Involvement of glyoxysomal lipase in the hydrolysis of storage triacylglycerols in the cotyledons of soybean seedlings

The total cotyledon extract of soybean (Glycine max [L.] Merr. var. Coker 136) seedlings underwent lipolysis as measured by the release of fatty acids. The highest lipolytic activity occurred at pH 9. This lipolytic activity was absent in the dry seeds and increased after germination concomitant with the decrease in total lipids. Using spherosomes (lipid bodies) isolated from the cotyledons during the peak stage of lipolysis (5-7 days) as substrates, about 40% of the lipase activity was found in the glyoxysomes after organelle breakage had been accounted for; the remaining activity was distributed among other subcellular fractions but none was found in the spherosomal fraction. The glyoxysomal lipase had maximal activity at pH 9, and catalyzed the hydrolysis of tri-, di-, and monoacylglycerols of linoleic acid, the most abundant fatty acid in soybean. The spherosomes contained a neutral lipase that could hydrolyze monolinolein and N-methylindoxylmyristate, but not trilinolein. This spherosomal lipase activity dropped off rapidly during early seedling growth, preceding lipolysis. Spherosomes isolated from either dry or germinated seeds did not possess lipolytic activity, and spherosomes from germinated seeds but not from dry seeds could serve as substrates for the glyoxysomal lipase. It is concluded that the glyoxysomal lipase is the enzyme catalyzing the initial hydrolysis of storage triacylglycerols.     

Reserve mobilization and starch formation in soybean (Glycine max) cotyledons in relation to seedling growth

Brown, C. S. and Huber, S. C. 1988. Reserve mobilization and starch formation in soybean ( Glycine max ) cotyledons in relation to seedling growth. - Physiol. Plant. 72: 518–524.
In germinating soybean ( Glycine max [L.] Merr.) seedlings, starch accumulated in the cotyledons during the first 5 days of seedling growth. Among 10 genotypes, the amount of starch accumulated after 5 days was relatively independent of light and appeared to be primarily related to the amount of sucrose depleted from the cotyledons during the same time period. Depletion of other reserves (e.g. protein and lipid) were not closely correlated with starch formation. In addition, the differences in starch formation were not related to differences in activities of certain enzymes that may be involved in the conversion of sucrose to starch, namely starch synthase (EC 2.4.1.21), ADP-glucose pyrophosphorylase (EC 2.7.7.27), sucrose synthase (EC 2.4.1.13), neutral invertase (EC 3.2.1.26), and PPi-linked phosphofructokinase (PFP) (EC 2.7.1.90). Starch synthesis did not compete with seedling growth, because among 10 genotypes, transient starch formation was correlated positively with seedling growth and cotyledonary photosynthetic rates. We postulate that starch is derived primarily from stored sucrose in the cotyledons and is not merely a result of 'overflow' carbon from other reserves. Starch formation also appeared to have a positive relationship with both early (0 to 5 days) and later (5 to 13 days) seedling growth, the latter perhaps due to enhanced cotyledon photosynthetic rates.     

Subcellular Localization Studies Indicate That Lipoxygenases 1 to 6 Are Not Involved in Lipid Mobilization during Soybean Germination

Soybean (Glycine max) lipoxygenase (LOX) has been proposed to be involved in reserve lipid mobilization during germination. Here, subcellular fractionation studies show that LOX1, -2, -3, -4, -5, and -6 isozymes were associated with the soluble fraction but not with purified oil bodies. The purified oil bodies contained small amounts of LOX1 (<0.01% total activity), which apparently is an artifact of the purification process. Immunogold labeling indicated that, in cotyledon parenchyma cells of LOX wild-type seeds that had soaked and germinated for 4 d, the majority of LOX protein was present in the cytoplasm. In 4-d-germinated cotyledons of a LOX1/2/3 triple null mutant (L0), a small amount of label was found in the cytoplasm. In epidermal cells, LOX appeared in vacuoles of both wild-type and L0 germinated seeds. No LOXs cross-reacting with seed LOX antibodies were found to be associated with the cell wall, plasma membrane, oil bodies, or mitochondria. Lipid analysis showed that degradation rates of total lipids and triacylglycerols between the wild type and L0 were not significantly different. These results suggest that LOX1, -2, -3, -4, -5, and -6 are not directly involved in reserve lipid mobilization during soybean germination.     

