Search

Transfer cells are characterized by an amplified plasma membrane area supported on a wall labyrinth composed of a uniform wall layer (UWL) from which wall ingrowth (WI) papillae arise. Adaxial epidermal cells of developing Vicia faba cotyledons, when placed in culture, undergo a rapid (hours) trans-differentiation to a functional epidermal transfer cell (ETC) phenotype. The trans-differentiation event is controlled by a signalling cascade comprising auxin, ethylene, apoplasmic reactive oxygen species (apoROS), and cytosolic Ca2+. Apoplasmic hydrogen peroxide (apoH2O2) was confirmed as the apoROS regulating UWL and WI papillae formation. Informed by an ETC-specific transcriptome, a pharmacological approach identified a temporally changing cohort of H2O2 biosynthetic enzymes. The cohort contained a respiratory burst oxidase homologue, polyamine oxidase, copper amine oxidase, and a suite of class III peroxidases. Collectively these generated two consecutive bursts in apoH2O2 production. Spatial organization of biosynthetic/catabolic enzymes was deduced from responses to pharmacologically blocking their activities on the cellular and subcellular distribution of apoH2O2. The findings were consistent with catalase activity constraining the apoH2O2 signal to the outer periclinal wall of the ETCs. Strategic positioning of class III peroxidases in this outer domain shaped subcellular apoH2O2 signatures that differed during assembly of the UWL and WI papillae., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over >60 years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction, and a variety of biosynthetic pathways in various species, tissues, and developmental stages. The complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question of how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here it is shown that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localized to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the ER could be necessary to bring auxin biosynthesis in closer proximity to ER-localized factors for transport, conjugation, and signalling, and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore, it might provide a link to ethylene action and be a factor in hormonal cross-talk as all five ethylene receptors are ER localized., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Isoflavone conjugates [7-O-β-D-glucosides and 7-O-(6″-malonyl-β-D-glucosides) of daidzein and genistein] accumulate in soybean roots and serve as the stored precursors of isoflavones (aglycons), which play very important roles in the rhizobia-mediated nodulation of this plant. Thus far, the isoflavone 7-O-glucosyltransferase (GmIF7GT or GmUGT1) has been biochemically characterized and is believed to be involved in isoflavone conjugate biosynthesis. The soybean genome encodes many other glycosyltransferase homologs (GmUGTs) that are related to GmUGT1; however, their catalytic properties, substrate specificities, and role(s) in isoflavone conjugation are unknown. In this study, nine different GmUGT1-related GmUGT cDNAs were isolated; six of these cDNAs belonged to two distinct phylogenetic subgroups (A and B), and these six were functionally characterized. The results showed that GmUGT4, a representative of subgroup A, encoded a UGT that was highly specific for isoflavones showing kcat and kcat/Km values for daidzein of 5.89 ± 0.65 s(-1) and 2.91 × 10(5) s(-1)M(-1), respectively. Moreover, GmUGT4 was expressed in the roots (mainly in lateral roots) of the 7-day-old seedlings and seeds, both of which contained abundant amounts of isoflavone conjugates. By contrast, GmUGT1 and GmUGT7, which were subgroup B members, encoded enzymes with broad glucosyl-acceptor specificities and were mainly expressed in the aerial portions (cotyledons and hypocotyls) of the seedlings. In the present study, we proposed that the role of isoflavone glucosylation in a soybean plant is assigned to different GmUGT members in an organ/tissue-dependent manner. We also established the functional importance of GmUGT4 in the biosynthesis of isoflavone conjugates in lateral roots that make a major contribution to overall N2 fixation., (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Chloroplasts perform many essential metabolic functions and their proper development is critically important in embryogenesis. However, little is known about how chloroplasts function in embryogenesis and more relevant components need to be characterized. In this study, we show that Arabidopsis Ribonuclease J (RNase J) is required for chloroplast and embryo development. Mutation of AtRNJ led to albino ovules containing aborted embryos; the morphological development of rnj embryos was disturbed after the globular stage. Observation of ultrastructures indicated that these aborted embryos may result from impaired chloroplast development. Furthermore, by analyzing the molecular markers of cell fate decisions (STM, FIL, ML1, SCR, and WOX5) in rnj embryos, we found that this impairment of chloroplast development may lead to aberrant embryo patterning along the apical-basal axis, indicating that AtRNJ is important in initiating and maintaining the organization of shoot apical meristems (SAMs), cotyledons, and hypocotyls. Moreover, the transport and response of auxin in rnj embryos was found to be disrupted, suggesting that AtRNJ may be involved in auxin-mediated pathways during embryogenesis. Therefore, we speculate that RNJ plays a vital role in embryo morphogenesis and apical-basal pattern formation by regulating chloroplast development., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Purine nucleotides can be fully catabolized by plants to recycle nutrients. We have isolated a urate oxidase (uox) mutant of Arabidopsis thaliana that accumulates uric acid in all tissues, especially in the developing embryo. The mutant displays a reduced germination rate and is unable to establish autotrophic growth due to severe inhibition of cotyledon development and nutrient mobilization from the lipid reserves in the cotyledons. The uox mutant phenotype is suppressed in a xanthine dehydrogenase (xdh) uox double mutant, demonstrating that the underlying cause is not the defective purine base catabolism, or the lack of UOX per se, but the elevated uric acid concentration in the embryo. Remarkably, xanthine accumulates to similar levels in the xdh mutant without toxicity. This is paralleled in humans, where hyperuricemia is associated with many diseases whereas xanthinuria is asymptomatic. Searching for the molecular cause of uric acid toxicity, we discovered a local defect of peroxisomes (glyoxysomes) mostly confined to the cotyledons of the mature embryos, which resulted in the accumulation of free fatty acids in dry seeds. The peroxisomal defect explains the developmental phenotypes of the uox mutant, drawing a novel link between uric acid and peroxisome function, which may be relevant beyond plants., (© 2014 American Society of Plant Biologists. All rights reserved.)

