Search

The expression patterns of the two distinct subfamilies of genes (designated porA and porB) encoding the light-dependent NADPH:protochlorophyllide oxidoreductases (PORs) in loblolly pine (Pinus taeda L.) were examined. Transcripts arising from both gene subfamilies were shown to be present at high levels in the cotyledons of dark-grown pine seedlings and to a lesser extent in their stems. Exposure of dark-grown seedlings to light resulted in increased levels of both porA and porB transcripts, as well as increased levels of mRNAs encoding other photosynthesis-related gene products, suggesting that they are under a common mode of regulation. Relative levels of the porA and porB transcripts were similar in seedling cotyledons and primary needles of two-month-old pine trees, whereas only porB transcripts were present at a significant level in mature secondary needles of two-year-old trees. Immunoblot analysis showed that the 37 kDa PORA protein was most abundant in dark-grown tissues, decreased dramatically upon exposure to light, but could still be detected at low levels in light-grown seedlings. In comparison, levels of the 38 kDa PORB protein were not significantly changed upon transfer of dark-grown tissues to light. While both PORA and PORB were detected in cotyledons and primary needles, only PORB could be detected in mature needles. Transcripts derived from the three plastid genes, chlL, chlN, and chlB, encoding subunits of the light-independent protochlorophyllide reductase were detected in the cotyledons and stems of dark-grown seedlings, and in mature needles. The highest levels of chlL, chlN, and chlB transcripts were detected within the top one-third of the stem and decreased gradually towards the stem/root transition zone. Correspondingly, the highest levels of light-independent chlorophyll formation took place near the top of the hypocotyl. A similar pattern of expression was observed for other photosynthesis-related gene products, including porA and porB. Our results suggest that many aspects of the light-dependent, tissue-specific and developmental regulation of POR expression first described in angiosperms were already established in the less evolutionarily advanced gymnosperms. However, unlike angiosperms, light is not the dominant regulatory factor controlling porA expression in these species.

Arginase (EC 3.5.3.1) transcript level and activity were measured in soybean (Glycine max L.) embryos from the reserve deposition stage to postgermination. Using a cDNA probe for a small soybean arginase gene family, no transcript was detected in developing embryos. However, arginase transcripts increased sharply on germination, reaching a maximum at 3 to 5 d after germination. There was low but measurable in vitro arginase specific activity in developing embryos (less than 6% of seedling maximum). During germination arginase specific activity increased in parallel with the sharply increasing arginase transcript level. Seedling arginase activity was largely localized in cotyledons. Arginase activity was assayed in vivo by measuring urea accumulation in a urease-deficient mutant. No urea was detected in developing embryos, whereas accumulated urea paralleled arginase specific activity and transcript level in germinating seedlings. As in planta embryos, cultured cotyledons did not accumulate urea when arginine (Arg) was provided with other amino acids in a "mock" seed-coat exudate. Arg as the sole nitrogen source was converted to urea but did not support cotyledon growth. There appeared to be a lack of recruitment of the low-level arginase activity to hydrolyze free Arg in developing embryos, thus avoiding a futile urea cycle.

Serotonin (5-HT) accumulation in walnut cotyledons is seen as a detoxification mechanism protecting the sensitive plant tissues of seeds from highly toxic ammonia concentrations following seed desiccation. Different metabolic pathways and cell compartments are involved in biosynthesis and storage of 5-HT. Ammonia fixation and incorporation into the indole moiety of tryptophan is followed by 5-HT biosynthesis via tryptamine in a two-step pathway with the adaptive tryptophan decarboxylase and the constitutive tryptamine 5-hydroxylase. Evidence is provided that tryptamine 5-hydroxylase is a member of the cytochrome P450 family which is involved in lipid hydroxylation processes in the very early period of seed development.

An asparaginyl-specific cysteine endopeptidase which was named 'legumain-like proteinase' (LLP) and has an apparent molecular mass of 38.1 kDa was isolated from cotyledons of kidney bean (Phaseolus vulgaris L.) seedlings and partially characterized. It is, to our knowledge, the first known proteinase which in vitro extensively degrades native phaseolin, the major storage globulin of this grain legume. Phaseolin that in vitro had been partially degraded by LLP (Pvitro) and phaseolin that was isolated after partial in vivo breakdown 6 days after the start of seed imbibition (Pvivo) showed similar fragment patterns on SDS/polyacrylamide gels. The fragments had identical cleavage sites in Pvitro and Pvivo as determined by partial amino acid sequencing. In both types of partially degraded phaseolin, these cleavage sites have asparagine in the P1 position. Two of the cleavage sites are located in the beta-barrel domain of the C-terminal module and only one cleavage site was found in the beta-barrel domain of the N-terminal module according to the consensus structural model of phaseolin subunits. These results suggest that very likely LLP could in vivo be responsible for the initiation of phaseolin proteolysis. Two different legumain-specific clones named cp6b and p21b were isolated from a cDNA library of germinated bean cotyledons. Cp6b encodes LLP, while p21b encodes a VPE-like enzyme. Southern-blot analysis revealed a single gene copy for Pv-VPE and, presumably, at least two gene copies for LLP in the kidney bean genome. Northern-blot analysis indicated that mRNAs for both clones appear de novo during seed germination. However, the developmental patterns of the transcript levels corresponding to the two clones differed significantly. The temporal pattern of phaseolin degradation and of LLP polypeptide levels agreed well with the suggestion that LLP plays a key role in the mobilization of phaseolin during and after kidney bean germination.