Expression of zein in long term endosperm cultures of maize

Continuous cultures, established 10 days after pollination from endosperms of inbred A636 Zea mays (L.) were extracted 21 months later with aqueous ethanol. The solubilized proteins were analyzed by poly-acrylamide-sodium dodecyl sulfate gel electrophoresis. Two protein bands co-migrated with zein, the major storage protein of maize. Immunoblotting of the gel followed by incubation of the immobilized proteins with anti-zein IgG provided evidence that the polypeptides were in fact zein. Electron microscopic studies showed that the cultures contained cells with protein bodies as found in developing endosperms. The protein bodies could be isolated from the cultures and were shown to contain zein. We conclude that the long term cultures described here synthesize zein and deposit it in the form of protein bodies of the type found in developing endosperms. Thus, certain endosperm characteristics and the production of tissue-specific proteins are retained in prolonged culture.     

beta-Amylases in Cereals : A Study of the Maize beta-Amylase System

β-Amylase of maize (Zea mays L.) caryopses was studied during development and germination by means of enzymic, electrophoretic, and immunochemical techniques. β-Amylase activity increased during caryopsis development to a maximum value at the beginning of the water content plateau (at this stage the enzyme was located primarily within the pericarp) and then decreased. Almost no β-amylase (activity or antigen) was found in either free or bound forms in the mature maize caryopsis. The activity increased again during seedling growth and reached much higher values. Both the aleurone layer (to a major extent) and the scutellum produced and secreted β-amylase during germination, the secretion being stimulated by Ca²⁺. No posttranslational modification of the enzyme was detected during germination. The molecular specific activity of the enzyme remained unchanged during the observed periods, indicating that the regulation of the activity is based essentially on protein turnover. The enzyme from developing and germinating caryopses was found to be identical in terms of antigenicity, isoelectric point, and molecular mass to the β-amylases extracted from the roots and the leaves of the maize seedling. The maize β-amylase resembles in all respects the ubiquitous β-amylase described for rye and wheat, whereas the major β-amylase of those cereals appears to be lacking in the maize caryopsis.     

Red Light-Regulated Growth : I. Changes in the Abundance of Indoleacetic Acid and a 22-Kilodalton Auxin-Binding Protein in the Maize Mesocotyl

We examined the changes in the levels of indoleacetic acid (IAA), IAA esters, and a 22-kilodalton subunit auxin-binding protein (ABP1) in apical mesocotyl tissue of maize (Zea mays L.) during continuous red light (R) irradiation. These changes were compared with the kinetics of R-induced growth inhibition in the same tissue. Upon the onset of continuous irradiation, growth decreased in a continuous manner following a brief lag period. The decrease in growth continued for 5 hours, then remained constant at 25% of the dark rate. The abundance of ABP1 and the level of free IAA both decreased in the mesocotyl. Only the kinetics of the decrease in IAA within the apical mesocotyl correlated with the initial change in growth, although growth continued to decrease even after IAA content reached its final level, 50% of the dark control. This decrease in IAA within the mesocotyl probably occurs primarily by a change in its transport within the shoot since auxin applied as a pulse moved basipetally in R-irradiated tissue at the same rate but with half the area as dark control tissue. In situ localization of auxin in etiolated maize shoots revealed that R-irradiated shoots contained less auxin in the epidermis than the dark controls. Irradiated mesocotyl grew 50% less than the dark controls even when incubated in an optimal level of auxin. However, irradiated and dark tissue contained essentially the same amount of radioactivity after incubation in [¹⁴C]IAA indicating that the light treatment does not affect the uptake into the tissue through the cut end, although it is possible that a small subset of cells within the mesocotyl is affected. These observations support the hypothesis that R causes a decrease in the level of auxin in epidermal cells of the mesocotyl, consequently constraining the growth of the entire mesocotyl.     