The major seed storage reserves in oilseeds are accumulated in protein bodies and oil bodies, and serve as an energy, carbon, and nitrogen source during germination. Here, the spatio-temporal relationships between protein bodies and several key enzymes (phospholipase A, lipase, and lipoxygenase) involved in storage lipid mobilization in cotyledon cells was analysed during in vitro seed germination. Enzyme activities were assayed in-gel and their cellular localization were determined using microscopy techniques. At seed maturity, phospholipase A and triacylglycerol lipase activities were found exclusively in protein bodies. However, after seed imbibition, these activities were shifted to the cytoplasm and the surface of the oil bodies. The activity of neutral lipases was detected by using α-naphthyl palmitate and it was associated mainly with protein bodies during the whole course of germination. This pattern of distribution was highly similar to the localization of neutral lipids, which progressively appeared in protein bodies. Lipoxygenase activity was found in both the protein bodies and on the surface of the oil bodies during the initial phase of seed germination. The association of lipoxygenase with oil bodies was temporally correlated with the appearance of phospholipase A and lipase activities on the surface of oil bodies. It is concluded that protein bodies not only serve as simple storage structures, but are also dynamic and multifunctional organelles directly involved in storage lipid mobilization during olive seed germination.

Papain-like cysteine protease activity that shows a unique transient expression profile in cotyledons of daikon radish during germination was detected. The enzyme showed a distinct elution pattern on DEAE-cellulose compared with cathepsin B-like and Responsive to dessication-21 cysteine protease. Although this activity was not detected in seed prior to imbibition, the activity increased markedly and reached a maximum at 2 days after imbibition and then decreased rapidly and completely disappeared after 5 days. Using cystatin-Sepharose, the 26 kDa cysteine protease (DRCP26) was isolated from cotyledons at 2 days after imbibition. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that DRCP26 is an orthologue of Arabidopsis unidentified protein, germination-specific cysteine protease-1, belonging to the C1 family of cysteine protease predicted from genetic information. In an effort to characterize the enzymatic properties of DRCP26, the enzyme was purified to homogeneity from cotyledons at 48 h after imbibition. The best synthetic substrate for the enzyme was carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide. All model peptides were digested to small peptides by the enzyme, suggesting that DRCP26 possesses broad cleavage specificity. These results indicated that DRCP26 plays a role in the mobilization of storage proteins in the early phase of seed germination.