In plants the carbohydrate state provides signals to adjust metabolism to specific physiological conditions. Storage-active sink organs like seeds often contain high levels of sucrose. In order to change the sugar status during seed development a yeast-derived invertase gene was expressed in Vicia narbonensis under control of the LeguminB4 promoter. A signal sequence targeted the invertase to the apoplast in maturing embryos. In the cotyledons, sucrose was decreased whereas hexoses strongly accumulated. There was a major reduction of starch whereas proteins were less affected. Vacuoles of cotyledon cells were enlarged and dry seeds wrinkled. Transcripts and enzyme activity of sucrose synthase, the small and large subunit of ADP-glucose pyrophosphorylase as well as vicilin were downregulated. Sucrose phosphate synthase and legumin-mRNAs were not affected. Analysing single seeds with different sucrose levels revealed a positive correlation of sucrose concentration to mRNA levels of sucrose synthase and most pronounced to ADP-glucose pyrophosphorylase-mRNA levels as well as to starch content. Glucose on the other hand did not show any correlation. After feeding 14C-sucrose in vitro, the invertase-expressing cotyledons partitioned less carbon into starch compared to the wild-type. In the transgenic cotyledons, a relatively higher amount was directed into proteins compared to starch. We conclude that starch accumulation in developing cotyledons could be a function of sucrose concentration. Our results are consistent with a possible sucrose-mediated induction of storage-associated differentiation indicated by upregulation of specific genes of the starch synthesis pathway.

A monocotyledonous tree, Roystonea regia, was found to bear root nodules. The root nodules contained a high amount (16.9 microg/g fresh mass) of indole acetic acid (IAA). A big tryptophan pool (1555.1 microg/g fresh mass) was found in the root nodules, which might serve as a source of IAA production. The presence of IAA-metabolizing enzymes IAA oxidase and peroxidase indicated metabolism of IAA in the root nodules. The symbiont isolated from the root nodules of R. regia, a Rhizobium sp., produced high amount of IAA in culture when supplemented with tryptophan. The possible role of this IAA production in the monocotyledonous tree-Rhizobium symbiosis is discussed.

Malate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate reversible malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C4 metabolism, pH balance, stomatal and pulvinal movement, respiration, beta-oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in various organelles. While antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule-enhanced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Four of the five forms could effectively complement an E. coli Mdh- mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules.

A beta-D-glucosidase has been purified to apparent homogeneity from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seedlings during the mobilization of the xyloglucan stored in the cotyledonary cell walls. The purified protein (Mr 76, 000; a glycoprotein; pl > 9.5; apparent pH optimum 4.5; temperature optimum 30 degrees C) catalysed the hydrolysis of p-nitrophenyl-beta-D-glucopyranoside, cello-oligosaccharides, beta-linked glucose disaccharides, and certain xyloglucan oligosaccharides. Glucose disaccharides with different linkages were hydrolysed at different rates [(1-->3) > (1-->4) > (1-->2) > (1-->6)] with significant transglycosylation occurring in the early stages of the reaction. Cello-oligosaccharide hydrolysis was also accompanied by extensive transglycosylation to give transitory accumulations of higher oligosaccharides. At least some of the glycosyl linkages formed during transglycosylation were (1-->6)-beta. Xyloglucan oligosaccharides xylose-substituted at the non-reducing terminal glucose residue (XXXG, XXLG, XLXG and XLLG, where G is an unsubstituted glucose residue, X is a xylose-substituted glucose residue, and L is a galactosylxylose-substituted glucose residue) were not hydrolysed. Some xyloglucan oligosaccharides with an unsubstituted non-reducing terminal glucose residue (GXXG, GXLG and GXG) were hydrolysed, but others (GLXG and GLLG) were not. This indicated steric hindrance by L but not X substitution at the glucose residue next to the one at the non-reducing end of the oligosaccharide. Hydrolysis of xyloglucan oligosaccharides was not accompanied by transglycosylation. Natural xyloglucan subunit oligosaccharides (XXXG, XXLG, XLXG, XLLG) were totally degraded to their monosaccharide components when treated with nasturtium beta-D-galactosidase. (Edwards et al (1988) J. Biol. Chem. 263, 4333-4337), followed by alternations of nasturtium xyloglucan-specific alpha-xylosidase (Fanutti et al (1991) Planta 184, 137-147) and this enzyme. Several extensively overlapping cDNA clones were obtained by RT-PCR and by screening cDNA libraries. A composite, full-length DNA had an open reading frame of 1962 bp, encoding a polypeptide of 654 amino acids, including all N-terminal and internal sequences obtained from the purified beta-glucosidase protein, and a motif resembling plant signal sequences thought to direct proteins to the cell wall. Database searches revealed homology with beta-glucosidases from several sources (plant, bacteria, yeast), notably with glycosylhydrolases of 'Family 3', according to the classification of Henrissat (Henrissat (1991) Biochem. J. 280, 309-316). There was strong sequence homology with a beta-glucan exo-hydrolase from barley (Hrmova et al. (1996) J. Biol. Chem. 271, 5277-5286). The nasturtium beta-glucosidase is ascribed a role in xyloglucan mobilization, and its interaction with the alpha-xylosidase and the beta-galactosidase is modelled.

Plastid biogenesis in etiolated lupine (Lupinus luteus L.) cotyledons is highly sensitive to cytokinins and abscisic acid. In the presence of the synthetic cytokinin N6-benzylaminopurine, greening and plastid biogenesis is substantially promoted as compared to untreated controls, whereas abscisic acid has an inhibitory effect. Faster greening in cytokinin-treated cotyledons is accompanied by a higher level and slower degradation of the light-sensitive protochlorophyllide-oxidoreductase (POR); while ABA has the opposite effect. The phytohormones appear to modulate POR gene expression, since the steady-state levels of POR mRNA, as well as transcripts of other nuclear genes for plastid proteins, are strongly increased by cytokinin and reduced by abscisic acid treatment. When etiolated lupine cotyledons were illuminated with far-red light prior to phytohormone application, the POR level substantially decreased; this was accompanied by the loss of the phytohormone's effect on greening. Based on these findings it is concluded that the level of POR and the integrity of the prolamellar body is crucial for cytokinin- and abscisic acid-controlled greening following transfer of etiolated lupine cotyledons into the light.