Ultrastructure of Mature Embryos in the Parasitic Flowering PlantCuscuta japonica

The ultrastructural features of embryos were studied from mature dry and soaked seeds of the parasitic angiospermCuscuta japonica. Outer tangential walls in the protoderm cells were thickened and covered by a thin cuticle layer. These walls could play important roles in preventing water loss from theCuscuta seedling surfaces after germination and in strengthening the surfaces against various environmental stresses. In the protoderm cells of soaked embryos, lipid materials were released into the thick outer walls through the fusion of lipid bodies with the plasma membrane. In the dry embryos were stored a large number of protein bodies with globoid crystals and lipid bodies. Numerous lipid bodies also were aligned under the plasma membrane. In both dry and soaked embryos, protein bodies were digested and transformed into small vacuoles. The degraded reserves of the lipid and protein bodies could then be mobilized to nourish subsequent germination and seedling growth. Proplastids in the soaked embryo cells contained a few thylakoids and electron-dense plastoglobuli, and crystallized phytoferritin. The phytoferritin, an iron-protein complex, would also be utilized in chloroplast development for autotrophic seedling growth.     

Acquisition of membrane lipids by differentiating glyoxysomes: role of lipid bodies

Glyoxysomes in cotyledons of cotton (Gossypium hirsutum, L.) seedlings enlarge dramatically within 48 h after seed imbibition (Kunce, C.M., R.N. Trelease, and D.C. Doman. 1984. Planta (Berl.). 161:156-164) to effect mobilization of stored cotton-seed oil. We discovered that the membranes of enlarging glyoxysomes at all stages examined contained a large percentage (36-62% by weight) of nonpolar lipid, nearly all of which were triacylglycerols (TAGs) and TAG metabolites. Free fatty acids comprised the largest percentage of these nonpolar lipids. Six uncommon (and as yet unidentified) fatty acids constituted the majority (51%) of both the free fatty acids and the fatty acids in TAGs of glyoxysome membranes; the same six uncommon fatty acids were less than 7% of the acyl constituents in TAGs extracted from cotton-seed storage lipid bodies. TAGs of lipid bodies primarily were composed of palmitic, oleic, and linoleic acids (together 70%). Together, these three major storage fatty acids were less than 10% of both the free fatty acids and fatty acids in TAGs of glyoxysome membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) constituted a major portion of glyoxysome membrane phospholipids (together 61% by weight). Pulse-chase radiolabeling experiments in vivo clearly demonstrated that 14C-PC and 14C-PE were synthesized from 14C-choline and 14C-ethanolamine, respectively, in ER of cotyledons, and then transported to mitochondria; however, these lipids were not transported to enlarging glyoxysomes. The lack of ER involvement in glyoxysome membrane phospholipid synthesis, and the similarities in lipid compositions between lipid bodies and membranes of glyoxysomes, led us to formulate and test a new hypothesis whereby lipid bodies serve as the dynamic source of nonpolar lipids and phospholipids for membrane expansion of enlarging glyoxysomes. In a cell-free system, 3H-triolein (TO) and 3H-PC were indeed transferred from lipid bodies to glyoxysomes. 3H-PC, but not 3H-TO, also was transferred to mitochondria in vitro. The amount of lipid transferred increased linearly with respect to time and amount of acceptor organelle protein, and transfer occurred only when lipid body membrane proteins were associated with the donor lipid bodies. 3H-TO was transferred to and incorporated into glyoxysome membranes, and then hydrolyzed to free fatty acids. 3H-PC was transferred to and incorporated into glyoxysome and mitochondria membranes without subsequent hydrolysis. Our data are inconsistent with the hypothesis that ER contributes membrane lipids to glyoxysomes during postgerminative seedling growth.(ABSTRACT TRUNCATED AT 400 WORDS)     