The mitochondrial ATP synthase (F(1)F(o) complex) is an evolutionary conserved multimeric protein complex that synthesizes the main bulk of cytosolic ATP, but the regulatory mechanisms of the subunits are only poorly understood in plants. In yeast, the δ-subunit links the membrane-embedded F(o) part to the matrix-facing central stalk of F(1). We used genetic interference and an inhibitor to investigate the molecular function and physiological impact of the δ-subunit in Arabidopsis thaliana. Delta mutants displayed both male and female gametophyte defects. RNA interference of delta resulted in growth retardation, reduced ATP synthase amounts, and increased alternative oxidase capacity and led to specific long-term increases in Ala and Gly levels. By contrast, inhibition of the complex using oligomycin triggered broad metabolic changes, affecting glycolysis and the tricarboxylic acid cycle, and led to a successive induction of transcripts for alternative respiratory pathways and for redox and biotic stress-related transcription factors. We conclude that (1) the δ-subunit is essential for male gametophyte development in Arabidopsis, (2) a disturbance of the ATP synthase appears to lead to an early transition phase and a long-term metabolic steady state, and (3) the observed long-term adjustments in mitochondrial metabolism are linked to reduced growth and deficiencies in gametophyte development.

Position-dependent patterning of hair and non-hair cells in the Arabidopsis thaliana root epidermis is a powerful system to study the molecular basis of cell fate specification. Here, we report an epidermal patterning mutant affecting the ADENOSINE DIMETHYL TRANSFERASE 1A (DIM1A) rRNA dimethylase gene, predicted to participate in rRNA posttranscriptional processing and base modification. Consistent with a role in ribosome biogenesis, DIM1A is preferentially expressed in regions of rapid growth, and its product is nuclear localized with nucleolus enrichment. Furthermore, DIM1A preferentially accumulates in the developing hair cells, and the dim1A point mutant alters the cell-specific expression of the transcriptional regulators GLABRA2, CAPRICE, and WEREWOLF. Together, these findings suggest that establishment of cell-specific gene expression during root epidermis development is dependent upon proper ribosome biogenesis, possibly due to the sensitivity of the cell fate decision to relatively small differences in gene regulatory activities. Consistent with its effect on the predicted S-adenosyl-l-Met binding site, dim1A plants lack the two 18S rRNA base modifications but exhibit normal pre-rRNA processing. In addition to root epidermal defects, the dim1A mutant exhibits abnormal root meristem division, leaf development, and trichome branching. Together, these findings provide new insights into the importance of rRNA base modifications and translation regulation for plant growth and development.

Genome analysis has indicated that plants, like animals, possess a variety of protease genes. However, bulk of putative proteases has not been characterized at the enzyme level. In this article, a novel enzyme that hydrolyses phenylalanyl-4-methylcoumaryl 7-amide (phenylalanyl-MCA) was purified from cotyledons of daikon radish by ammonium sulphate fractionation and successive chromatography with DEAE-cellulose, phenyl-Sepharose, Sephacryl S-200 and Mini-Q. The molecular mass of the enzyme was estimated to be 78 kDa by SDS-PAGE under reducing conditions and 74 kDa by gel filtration, indicating that the enzyme is a monomer. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that the enzyme is an orthologue of Arabidopsis unidentified protein, acylpeptide hydrolase-like protein (AHLP; UniProt ID: Q9FG66) belonging to the prolyl oligopeptidase (POP) family of a serine-type peptidase predicted from genetic information. Good substrates identified for the enzyme include phenylalanyl-MCA, tyrosyl-MCA and enkephalin. Neither acylamino acid-releasing activity nor endopeptidase activity was detected. The enzyme cleaved enkephalin (YGGFM, YGGFL), whereas, BAM-12 P (YGGFMRRVGRPE) and dynorphin A (YGGFLRRIRPKLK) were not digested. These results suggested that the enzyme possesses strict size selectivity of substrate. We propose the name 'tyrosyl aminopeptidase' for the uncharacterized protein AHLP.