Spinach (Spinacia oleracea) cytosolic fructose-1,6-bisphosphatase (FBPase) was purified and the final preparation of protein has a specific activity of about 45 units/mg protein and a single band of molecular mass of 39 kDa. Polyclonal antibody against the protein did not cross-react with chloroplast FBPase, but showed strong cross-reactivity with all plant cytosolic FBPases tested. Studies of the FBPase expression characteristics at early stages of development demonstrated that it was controlled at both the transcriptional and translational levels, and its mRNA was detected even in etiolated cotyledons. This suggests that the expression is not light-inducible. A single transcript was detected in all spinach tissues tested. Western blot analysis revealed two protein bands in the etiolated cotyledons: one was the same size as that present in the mature leaf, and the other slightly smaller. A high enzyme activity was detected in etiolated cotyledons, especially compared to protein levels in Western blots. Expression of the cytosolic FBPase gene during leaf development showed no change in the steady-state level of mRNA, but the protein level and enzyme activity were higher in mature leaves than in young ones, suggesting that the increase in FBPase activity during development is due to an increase in protein synthesis. Young roots showed low enzyme activity, but an unexpectedly high activity was detected in old fiber roots.

Asymmetric distribution of acetylcholinesterase (AChE) activity has previously been demonstrated to occur in the lower side of the gravity-stimulated maize shoot. The localization of immunoreacted IAA-inositol synthase, AChE and safranin was detected in selected organs of gravistimulated dark grown maize seedlings using a light microscope. Immunoreacted IAA-inositol synthase was asymmetrically distributed in the lower side of the stele of coleoptile node and mesocotyl in maize seedlings placed horizontally. The positive AChE spots in the coleoptile node and mesocotyl were apparently localized in the lower half of the gravistimulated seedlings. Safranin was also asymmetrically distributed in the lower half of the endodermis and stele cells of coleoptile node and mesocotyl. Namely, transport of safranin in the upper half of the coleoptile node and mesocotyl was blocked by gravistimulation. Furthermore, the asymmetric distribution of immunoreacted IAA-inositol synthase was inhibited by neostigmine bromide, AChE inhibitor. These results show that an asymmetric environmental stimulus induces changes in AChE activity, affecting IAA-inositol synthase localization and safranin transport.

The alternative oxidase (AOX) of the soybean (Glycine max L.) inner mitochondrial membrane is encoded by a multigene family (Aox) with three known members. Here, the Aox2 and Aox3 primary translation products, deduced for cDNA analysis, were found to be 38.1 and 36.4 Kd, respectively. Direct N-terminal sequencing of partially purified AOX from cotyledons demonstrates that the mature proteins are 31.8 and 31.6 KD, respectively, implying that processing occurs upon import of these proteins into the mitochondrion. Sequence comparisons show that the processing of plant AOX proteins occurs at a characteristic site and that the AOX2 and AOX3 proteins are more similar to one another than to other AOX proteins, including soybean AOX1. Transcript analysis using a polymerase chain reaction-based assay in conjunction with immunoblot experiments indicates that soybean Aox genes are differentially expressed in a tissue-dependent manner. Moreover, the relative abundance of both Aox2 transcripts and protein in cotyledons increase upon greening of dark-grown seedlings. These results comprehensively explain the multiple AOX-banding patterns observed on immunoblots of mitochondrial proteins isolated from various soybean tissues by matching protein bands with gene products.

Acetylcholinesterase (AChE) activity has previously been studied by this laboratory and shown to occur at the interface between the stele and cortex of the mesocotyl of maize (Zea mays L.) seedlings. In this work we studied the distribution of AChE activity in 5-d-old maize seedlings following a gravity stimulus. After the stimulus, we found an asymmetric distribution of the enzyme in the coleoptile, the coleoptile node, and the mesocotyl of the stimulated seedlings using both histochemical and colorimetric methods for measuring the hydrolysis of acetylthiocholine. The hydrolytic capability of the esterase was greater on the lower side of the horizontally placed seedlings. Using the histochemical method, we localized the hydrolytic capability in the cortical cells around the vascular stele of the tissues. The hydrolytic activity was inhibited 80 to 90% by neostigmine, an inhibitor of AChE. When neostigmine was applied to the corn kernel, the gravity response of the seedling was inhibited and no enzyme-positive spots appeared in the gravity-stimulated seedlings. We believe these results indicate a role for AChE in the gravity response of maize seedlings.

Microsomal membranes from growing tissue of pea (Pisum sativum L.) epicotyls were incubated with the substrate UDP-[14C]galactose (Gal) with or without tamarind seed xyloglucan (XG) as a potential galactosyl acceptor. Added tamarind seed XG enhanced incorporation of [14C]Gal into high-molecular-weight products (eluted from columns of Sepharose CL-6B in the void volume) that were trichloroacetic acid-soluble but insoluble in 67% ethanol. These products were hydrolyzed by cellulase to fragments comparable in size to XG subunit oligosaccharides. XG-dependent galactosyltransferase activity could be solubilized, along with XG fucosyltransferase, by the detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. When this enzyme was incubated with tamarind (Tamarindus indica L.) seed XG or nasturtium (Tropaeolum majus L.) seed XG that had been partially degalactosylated with an XG-specific beta-galactosidase, the rates of Gal transfer increased and fucose transfer decreased compared with controls with native XG. The reaction products were hydrolyzed by cellulase to 14C fragments that were analyzed by gel-filtration and high-performance liquid chromatography fractionation with pulsed amperometric detection. The major components were XG subunits, namely one of the two possible monogalactosyl octasaccharides (-XXLG-) and digalactosyl nonasaccharide (-XLLG-), whether the predominant octasaccharide in the acceptor was XXLG (as in tamarind seed XG) or XLXG (as in nasturtium seed XG). It is concluded that the first xylosylglucose from the reducing end of the subunits was the Gal acceptor locus preferred by the solubilized pea transferase. These observations are incorporated into a model for the biosynthesis of cell wall XGs.