Divergent properties of prolamins in wheat and maize

Cereal grains are an important nutritional source of amino acids for humans and livestock worldwide. Wheat, barley, and oats belong to a different subfamily of the grasses than rice and in addition to maize, millets, sugarcane, and sorghum. All their seeds, however, are largely devoid of free amino acids because they are stored during dormancy in specialized storage proteins. Prolamins, the major class of storage proteins in cereals with preponderance of proline and glutamine, are synthesized at the endoplasmic reticulum during seed development and deposited into subcellular structures of the immature endosperm, the protein bodies. Prolamins have diverged during the evolution of the grass family in their structure and their properties. Here, we used the expression of wheat glutenin-Dx5 in maize to examine its interaction with maize prolamins during endosperm development. Ectopic expression of Dx5 alters protein body morphology in a way that resembles non-vitreous kernel phenotypes, although Dx5 alone does not cause an opaque phenotype. However, if we lower the amount of γ-zeins in Dx5 maize through RNAi, a non-vitreous phenotype emerges and the deformation on the surface of protein bodies is enhanced, indicating that Dx5 requires γ-zeins for its proper subcellular organization in maize.     

The biosynthesis of ribonuclease and its accumulation in protein bodies in the cotyledons of mung bean seedlings

Germination and seedling growth of mung bean are accompanied by a 7- to 10-fold increase in the ribonuclease content of the cotyledons. The increase occurs during the first 4 days of seedling growth and precedes the senescence of the cotyledons. Separation of the RNases in the cotyledons by polyacrylamide gel electrophoresis indicates the presence of several minor bands in seeds imbibed for 24 hr. On the second day of seedling growth a new major band with an R_f of 0.76 is present. In 4- to 5-day old seedlings this major band accounts for nearly all the RNase activity in the tissue. The characteristics of this RNase show that it is a plant ribonuclease I (pH optimum of 5.0; MW 16,000; activity preferentially inhibited by purine nucleotides; no activity toward DNA; no phosphodiesterase activity). When the seedlings are grown in 66% D₂O the RNase activity undergoes a density shift of 0.61% indicating that the increase in enzyme activity is due to the de novo synthesis of the enzyme molecules. A method is described for the isolation of protein bodies from protoplasts of storage parenchyma cells. Fractionation of protoplast lysates on Ficoll gradients results in the recovery of a high proportion (75%) of intact protein bodies. On these gradients RNase activity comigrates with α-mannosidase, a protein body marker enzyme indicating that the newly synthesized RNase accumulates in the protein bodies. We suggest that the synthesis of RNase in the cotyledons and its accumulation in the protein bodies indicates that protein bodies may function in the degradation of cellular macromolecules other than the reserves stored within them.     

Regulation of Mitochondrial Function and Biogenesis in Cucumber (Cucumis sativus L.) Cotyledons during Early Seedling Growth

The aim of this work was to characterize the respiratory metabolism of the greening cotyledons of cucumber (Cucumis sativus L.) during early seedling growth and to investigate how this is integrated with changes in mitochondrial biogenesis and function. In light-grown cotyledons, lipid mobilization extended from germination to 6 days postimbibition, reaching a maximum at 3 to 4 days postimbibition. The rate of dark oxygen uptake reached a maximum at 2 days postimbibition in dark-grown and 3 days postimbibition in light-grown cotyledons. Development of photosynthetic capacity occurred from 4 to 7 days postimbibition. In dark-grown cotyledons, lipid mobilization extended beyond 7 days postimbibition, and there was no greening or acquisition of photosynthetic competence. Measurements of mitochondrial function indicated that the respiratory capacity of the tissue changed such that during lipid mobilization there was a much greater capacity for the operation of the nondecarboxylating portion of the citric acid cycle (succinate to oxaloacetate), whereas during the development of photosynthetic function the activity of the remainder of the cycle (oxaloacetate to succinate) was induced. Comparison of the maximum capacities for mitochondrial substrate oxidations in vitro with the rates of in vivo substrate oxidations, predicted from the rate of lipid breakdown, indicated that mitochondria in this tissue operate at or below state 4 rates, suggesting limitation by both availability of ADP and substrate.     