Plant thioredoxins (Trxs) participate in two redox systems found in different cellular compartments: the NADP-Trx system (NTS) in the cytosol and mitochondria and the ferredoxin-Trx system (FTS) in the chloroplast, where they function as redox regulators by regulating the activity of various target enzymes. The identities of the master regulators that maintain cellular homeostasis and modulate timed development through redox regulating systems have remained completely unknown. Here, we show that proteins consisting of a single cystathionine β-synthase (CBS) domain pair stabilize cellular redox homeostasis and modulate plant development via regulation of Trx systems by sensing changes in adenosine-containing ligands. We identified two CBS domain-containing proteins in Arabidopsis thaliana, CBSX1 and CBSX2, which are localized to the chloroplast, where they activate all four Trxs in the FTS. CBSX3 was found to regulate mitochondrial Trx members in the NTS. CBSX1 directly regulates Trxs and thereby controls H(2)O(2) levels and regulates lignin polymerization in the anther endothecium. It also affects plant growth by regulating photosynthesis-related [corrected] enzymes, such as malate dehydrogenase, via homeostatic regulation of Trxs. Based on our findings, we suggest that the CBSX proteins (or a CBS pair) are ubiquitous redox regulators that regulate Trxs in the FTS and NTS to modulate development and maintain homeostasis under conditions that are threatening to the cell.

Postgerminative growth of seed plants requires specialized metabolism, such as gluconeogenesis, to support heterotrophic growth of seedlings until the functional photosynthetic apparatus is established. Here, we show that the Arabidopsis thaliana fugu5 mutant, which we show to be defective in AVP1 (vacuolar H(+)-pyrophosphatase), failed to support heterotrophic growth after germination. We found that exogenous supplementation of Suc or the specific removal of the cytosolic pyrophosphate (PPi) by the heterologous expression of the cytosolic inorganic pyrophosphatase1 (IPP1) gene from budding yeast (Saccharomyces cerevisiae) rescued fugu5 phenotypes. Furthermore, compared with the wild-type and AVP1(Pro):IPP1 transgenic lines, hypocotyl elongation in the fugu5 mutant was severely compromised in the dark but recovered upon exogenous supply of Suc to the growth media. Measurements revealed that the peroxisomal β-oxidation activity, dry seed contents of storage lipids, and their mobilization were unaffected in fugu5. By contrast, fugu5 mutants contained ~2.5-fold higher PPi and ~50% less Suc than the wild type. Together, these results provide clear evidence that gluconeogenesis is inhibited due to the elevated levels of cytosolic PPi. This study demonstrates that the hydrolysis of cytosolic PPi, rather than vacuolar acidification, is the major function of AVP1/FUGU5 in planta. Plant cells optimize their metabolic function by eliminating PPi in the cytosol for efficient postembryonic heterotrophic growth.