This paper describes a biochemical study on the protein composition of crystalline inclusions (cores) from plant peroxisomes. By SDS/PAGE and immunoblotting, a catalase of 59 kDa was identified as the predominant protein component in purified cores from sunflower (Helianthus annuus L.) cotyledons. A 55-kDa catalase was the only additional peptide detected. In contrast to in cores, the 55-kDa catalase was the major catalase protein in matrix fractions obtained from lysed peroxisomes. These findings suggested two peroxisomal populations of catalase differing in molecular structure and subperoxisomal compartmentation in sunflower cotyledons. Evidence for different amino acid sequences of the two catalases was found by peptide mapping with endoproteinase Glu-C, by expressing a cDNA encoding matrix catalase in Escherichia coli, and by partial amino acid sequencing of peptide fragments from 59-kDa core catalase. These results contradict the previous view that the formation of cores occurred via condensation of matrix catalase, and indicate that new concepts on the biogenesis and physiological function of plant peroxisomal cores need to be developed.

In previous works using cell fractionation methods we demonstrated the presence of a Cu,Zn-containing superoxide dismutase in peroxisomes from watermelon cotyledons. In this work, this intracellular localization was evaluated by using western blot and EM immunocytochemical analysis with a polyclonal antibody against peroxisomal Cu,Zn-SOD II from watermelon cotyledons. In crude extracts from 6-day old cotyledons, analysis by western blot showed two cross-reactivity bands which belonged to the isozymes Cu,Zn-SOD I and Cu,Zn-SOD II. In peroxisomes purified by sucrose density-gradient centrifugation only one cross-reactivity band was found in the peroxisomal matrix which corresponded to the isozyme Cu,Zn-SOD II. When SOD activity was assayed in purified peroxisomes two isozymes were detected, Cu,Zn-SOD II in the matrix, and a Mn-SOD in the membrane fraction which was removed by sodium carbonate washing. EM immunocytochemistry of Cu,Zn-SOD on sections of 6-day old cotyledons, showed that gold label was mainly localized over plastids and also in peroxisomes and the cytosol, whereas mitochondria did not label for Cu,Zn-SOD.

Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

Two nodule cDNA clones representing genes involved in Alnus glutinosa nitrogen metabolism were analysed. ag11 encoded glutamine synthetase (GS), the enzyme responsible for ammonium assimilation, while ag118 encoded acetylornithine transaminase (AOTA), an enzyme involved in the biosynthesis of citrulline, the nitrogen transport form in Alnus. GS mRNA was found at highest levels in root nodules, where it was present in the infected cells as well as in the cells of the pericycle of the vascular system. AOTA transcripts were found at high levels in nodules, confined to the infected cells, suggesting that in nodules of A. glutinosa, citrulline biosynthesis takes place mainly in the infected cells.

Ch3, an endochitinase of 32 kDa present in Castanea sativa cotyledons, showed in vitro antifungal properties when assayed against Trichoderma viride. The characterization of a cDNA clone corresponding to this protein indicated that Ch3 is a class Ib endochitinase that is synthesized as a preprotein with a signal sequence preceding the mature polypeptide. Bacterial expression of mature Ch3 fused to the leader peptide of the periplasmic protein ompT resulted in active Ch3 enzyme. A plate assay was adapted for semi-quantitative determination of chitinase activity secreted from cultured bacteria, which should facilitate the identification of mutants with altered capacity to hydrolyse chitin.

The cyanide-resistant alternative oxidase of plant mitochondria is known to be activated by alpha-keto acids, such as pyruvate, and by the reduction of a disulfide bond that bridges the two subunits of the enzyme homodimer. When the regulatory cysteines are oxidized, the inactivated enzyme is much less responsive to pyruvate than when these groups are reduced. When soybean cotyledon mitochondria were isolated in the presence of iodoacetate or N-ethylmaleimide, the intermolecular disulfide bond did not form and the alternative oxidase was present only as a noncovalently associated dimer. N-Ethylmaleimide inhibited alternative oxidase activity, but iodoacetate was found to stimulate activity much like pyruvate, including enhancing the enzyme's apparent affinity for reduced ubiquinone. The presence of pyruvate or iodoacetate blocked inhibition of the enzyme by N-ethylmaleimide, indicating that all three compounds acted at the same sulfhydryl group on the alternative oxidase protein. The site of pyruvate and iodoacetate action was shown to be a different sulfhydryl than that involved in the redox-active regulatory disulfide bond, because iodoacetate bound to the alternative oxidase at the activating site even when the redox-active regulatory sulfhydryls were oxidized. Given the nature of the covalent adduct formed by the reaction of iodoacetate with sulfhydryls, the activation of the alternative oxidase by alpha-keto acids appears to involve the formation of a thiohemiacetal.

The first committed step of porphyrin synthesis in higher plants is the reduction of glutamyl-tRNA to glutamate 1-semialdehyde. This reaction is catalyzed by glutamyl-tRNA reductase, which is encoded by hemA genes. Two hemA cDNA clones (hemA1 and hemA2) were obtained from cucumber (Cucumis sativus) cotyledons by the PCR and cDNA library screening. They showed significant homology with published hemA sequences. Southern blot analysis of cucumber genomic DNA revealed that these genes are located at different loci and that there is another gene similar to the hemA genes. Accumulation of hemA1 mRNA was detected primarily in cotyledons and hypocotyls of greening cucumber seedlings, whereas that of hemA2 mRNA was detected in all tissues examined. Illumination of cucumber seedlings increased markedly the accumulation of hemA1 mRNA, but it did not induce remarkable changes in that of hemA2 mRNA. These findings suggest that hemA1 mRNA was accumulated in response to the demand of Chl synthesis in photosynthesizing tissues, whereas hemA2 mRNA was expressed in response to the demand of the synthesis of porphyrins other than chlorophylls.