Molecular Cloning of Osmotin and Regulation of Its Expression by ABA and Adaptation to Low Water Potential

In response to adaptation to NaCl, cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) synthesize a major 26 kilodalton protein which has been named osmotin due to its induction by low water potentials. To help characterize the expression of osmotin in adapted cells, a cDNA clone for osmotin has been isolated. Abscisic acid induces messenger RNA encoding osmotin. Levels of this mRNA in adapted cells are approximately 15-fold higher than in unadapted cells. Message for osmotin is present at constant levels through the growth cycle of adapted cells, while in unadapted cells, the level decreases during exponential phase of growth and increases again when the cells approach stationary phase. While abscisic acid induces the message for osmotin, a low water potential environment appears to be required for accumulation of the protein. An osmotic shock to unadapted cells does not increase the amount of message or protein present most likely because this treatment does not induce immediately the accumulation of abscisic acid. The increased expression of osmotin in adapted cells is not correlated with an increase in osmotin gene copy number. Osmotin is homologous to a 24 kilodalton NaCl-induced protein in tomato, as well as thaumatin, maize α-amylase/trypsin inhibitor and a tobacco mosaic virus-induced pathogenesis-related protein.     

Effects of glucose starvation on mitochondrial subpopulations in the meristematic and submeristematic regions of maize root

Mitochondria isolated from 3-mm long maize (Zea mays L. var Dea) root tips were found to be heterogeneous on Percoll density gradients. The ultrastructure of these isolated mitochondria correlated well with that of mitochondria observed in situ and was consistent with the existence of mitochondria at different stages of maturation during cell development. The mitochondria of higher density presented an ultrastructure with many cristae and a dense matrix. These mitochondria showed classic respiratory properties, although with low ADP/O ratios. In contrast, the mitochondria of lower density showed few cristae and a clear matrix and did not seem to be fully functional because their rate of respiration was low and showed weak respiratory control. Lower- and higher- density mitochondria were shown to be differentially affected during the first stages of glucose starvation. The higher-density mitochondria from glucose-starved maize root tips retained the ultrastructure and most of the respiratory properties of nonstarved mitochondria, whereas lower- and intermediate-density mitochondria were absent in the mitochondrial preparations from glucose-starved maize root tips and were not observed in situ. Quantitatively, there was a decrease of the total mitochondrial pool when expressed as the amount of mitochondrial protein per root tip. However, this decrease affected low- and intermediate-density mitochondria, but not higher-density mitochondria. Thus, it was shown that a significant pool of functional mitochondria is maintained in maize root tips during the first stages of glucose starvation. The reasons for these apparently selective effects of glucose starvation on mitochondria are discussed in relation to effects on mitotic and differentiation processes.     

Soil nitrification and foliar δ15N declined with stand age in trembling aspen and jack pine forests in northern Alberta,Canada

Aims

Seed germination and seedling emergence are vulnerable to water stress in arid environments. When precipitation is low and unpredictable during the early growing season, seeds near the sand surface often suffer from hydration/dehydration during germination. We investigated the responses of seedling emergence and survival of a sand dune grass with high sand stabilization value to amount and frequency of precipitation and depth of burial in sand.

Methods

Effects of amount and frequency of precipitation, burial and hydration/dehydration on seedling emergence of Leymus secalinus, were examined using standard procedures.

Results

Seedling emergence was affected by amount and frequency of monthly precipitation and depth of burial, and it decreased as precipitation frequency decreased with same amount of precipitation. Highest emergence percentage was obtained with 100 or 150 mm precipitation at 1–4 cm depth. Hydration/dehydration treatments decreased germination and increased dormancy percentage. Young seedlings with root lengths of 0–1 mm desiccated up to 30 days revived after rehydration.

Conclusions

Seedling emergence of L. secalinus is adapted to 150 mm monthly precipitation with frequency of 10–30 times per month, 1–4 cm burial depth and dehydration interval of 1–2 days. Alteration of amount and/or frequency of precipitation caused by climate change could markedly affect seedling emergence and population regeneration of this species.     

Compositional changes in germinating spores ofAdiantum capillus-veneris L.