Background and Aims: When rice seeds germinate under complete submergence, only the coleoptile elongates efficiently. It has been reported previously that coleoptile elongation is reduced in the rice alcohol dehydrogenase 1 (ADH1)-deficient mutant, reduced adh activity (rad). The aim of this study was to elucidate how expressions of genes responsible for coleoptile elongation are affected by the ADH1 deficiency in the rad mutant under submergence., Methods: To identify genes whose expressions are changed in the rad coleoptile at an early stage in germination (i.e. 1 d after imbibition), coleoptiles of the rad mutant and its wild type (WT) were isolated by laser microdissection, and their mRNA levels were examined with a microarray., Key Results: The microarray analysis identified 431 genes whose transcript levels were different between rad and WT. Interestingly, among the down-regulated genes in the rad coleoptile, 17·5 % were cell division-related genes and 5·1 % were cell elongation-related genes. It was found that cell division started at 1 d after imbibition and then gradually ceased, whereas in the WT coleoptile cell elongation started between 1 d and 2 d after imbibition and then continued. However, neither cell division nor cell elongation actively occurred in the rad coleoptile, in which the amounts of ATP were reduced., Conclusions: These results indicate that cell division, as well as cell elongation, occur during coleoptile elongation in rice under complete submergence, and that the reduced ATP levels caused by the ADH1 deficiency repress both of them, thereby impairing coleoptile elongation in the rad mutant under submerged conditions.

Arabidopsis thaliana contains two GDP-L-galactose phosphorylase genes, VTC2 and VTC5, which are critical for ascorbate (AsA) biosynthesis. We investigated the expression levels of both VTC2 and VTC5 genes in wild-type A. thaliana and the AsA deficient mutants during early seedling growth. Ascorbate accumulated to an equal extent in all genotypes up to 5 d post-germination (DPG). The transcript level of VTC2 was dominant, and increased in parallel with AsA accumulation in the wild type. On the other hand, the expression of VTC5 compensated for the reduced VTC2 transcription levels in the AsA deficient mutant vtc2-1 in young seedlings. A luciferase activity assay indicated that the VTC5 promoter was more active in young (2 DPG) cotyledons and that the VTC2 and VTC5 promoters drove a day-to-night variation in expression. The present work provides clues to the precise roles of VTC2 and VTC5 in AsA biosynthesis in A. thaliana at the young seedling stage.

Oilseed germination is characterized by the mobilization of storage lipids as a carbon and energy source for embryonic growth. In addition to storage lipid degradation in germinating oilseeds via the direct action of a triacylglycerol lipase (TGL) on the storage lipids, a second degradation pathway that is dependent on a specific lipid body trilinoleate 13-lipoxygenase (13-LOX) has been proposed in several plant species. The activity of this specific 13-LOX leads first to the formation of ester lipid hydroperoxides. These hydroperoxy fatty acids are then preferentially cleaved off by a TGL and serve as a substrate for glyoxysomal β-oxidation. As a prerequisite for triacylglycerol (TAG) mobilization, a partial degradation of the phospholipid monolayer and/or membrane proteins of the oil body has been discussed. Evidence has now been found for both processes: partial degradation of the proteins caleosin and oleosin was observed and simultaneously a patatin-like protein together with transient phospholipase (PLase) activity could be detected at the oil body membranes during germination. Moreover, in vitro experiments with isolated oil bodies from mature seeds revealed that the formation of 13-LOX-derived lipid peroxides in lipid body membranes is increased after incubation with the purified recombinant patatin-like protein. These experiments suggest that in vivo the degradation of storage lipids in cucumber cotyledons is promoted by the activity of a specific oil body PLase, which leads to an increased decomposition of the oil body membrane by the 13-LOX and thereby TAGs may be better accessible to LOX and TGL.