The blue-light-sensing apical part of coleoptiles of grasses is responsible for the first positive phototropic bending reaction of this organ. The photoreceptor responsible has been shown to be localized to the plasma membrane (PM) of this tip region. An approximately 100-kDa protein moiety of this receptor is rapidly phosphorylated upon irradiation. Properties of this protein kinase reaction were studied in vitro by using PMs from the maize (Zea mays L.) coleoptile tip region; (i) The substrate for the blue-light-triggered phosphorylation of the 100-kDa protein was found to be ATP as well as GTP. However, the affinity of the involved protein kinase for the substrate GTP was lower than for ATP. (ii) Experiments were undertaken to find out whether a photoreceptor moiety acts as an autophosphorylating protein kinase or whether the photoreceptor protein, when activated by light, becomes the target of an extrinsic protein kinase. Two studied extrinsic protein kinases (50 and 55 kDa) of the coleoptile tip were found not to be involved in the light-dependent protein phosphorylation. The degree of phosphorylation of the 100-kDa protein on isolated plasma membranes upon irradiation at 0 degrees C was scarcely different from a reaction at 30 degrees C, in contrast to the background protein phosphorylations which decreased with decreasing temperature. This result points to an autophosphorylation mechanism at the receptor. (iii) In mixing experiments, solubilized membranes from maize coleoptiles were irradiated and added to unirradiated membrane proteins from pea (Pisum sativum L.) epicotyls followed by addition of [gamma-32P]ATP. Unirradiated proteins from pea were not phosphorylated by light-activated (autophosphorylatable) maize protein kinases. (iv) It is suggested that the blue-light-sensitive photoreceptor localized to the PM of the phototropically active tip region of coleoptiles has an autophosphorylatable kinase domain which is able to use ATP or GTP as substrate.

Two full-length cDNAs encoding hydroxypyruvate reductase were isolated from a cDNA library constructed with poly(A)+ RNA from pumpkin green cotyledons. One of the cDNAs, designated HPR1, encodes a polypeptide of 386 amino acids, while the other cDNA, HPR2 encodes a polypeptide of 381 amino acids. Although the nucleotide and deduced amino acid sequences of these cDNAs are almost identical, the deduced HPR1 protein contains Ser-Lys-Leu at its carboxy-terminal end, which is known as a microbody-targeting signal, while the deduced HPR2 protein does not. Analysis of genomic DNA strongly suggests that HPR1 and HPR2 are produced by alternative splicing.

We have investigated the substrate subsite recognition requirement of the xyloglucan endo-transglycosylase/xyloglucan-specific endo-(1-->4)-beta-D-glucanase (NXET) from the cotyledons of nasturtium seedlings. Seed xyloglucans are composed almost entirely of the Glc4 subunits XXXG, XLXG, XXLG and XLLG, where G represents an unsubstituted glucose residue, X a xylose-substituted glucose residue and L a galactosyl-xylose-substituted glucose residue. Thus in the xyloglucan sequence shown below, the xylose (Xyl) residues at the backbone glucose (Glc) residues numbered -3, -2, +2 and +3 may be galactose-substituted, and NXET cleaves between the unsubstituted glucose at -1 and the xylose-substituted glucose at +1, which never carries a galactosyl substituent. [formula: see text] We have isolated the xyloglucan oligosaccharides XXXGXXXG and XLLGXLLG from NXET digests of tamarind seed xyloglucan, have modified them enzymatically using a pure xyloglucan oligosaccharide-specific alpha-xylosidase from nasturtium seeds to give GXXGXXXG and GLLGXLLG, and have identified and compared the products of NXET action on XXXGXXXG, GXXGXXXG, XLLGXLLG and GLLGXLLG. We have also compared the molar proportions of XXXG, XLXG, XXLG and XLLG in native tamarind and nasturtium seed xyloglucans with those in NXET digests of these polysaccharides. Using these and existing data we have demonstrated that NXET action does not require xylose-substitution at glucose residues -4, -2, +1 and +3 and that xylose substitution at +2, is a requirement. There may also be a requirement for xylose substitution at -3. We have demonstrated also that galactosyl substitution of a xylose residue at +1 prevents, and at -2 modifies, chain-cleavage. A partial model for the minimum substrate binding requirement of NXET is proposed.

A rapid and efficient method for the purification of malate synthase, an enzyme uniquely confined to glyoxysomes, from cotyledons of Brassica napus L. has been developed. The two step purification procedure is based on the consequent utilization of the tendency of malate synthase to form high molecular weight aggregates. Malate synthase was purified 75-fold to apparent homogeneity with a specific activity of 180 nkat/mg protein. The estimated molecular weight of malate synthase subunits was 63 kDa. Polyclonal antibodies raised against malate synthase in rabbits detect on Western blots only one single polypeptide with an identical molecular weight.

The reactivity of cucumber cotyledon lipoxygenase with trilinolein was examined. The activity of the enzyme against linoleic acid rapidly decreased with increasing pH of the assay solution, and essentially no activity could be detected above pH 8.5. The rapid decrease in activity was not the result of an inactiveness of the enzyme at alkaline pH, because with trilinolein, the enzyme showed a broad pH-activity profile, and substantial activity could be detected even at pH 9.0. Rather, the decrease in activity was due to the dissociation of the linoleic acid emulsion into acid-soap aggregates and/or the monomeric form, depending on the ionization of the terminal carboxylic group. This suggests that cucumber cotyledon lipoxygenase acts only on an insoluble substrate at the lipid/water interface but not on a soluble one. High-performance liquid chromatography analyses of the products formed from trilinolein revealed that the enzyme inserted oxygen into the acyl moiety of trilinolein without hydrolysis of the ester bonds. Preincubation of the enzyme with triolein emulsions effectively abolished its activity against trilinolein added afterward. Furthermore, the enzyme was adsorbed on the trilinolein or triolein emulsion droplets in an essentially irreversible manner. A reaction velocity curve of the enzyme with trilinolein showed saturation kinetics. This is thought to be due to a regional substrate deficiency as the reaction proceeds. These lines of evidence indicate that the enzyme, once bound to the lipid/water interface, is unable to break free and bind to other emulsions.