Spores ofAdiantum capillus-veneris L. incubated at 25 C for 3 days in the dark were irradiated with continuous red light to induce spore germination and cell growth during following 7 days. A portion of spores were cultured for 8 days in the dark as non-irradiated control. Rhizoidal and protonemal cells were observed at 3 days after transferring spores to the irradiation conditions. During 10 days of the experimental period, changes in the contents of following cell constituents were investigated: total lipid, total soluble sugar, reducing sugar, insoluble glucan, organic acid, protein, soluble α-amino N, and major free amino acids. A large part of nutrient reserves of spores was found to be lipid, whose content decreased markedly as spores germinated. Soluble and insoluble carbohydrates also provided carbon and energy sources during imbibition and germination. Two main reserve proteins were detected by SDS-polyacrylamide gel electrophoresis. These proteins disappeared mostly during germination. Major free amino acids could be assorted into three groups by their patterns of fluctuation during the germination.     

Relationships Between Seed Respiration During Imbibition and Subsequent Seedling Growth in Zea mays L

Corn (Zea mays L.) seed respiration rates during the first 30 hours of germination were compared with seedling growth 3 to 5 days after planting. Significant positive correlations were observed between rates of O₂ uptake during imbibition and later stages of germination and seedling growth. Glutamic acid decarboxylase activity also was positively correlated with seedling growth. The highly significant correlations between respiratory quotients and seedling growth were negative.

Seed metabolism during the initial hours of germination is evidently related somehow to seedling growth rates several days later. Whether this relationship is due to the dependence of synthetic processes and growth on respiratory energy, the fact that high respiration rates reflect high levels of metabolic activity, or to some other cause, remains to be determined.

     

Quantitative real-time expression profiling of aquaporins-isoforms and growth response of Brassica juncea under arsenite stress

The present study analyzed the expression level of aquaporins of plasma membrane intrinsic protein (PIP) class in response to arsenite (AsIII) exposure of 100 μM from 0.5 h to 8 days in Brassica juncea. The expression levels of most of the PIPs were down-regulated during the course of AsIII exposure. This led to decrease in total water content of plants, which in turn hampered seedling growth. The level of reactive oxygen species (superoxide radicals and hydrogen peroxide), lipid peroxidation and root oxidizability increased significantly upon exposure to AsIII as compared to that of control leading to an increase in cell death. The study proposes that the down-regulation of PIPs happened presumably to regulate AsIII levels, which, however, occurred at the cost of reduced growth, disturbed water balance and induced oxidative stress.     

Lipase in lipid bodies of cotyledons of rape and mustard seedlings

Lipolytic activity was absent in the crude cotyledon extract of ungerminated rapeseed (Brassica napm L. var. Dwarf Essex), and increased to a peak at day 4 in seedling growth, concomitant with the decrease in total lipids. About 50% of the lipase activity was recovered in the lipid bodies isolated from the cotyledon extract by flotation centrifugation. Isolated lipid bodies underwent autolysis of internal triacylglycerols resulting in the release of fatty acids. After the triacylglycerols in isolated lipid bodies had been extracted with diethyl ether, the lipase was recovered in the remaining membrane fraction. The lipase had a maximal activity at pH 6.5 on trierucin, trilinolein, or endogenous triacylglycerols, and at pH 8.0 on N-methylindoxylmyristate. The lipase was most active on trierucin and trilinolein, and hydrolyzed the related di- and monoacylglycerols at lower rates. There was little enhancement of the lipase activity in the presence of NaCl, CaCl₂, or detergents, and detergents in general reduced the activity. The hydrolysis of trierucin was linear until about 50% of the trierucin had been converted to erucic acid, and there was little accumulation of dierucin and monoerucin. Lipase extracted from lipid bodies isolated from germinated rapeseed of the variety Tower, which contains little or no erucic acids in the storage triacylglycerols, also had the highest activities on trierucin and trilinolein. A comparative study on mustard seed (Brassica juncea) revealed that the mustard lipase possessed characteristics very similar to those of the rapeseed lipase.