Most trichomes on the surface of cucumber (Cucumis sativus L.) cotyledons consist of three cells. We previously showed that continuous UV-B (290-320 nm) irradiation induces rapid cellular expansion and the accumulation of polyphenolic compounds, possibly stress lignin, in epidermal cells around these trichomes.(1)) To examine the mechanism of the UV-B-induced cellular expansion and to determine which step is stimulated by UV-B irradiation in the lignin synthesis pathway, we investigated relative DNA contents in epidermal cells, including trichomes, and enzyme activity and gene expression in the phenylpropanoid pathway. UV-B irradiation increased the ploidy level over 15 days, specifically in the epidermal cells surrounding trichomes, but not in the other epidermal cells or trichomes. In epidermal cells surrounding trichomes, UV-B irradiation induced peroxidase (POX) activity from days 7 to 15. In cotyledons, UV-B exposure induced CS-POX1 and CS-POX3 gene expression within 2 days, and it also induced two other enzymes in the phenylpropanoid pathway, sinapyl alcohol dehydrogenase and coniferyl alcohol dehydrogenase, from days 9 to 11. Thus, exposure to UV-B induces expansion, endoreduplication, POX activity, and the accumulation of polyphenolic compounds in epidermal cells surrounding the trichomes of cucumber cotyledons. Because polyphenolic compounds such as lignin absorb UV-B, our data indicate a physiological protective mechanism against UV-B irradiation in cucumber.

A novel enzyme that catalyzes the efficient hydrolysis of Glu-Glu was isolated from soybean cotyledons by ammonium sulfate fractionation and successive column chromatographies of Q-sepharose, Phenyl sepharose, and Superdex 200. The apparent molecular mass of this enzyme was found to be 56 kDa and 510 kDa by SDS-polyacrylamide gel electrophoresis and Superdex 200 HR 10/30 column chromatography respectively. The enzyme had high activity against Glu-p-nitroanilide (pNA) and Asp-pNA, whereas Leu-pNA, Phe-pNA, Ala-pNA, and Pro-pNA were not hydrolyzed. The synthetic dipeptides Glu-Xxx and Asp-Xxx were hydrolyzed, but Xxx-Glu was not. The digestion of a Glu-rich oligopeptide, chromogranin A (Glu-Glu-Glu-Glu-Glu-Met-Ala-Val-Val-Pro-Gln-Gly-Leu-Phe-Arg-Gly-NH(2)) using this purified enzyme was also investigated. Glutamic acid residues were cleaved one by one from the N-terminus. These observations indicate that the enzyme removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. It is thought that it was an N-terminal acidic amino acid-specific aminopeptidase from a plant.

RD21 (Responsive to desiccation-21) is an Arabidopsis cysteine protease which possesses a granulin-like domain at the C-terminus. Although two forms of RD21 have been identified, consisting of an intermediate form (iRD21) containing a granulin domain and a mature form (mRD21) lacking this domain, the enzymatic properties of these enzymes remain poorly understood. In this study, mRD21 orthologue was purified to homogeneity from the cotyledons of daikon radish (Raphanus sativus). RD21 preferentially cleaved peptide bond that had an aromatic or hydrophobic amino acid at the P2 position. Furthermore, the presence of a polar amino acid at the P1 position enhanced the cleavage susceptibility of the peptide bond, although the importance of the type of amino acid residue at the P1 and P1' positions was not as significant as the residue located at the P2 position. The iRD21 was also identified as an oligomeric form by gel filtration and sedimentation analyses. The expression of RD21 mRNA was initiated by imbibition and continued at almost constant levels during germination. On the other hand, the enzyme activity increased markedly 5 days after imbibition. These results indicate that this elevation of RD21 activity is generated post-transcriptionally.