A cysteine endopeptidase, designated SH-EP, occurs in the cotyledons of germinated seeds of Vigna mungo and acts to degrade the seed storage protein in protein storage vacuoles. SH-EP is synthesized on membrane-bound ribosomes as an inactive 45-kDa precursor, which is cotranslationally processed to a 43-kDa intermediate through cleavage of the signal sequence; the 43-kDa intermediate of SH-EP is further processed to the 33-kDa mature enzyme via 39-kDa and 36-kDa intermediates [Mitsuhashi, W. & Minamikawa, T. (1989) Plant Physiol. 89, 274-279]. The present in vitro processing experiments indicated that at least two processing enzymes, designated VmPE-1 and VmPE-2 (V. mungo processing enzymes 1 and 2), were involved in post-translational processing of SH-EP in cotyledons of V. mungo seedlings. VmPE-1 was purified from the cotyledons as a protease that was involved in the processing of the 43-kDa intermediate to the 36-kDa intermediate. The enzyme has a molecular mass of 33 kDa as estimated by SDS/polyacrylamide gel electrophoresis, and showed high similarity to the jackbean asparaginyl endopeptidase in terms of the primary structure and substrate specificity. We discuss the function of VmPE-1 in the processing of SH-EP and related proteases in the cotyledons of germinated seeds.

Two different aconitases are known to be expressed after the germination of oil-seed plants. One is a mitochondrial aconitase that is involved in the tricarboxylic acid cycle. The other participates in the glyoxylate cycle, playing a role in gluconeogenesis from stored oil. We isolated and characterized a cDNA for an aconitase from etiolated pumpkin cotyledons. The cDNA was 3,145 bp long and capable of encoding a protein of 98 kDa. N-terminal and C-terminal amino acid sequences deduced from the cDNA did not contain mitochondrial or glyoxysomal targeting signals. A search of protein databases suggested that the cDNA encoded a cytosolic aconitase. Immunoblotting analysis with a specific antibody against the aconitase expressed in Escherichia coli revealed that developmental changes in the amount of the aconitase were correlated with changes in levels of other enzymes of the glyoxylate cycle during growth of seedlings. Further analysis by subcellular fractionation and immunofluorescence microscopy revealed that aconitase was present only the cytosol and mitochondria. No glyoxysomal aconitase was found in etiolated cotyledons even though all the other enzymes of the glyoxylate cycle are known to be localized in glyoxysomes. Taken together, the data suggest that the cytosolic aconitase participates in the glyoxylate cycle with four glyoxysomal enzymes.

Phosphoenolpyruvate carboxykinase (PEPCK) with a subunit molecular mass of 74 kDa has been purified 450-fold to homogeneity from the cotyledons of cucumber (Cucumis sativus L.). This is the first purification of the native form of the enzyme from any plant tissue. Incubation of the purified enzyme with [gamma-32P]ATP and either phosphoenolpyruvate-carboxylase kinase or mammalian cAMP-dependent protein kinase led to labelling of the enzyme in a part of the molecule separate from the active site. This was reversed by incubation with protein phosphatase 2A. Cotyledons of cucumber seedlings were also supplied with 32Pi. Homogenates of such cotyledons contained a heavily labelled polypeptide which was confirmed as PEPCK by immunoprecipitation. Labelling of PEPCK by 32Pi in darkened cotyledons was reversed by illumination.

Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) catalyzes the conversion of allantoin to allantoic acid in the final step of ureide biogenesis. We have purified allantoinase more than 4000-fold by immunoaffinity chromatography from root nodules and cotyledons of soybean (Glycine max [L] Merr.). We characterized and compared properties of the enzyme from the two sources. Seed and nodule allantoinases had 80% identity in the first 24 amino acid residues of the N terminus. Two-dimensional gel electrophoresis of the purified enzymes showed that multiple forms were present in each. Allantoinases from nodules and cotyledons had very low affinity for allantoin with a Km for allantoin of 17.3 mM in cotyledons and 24.4 mM in nodules. Both had activity in a broad range of pH values from 6.5 to 7.5. In addition, purified allantoinase from both sources was very heat stable. Enzyme activity was stable after 1 h at 70 degrees C, decreased gradually with heating to 85 degrees C, and was lost at 90 to 95 degrees C. Although these studies have revealed some differences between allantoinases in seeds and nodules, the differences were not reflected in key enzyme properties. The immunoaffinity approach enabled purification of allantoinase from soybean root nodules and simplified its purification from cotyledons, thereby allowing characterization and comparison of the enzyme from the two sources.

In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD+ oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (Mr) of 670,000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M(r) 510,000 and of gMDH as a dimer of M(r) 73,000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X6)HL motif also found in plant and mammalian thiolases.

Chloroplastic NADP(+)-malate dehydrogenase (cpMDH, EC 1.1.1.82) is a key enzyme in the carbon-fixation pathway of some C4 plants such as the monocotyledons maize or Sorghum. We have expressed cpMDH from Sorghum vulgare Pers. in transgenic tobacco (Nicotiana tabacum L.) (a dicotyledonous C3 plant) by using a gene composed of the Sorghum cpMDH cDNA under the control of cauliflower mosaic virus 35S promoter. High steady-state levels of cpMDH mRNA were observed in isogenic dihaploid transgenic tobacco lines. Sorghum cpMDH protein was detected in transgenic leaf extracts, where a threefold higher cpMDH activity could be measured, compared with control tobacco leaves. The recombinant protein was identical in molecular mass and in N-terminal sequence to Sorghum cpMDH. The tobacco cpMDH protein which has a distinct N-terminal sequence, could not be detected in transgenic plants. Immunocytochemical analyses showed that Sorghum cpMDH was specifically localized in transgenic tobacco chloroplasts. These data indicate that Sorghum cpMDH preprotein was efficiently synthesized, transported into and processed in tobacco chloroplasts. Thus, C3-C4 photosynthesis specialization or monocotyledon-dicotyledon evolution did not affect the chloroplastic protein-import machinery. The higher levels of cpMDH in transgenic leaves resulted in an increase of L-malate content, suggesting that carbon metabolism was altered by the expression of the Sorghum enzyme.

