dormant and after-ripened Vaccaria pyramidata seeds.

The regulation of nucleic acid and protein synthesis in dormant, thermodormant, and after-ripened embryos of Vaccaria pyramidata (Caryophyllaceae) has been studied. Germination of after-ripened V. pyramidata seeds is prevented by inhibitors of protein, RNA, and DNA synthesis. The synthesis of both protein and RNA is activated at the beginning of imbibition, whereas [³H]thymidine incorporation does not start until the second period of the imbibition phase. [³H]Thymidine incorporation is greatly reduced in embryos treated with cycloheximide or 6-methylpurine. There is no correlation between the level of [³H]uracil and l-[¹⁴C]leucine incorporation into macromolecules and the physiological state of the seeds: tRNA, ribosomal RNA, and poly(A)-containing RNA (probably mRNA) as well as proteins are synthesized at the same rate in both dormant and thermodormant embryos as in after-ripened embryos. The protein patterns of dormant and after-ripened embryos are similar, as shown by electrophoresis and electrofocusing of double-labeled proteins. The level of DNA synthesis, measured as [³H]thymidine incorporation, may, on the other hand, indicate the physiological activity of the seeds: [³H]Thymidine is incorporated at a high rate in after-ripened embryos only and remains at a low level in dormant or thermodormant embryos. This correlation is, however, observed only in the axes. DNA synthesis in the cotyledons does not show any relation to the developmental stage of the seeds. These results are discussed in relation to the regulation of dormancy and after-ripening of seeds.     

Rapid endocytosis is triggered upon imbibition in Arabidopsis seeds

During seed imbibition and embryo activation, rapid change from a metabolically resting state to the activation of diverse extracellular and/or membrane bound molecules is essential and, hence, endocytosis could be activated too. In fact, we have documented endocytic internalization of the membrane impermeable endocytic tracer FM4–64 already upon 30 min of imbibition of Arabidopsis seeds. This finding suggest that endocytosis is activated early during seed imbibition in Arabidopsis. Immunolocalization of rhamnogalacturonan-II (RG-II) complexed with boron showed that whereas this pectin is localized only in the cell walls of dry seed embryos, it starts to be intracellular once the imbibition started. Brefeldin A (BFA) exposure resulted in recruitment of the intracellular RG-II pectin complexes into the endocytic BFA-induced compartments, confirming the endocytic origin of the RG-II signal detected intracellularly. Finally, germination was significantly delayed when Arabidopsis seeds were germinated in the presence of inhibitors of endocytic pathways, suggesting that trafficking of extracellular molecules might play an important role in the overcome of germination. This work constitutes the first demonstration of endocytic processes during germination and opens new perspectives about the role of the extracellular matrix and membrane components in seed germination.     

Investigations on protein- and ribonucleic acid synthesis during imbibition of inhibited or light-induced lettuce seeds

The relationships between protein- and RNA synthesis and the germination behaviour of lettuce seeds were studied. Protein synthesis starts right at the beginning of imbibition and increases until the radicle protrudes. According to our results the causes for a blocked development of scotodormant lettuce seeds cannot be seen in a generally reduced protein or RNA synthesis.     

Induction of acid phosphatase activity during germination of maize (Zea mays) seeds.

Acid phosphatase activity (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) increased during the first 24 h of maize (Zea mays) seed germination. The enzyme displayed a pH optimum of 4.5-5.5. Catalytic activity in vitro displayed a linear time course (60 min) and reached its half maximum value at 0.47 mM p-nitrophenyl phosphate (pNPP). Phosphatase activity towards phosphoamino acids was greatest for phosphotyrosine. The phosphatase activity was strongly inhibited by ammonium molybdate, vanadate and NaF and did not require divalent cations for the catalysis. The temperature optimum for pNPP hydrolysis was 37 degrees C. Under the same conditions, no enzyme activity was detected with phytic acid as substrate. Western blotting of total homogenates during seed germination revealed proteins/polypeptides that were phosphorylated on tyrosine residues; a protein of approximately 14 kDa is potentially a major biological substrate for the phosphatase activity. The results presented in this study suggest that the acid phosphatase characterized under the tested conditions is a member of the phosphotyrosine phosphatase family.     

Embryonic protein composition and synthesis during imbibition of white lupin seeds: effect of low temperatures

White lupin seed imbibition under low temperature conditions (8 °C) and their subsequent effects on embryonic protein composition and synthesis were investigated. The response to low temperatures was accompanied by changes in polypeptide composition and synthesis. The embryonic axes labelled in vivo with (³⁵S)-methionine retained their capacity to synthesize proteins during imbibition at 8 °C. The synthesis of some polypeptides was increased during low temperature treatment as compared to that at 25 °C. These cold-induced polypeptides were essentially detected in a molecular mass range from 15 to 35 kDa and a pI range from 6.3 to 8.7.     

Differential specificity in water imbibition of indian arid zone seeds

Studies on seeds of 40 arid zone plant species have revealed that seeds exhibit differential specificity in water inhibition percentage. Most of these seeds show very poor imbibition capacity and that takes place within the first three hours, after which this uptake becomes very slow. This low hydrature and quick water uptake wherever it takes place is correlated with the erratic rainfall in the Indian arid zone. Most of the leguminous seeds are impermeable to water because of hard seed coatedness. However, those seeds which imbibed showed a very high percentage of water uptake.