Background and Aims: The metabolism of beta-1,3 : 1,4-glucan regulates the mechanical properties of cell walls, and thereby changes the elongation growth of Poaceae plants. A previous study has shown that elongation growth of rice coleoptiles under water is enhanced by increased activity of beta-1,3 : 1,4-glucan hydrolases; however, the involvement of beta-1,3 : 1,4-glucan synthase activity in elongation growth under water has not yet been clarified., Methods: The beta-1,3 : 1,4-glucan synthase activity in a microsomal fraction prepared from rice seedlings grown under water was compared with that from control seedlings grown in air. The change under water in the relative expression level of CslF6, a major isoform of the beta-1,3 : 1,4-glucan synthase genes, was examined by quantitative reverse-transcriptase PCR., Key Results: The level of beta-1,3 : 1,4-glucan synthase activity in submerged seedlings decreased to less than 40 % of that of the control seedlings and was accompanied by a significant reduction in the amount of beta-1,3 : 1,4-glucan in the cell walls. Under water, the expression of CslF6 was reduced to less than 20 % of the unsubmerged control. Bubble aeration partially restored both beta-1,3 : 1,4-glucan synthase activity and the expression of CslF6 under water, correlating with suppression of the submergence-induced elongation growth of coleoptiles., Conclusions: Submergence down-regulates the expression of the CslF6 gene, leading to a decreased level of beta-1,3 : 1,4-glucan synthase activity. Together with the increased activity of beta-1,3 : 1,4-glucan hydrolases, the decreased activity of beta-1,3 : 1,4-glucan synthase contributes to the decrease in the amount of beta-1,3 : 1,4-glucan in the cell walls under water. The suppression of beta-1,3 : 1,4-glucan synthesis under water may be mainly due to oxygen depletion.

Dipeptidase activity was detected in the soluble fraction of radish (Raphanus sativus L.) cotyledon, and the purified enzyme had a specific activity of 7.32 nkat/mg protein for hydrolyzing L-cysteinylglycine. The dipeptidase was found to be a hexameric metalloenzyme, composed of homological 55 kDa-subunits. This is the first glutathione catabolism-related dipeptidase isolated from higher plants.

Plant aspartate aminotransferase (AAT, EC 2.6.1.1) plays a key role in primary nitrogen assimilation, the transfer of reducing equivalents and the interchanges of carbon and nitrogen pools between subcellular compartments. We investigated the AAT family in conifers using maritime pine as the experimental model. Genes for cytosolic, mitochondrial and two plastidic isoenzymes (eukaryotic- and prokaryotic-types) were identified and their deduced amino acid sequences compared. The primary structure of the eukaryotic-type enzymes is quite well conserved, whereas the prokaryotic-type AAT is highly divergent (15% of identity). These molecular data were confirmed by the absence of immunological cross-reactivity between the two types of native AATs. The mature prokaryotic-type polypeptide was overexpressed in Escherichia coli, and the native enzyme was purified to apparent homogeneity and its molecular properties determined. The fully active recombinant holoenzyme showed highest catalytic activity at 50-60 degrees C and was moderately thermostable, retaining about 50% of its activity after incubation at 70 degrees C for 5-10 min. The presence of pyridoxal 5'-phosphate significantly increased the thermostability of the enzyme. These molecular characteristics were exploited to develop a rapid protocol for the purification of this prokaryotic-type enzyme from pine cotyledons. The results will be useful for studying aspartate and amino acid metabolism in trees.

In this work the manganese superoxide dismutase (Mn-SOD) bound to peroxisomal membranes of watermelon cotyledons (Citrullus lanatus Schrad.) was purified to homogeneity and some of its molecular properties were determined. The stepwise purification procedure consisted of ammonium sulphate fractionation, batch anion-exchange chromatography, and anion-exchange and gel-filtration column chromatography using a fast protein liquid chromatography system. Peroxisomal membrane Mn-SOD (perMn-SOD; EC 1.15.1.1) was purified 5600-fold with a yield of 2.6 mug of enzyme g(-1) of cotyledons, and had a specific activity of 480 U mg(-1) of protein. The native molecular mass determined for perMn-SOD was 108 000 Da, and it was composed of four equal subunits of 27 kDa, which indicates that perMn-SOD is a homotetramer. Ultraviolet and visible absorption spectra of the enzyme showed a shoulder at 275 nm and two absorption maxima at 448 nm and 555 nm, respectively. By isoelectric focusing, a pI of 5.75 was determined for perMn-SOD. In immunoblot assays, purified perMn-SOD was recognized by a polyclonal antibody against Mn-SOD from pea leaves, and the peroxisomal enzyme rapidly dissociated in the presence of dithiothreitol and SDS. The potential binding of the Mn-SOD isozyme to the peroxisomal membrane was confirmed by immunoelectron microscopy analysis. The properties of perMn-SOD and the mitMn-SOD are compared and the possible function in peroxisomal membranes of the peripheral protein Mn-SOD is discussed.