A cDNA clone for glyoxysomal citrate synthase (gCS) was isolated from a lambda gt11 cDNA library prepared from etiolated pumpkin cotyledons. The cDNA of 1989 bp consisted of a 1548 bp open reading frame that encoded 516 amino acid residues. The deduced amino acid sequence of gCS did not have a typical peroxisomal targeting signal at its carboxyl terminal. A study of expression in vitro of the cDNA and an analysis of the amino-terminal sequence of the citrate synthase indicated that gCS is synthesized as a larger precursor that has a cleavable amino-terminal presequence of 43 amino acids. The predicted amino-terminal sequence of pumpkin gCS was highly homologous to those of other microbody enzymes, such as 3-ketoacyl-CoA thiolase of rat and malate dehydrogenase of watermelon that are also synthesized as precursors of higher molecular mass. Immunoblot analysis showed that the level of gCS protein increased markedly during germination and decreased rapidly during the light-induced transition of microbodies from glyoxysomes to leaf peroxisomes. By contrast, the level of mRNA for gCS reached a maximum earlier than that of the protein and declined even in darkness. The level of the mRNA was low during the microbody transition. These results indicate that the accumulation of the gCS protein does not correspond to that of the mRNA and that degradation of gCS is induced during the microbody transition, suggesting that post-transcriptional regulation plays an important role in the microbody transition.

A full-length cDNA clone encoding microbody NAD(+)-dependent malate dehydrogenase (MDH) of cucumber has been isolated. The deduced amino acid sequence is 97% identical to glyoxysomal MDH (gMDH) of watermelon, including the amino terminal putative transit peptide. The cucumber genome contains only a single copy of this gene. Expression of this mdh gene increases dramatically in cotyledons during the few days immediately following seed imbibition, in parallel with genes encoding isocitrate lyase (ICL) and malate synthase (MS), two glyoxylate cycle enzymes. The level of MDH, ICL and MS mRNAs then declines, but then MDH mRNA increases again together with that of peroxisomal NAD(+)-dependent hydroxypyruvate reductase (HPR). The mdh gene is also expressed during cotyledon senescence, together with hpr, icl and ms genes. These results indicate that a single gene encodes MDH which functions in both glyoxysomes and peroxisomes. In contrast to icl and ms genes, expression of the mdh gene is not activated by incubating detached green cotyledons in the dark, nor is it affected by exogenous sucrose in the incubation medium. The function of this microbody MDH and the regulation of its synthesis are discussed.

Three structurally distinct cDNA clones for cytosolic glutamine synthetase (GS1) were isolated from libraries prepared from senescing radish cotyledons. Northern blot analysis showed that transcripts from two of the three genes encoding GS1, Gln1;1 and Gln1;3, accumulated in the cotyledons during both dark-induced and natural senescence. Transcripts from the last gene, Gln1;2, remained at a low level during both processes. Transcripts from all three Gln1 genes accumulated in cotyledons of germinating seeds. We infer from these findings that GS1 enzymes function in both germination and senescence to convert ammonium to glutamine to remobilize nitrogen from source to sink organs. We have also examined the pattern of expression of these genes in different tissues. All three genes are expressed in roots. A large amount of transcripts from Gln1;1 accumulated in hypocotyls. Whereas none were transcribed in flowers. During dark-induced senescence of cotyledons, application of inorganic nitrogen delayed chlorophyll degradation. Inorganic nitrogen enhanced the accumulation of Gln1;1 transcripts, but decreased those of Gln1;3. In contrast, application of glutamine promoted yellowing of cotyledons during the dark treatment, and slightly increased the amounts of transcripts from Gln1;3 but decreased those of Gln1;1. Transcription of the three Gln1 genes appears, therefore, to be differentially regulated in radish cotyledons during senescence and germination.

cDNA clones encoding cysteine proteinases from cotyledons of germinated seeds of Vicia sativa L. have been obtained by means of PCR. Degenerate oligonucleotide primers were designed according to conserved amino acid regions of known cysteine proteinases. The deduced amino acid sequences of the cDNA clones encoding VSCYSPR1 and VSCYSPR2 display strong homology to cysteine proteinases of the so called papain superfamily. Northern analyses revealed developmentally regulated expression of both the mRNAs in germinating seeds. The transcripts were shown to be products of two distinct single genes, each exhibiting structural polymorphisms as exposed in few nucleotide substitutions.

Extracts of the cotyledons of germinated honey locust (Gleditsia triacanthos) seeds, which contain galactomannan as a reserve polysaccharide in the endosperm, were fractionated by chromatography and the fractions examined for the presence of a specific manno-6-kinase which could phosphorylate the D-mannose released by hydrolysis of galactomannan. One particulate hexokinase (the major hexose-6-kinase fraction) and two soluble hexokinase fractions (the minor portion), as well as a soluble fructo-6-kinase fraction, were initially separated. From chromatography, electrophoresis and kinetic studies, no evidence for a specific manno-kinase was obtained. This and the level and kinetic behaviour of the particulate hexokinase implicated it as the enzyme catalysing the phosphorylation of released D-mannose. The fructo-kinase activity was further separated into three fractions. Kinetic studies on one of these with native and synthetic substrates indicated that the structural requirements for the monosaccharide substrate were a beta-D-anomeric 2-OH in the furanose ring, a 4-OH trans to the D-5-CH2OH and a -CH2OH substituent on C2 (trans to the 5-CH2OH) which could be modified. The orientation of the hydroxyl on C-3 had only a limited effect.

Protein kinase activity has repeatedly been found to co-purify with the plant photoreceptor phytochrome, suggesting that light signals received by phytochrome may be transduced or modulated through protein phosphorylation. In this study immunoprecipitation techniques were used to characterize protein kinase activity associated with phytochrome from maize (Zea mays L.). A protein kinase that specifically phosphorylated phytochrome was present in washed anti-phytochrome immunoprecipitates of etiolated coleoptile proteins. No other substrate tested was phosphorylated by this kinase. Adding salts or detergents to disrupt low-affinity protein interactions reduced background phosphorylation in immunoprecipitates without affecting phytochrome phosphorylation, indicating that the protein kinase catalytic activity is either intrinsic to the phytochrome molecule or associated with it by high-affinity interactions. Red irradiation (of coleoptiles or extracts) sufficient to approach photoconversion saturation reduced phosphorylation of immunoprecipitated phytochrome. Subsequent far-red irradiation reversed the red-light effect. Phytochrome phosphorylation was stimulated about 10-fold by a co-immunoprecipitated factor. The stimulatory factor was highest in immunoprecipitates when Mg2+ was present in immunoprecipitation reactions but remained in the supernatant in the absence of Mg2+. These observations provide strong support for the hypothesis that phytochrome-associated protein kinase modulates light responses in vivo. Since only phytochrome was found to be phosphorylated, the co-immunoprecipitated protein kinase may function to regulate receptor activity.

Six different putative UDP-glucose glucosyltransferase clones were isolated from a cassava cotyledon cDNA library probed with an Acc I-Bgl II restriction fragment from a UDP-glucose flavonoid 3-O-glucosyltransferase from Antirrhinum majus. The heterologous probe contained a glucosyltransferase consensus signature amino acid sequence which was also present in the cassava cDNA clones. Nucleotide and derived amino acid sequences are presented for two of the clones. Northern analysis showed different patterns of expression for the six genes in developing seedling tissues, indicating temporal and tissue-specific regulation. A comparative analysis was made of the six cassava clone derived amino acid sequences and other reported UDP-glucosyltransferase genes. Highly conserved residues in plant genes from three species allow redefinition of essential residues within the signature sequence for secondary plant product metabolism glucosyltransferase genes.

We have demonstrated that etiolated soybean seedlings grown under the altered gravity conditions of clinorotation (1 rpm) and centrifugation (5xg) exhibit changes in starch metabolism. Cotyledon starch concentration was lower (-28%) in clinorotated plants and higher (+24%) in centrifuged plants than in vertical control plants. The activity of ADP-glucose pyrophosphorylase in the cotyledons was affected in a similar way, i.e. lower (-37%) in the clinorotated plants and higher (+22%) in the centrifuged plants. Other starch metabolic enzyme activities, starch synthase, starch phosphorylase and total hydrolase were not affected by the altered gravity treatments. We conclude that the observed changes in starch concentrations were primarily due to gravity-mediated differences in ADP-glucose pyrophosphorylase activity.

Soybean (Glycine max [L.] Merr. cv. McCall) seedlings germinated and grew for 6d under the altered gravity conditions of horizontal clinorotation and centrifugation. Both of these conditions resulted in decreased growth relative to the control (vertically rotated) plants. Starch concentration in the cotyledons was lower in the clinorotated plants and was higher in the centrifuged plants compared to the controls. The opposite relationship was noted for total lipid concentration. Of the six starch metabolic enzyme activities measured, only ADP glucose pyrophosphorylase was affected by the gravity treatments; being lower in the cotyledons of the horizontally rotated plants and higher in the cotyledons of the centrifuged plants relative to the control values.

The total lipid composition of plasma membranes (PM), isolated by the phase partitioning method from two different oat (Avena sativa L.) tissues, the root and coleoptile, was compared. In general, the PM lipid composition was not conserved between these two organs of the oat seedling. Oat roots contained 50 mole percent phospholipid, 25 mole percent glycolipid, and 25 mole percent free sterol, whereas comparable amounts in the coleoptile were 42, 39, and 19 mole percent, respectively. Individual lipid components within each lipid class also showed large variations between the two tissues. Maximum specific ATPase activity in the root PM was more than double the activity in the coleoptile. Treatment of coleoptile with auxin for 1 hour resulted in no detectable changes in PM lipids or extractable ATPase activity. Differences in the PM lipid composition between the two tissues that may define the limits of ATPase activity are discussed.

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a Mr 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a Mr 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.

The regulatory pigment phytochrome induces rapid and opposite growth changes in different regions of etiolated maize seedlings: it stimulates the elongation rate of coleoptiles and inhibits that of mesocotyls. As measured by a quantitative immunoassay, phytochrome also promotes rapid and opposite changes in the extractable content of a Mr 98,000 anionic isoperoxidase in the cell walls of these same organs: it induces a decrease of this peroxidase in coleoptiles and an increase in mesocotyls. The peroxidase changes precede the growth changes. As measured by video stereomicroscopy or a position transducer, red light (R), which photoactivates phytochrome, stimulates coleoptile elongation with a lag of about 15-20 min and suppresses mesocotyl growth with a lag of 45-50 min. R also induces a 50% reduction in the extractable level of the anionic peroxidase in coleoptile walls in less than 10 min and a 40% increase in the level of this peroxidase in mesocotyl walls within 30 min. Ascorbic acid, an inhibitor of peroxidase activity, blocks the effects of R on mesocotyl section growth. These results are relevant to hypotheses that postulate that certain wall peroxidases can participate in light-induced changes in growth rate by their effects on wall extensibility.

An early gravity-transduction event in oat coleoptiles which precedes any noticeable bending is the accumulation of calcium on their prospective slower-growing side. Sub-cellular calcium localization studies indicate that the gravity-stimulated redistribution of calcium results in an increased concentration of calcium in the walls of responding cells. Since calcium can inhibit the extension growth of plant cell walls, this selective accumulation of calcium in walls may play a role in inducing the asymmetry of growth which characterizes gravitropism. The active transport of calcium from cells into walls is performed by a calcium-dependent ATPase localized in the plasma membrane. Evidence is presented in support of the hypothesis that this calcium pump is regulated by a feed-back mechanism which includes the participation of calmodulin.

Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.