An alkaline proteinase activity is present in quiescent seeds and up to the 24th day of development of Canavalia ensiformis DC (L.) plants. By a simple protocol consisting of cation exchange chromatography, followed by an anion exchange column, a serine proteinase (Q-SP) was purified to homogeneity from quiescent seeds. Q-SP consists of a 33 kDa chain with an optimum pH between 8.0 and 9.0. Arginine residues at P1 and P2 subsites favour binding to the substrate, as shown by the KM assay with N-alpha-benzoyl-DL-arginine-4-nitroanilide-hydrochloride and N-benzoylcarboxyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin. The same protocol was used for partial purification of benzamidine-sensitive enzymes from the developing plant. On the 7th day, a new benzamidine-sensitive enzyme is synthesized in the seedling, seen as the second active peak appearing in anion exchange chromatography. A benzamidine-sensitive enzyme purified from cotyledons presented a similar gel filtration profile as Q-SP, although it was eluted at different salt concentrations in the anion exchange chromatography. None of the enzymes was inhibited by PMSF, APMSF, or SBTI, but they were inactivated by benzamidine, TLCK, and leupeptin. Q-SP did not cleave in vitro C. ensiformis urease, concanavalin A, or its main storage protein, canavalin. In conclusion, a ubiquitous benzamidine-sensitive proteolytic activity was found in C. ensiformis from quiescent seeds up to 24 d of growth, which apparently is not involved in the hydrolysis of storage proteins and might participate in an as yet unidentified limited proteolysis event.

Brassinosteroids (BRs) are biosynthesized from campesterol via several cytochrome P450 (P450)-catalyzed oxidative reactions. We report the functional characterization of two BR-biosynthetic P450s from Arabidopsis thaliana: CYP90C1/ROTUNDIFOLIA3 and CYP90D1. The cyp90c1 cyp90d1 double mutant exhibits the characteristic BR-deficient dwarf phenotype, although the individual mutants do not display this phenotype. These data suggest redundant roles for these P450s. In vitro biochemical assays using insect cell-expressed proteins revealed that both CYP90C1 and CYP90D1 catalyze C-23 hydroxylation of various 22-hydroxylated BRs with markedly different catalytic efficiencies. Both enzymes preferentially convert 3-epi-6-deoxocathasterone, (22S,24R)-22-hydroxy-5alpha-ergostan-3-one, and (22S,24R)-22-hydroxyergost-4-en-3-one to 23-hydroxylated products, whereas they are less active on 6-deoxocathasterone. Likewise, cyp90c1 cyp90d1 plants were deficient in 23-hydroxylated BRs, and in feeding experiments using exogenously supplied intermediates, only 23-hydroxylated BRs rescued the growth deficiency of the cyp90c1 cyp90d1 mutant. Thus, CYP90C1 and CYP90D1 are redundant BR C-23 hydroxylases. Moreover, their preferential substrates are present in the endogenous Arabidopsis BR pool. Based on these results, we propose C-23 hydroxylation shortcuts that bypass campestanol, 6-deoxocathasterone, and 6-deoxoteasterone and lead directly from (22S,24R)-22-hydroxy-5alpha-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol.

In the present work, the promoter of the Arabidopsis thaliana nuclear gene COX5b-1, encoding subunit 5b of the mitochondrial cytochrome c oxidase, has been analysed. For this purpose, plants, stably transformed with different promoter fragments fused to the beta-glucuronidase reporter gene, have been obtained. Histochemical staining indicated that the COX5b-1 promoter directs expression in meristems and in vascular tissues of cotyledons, roots, and hypocotyls, as well as in anthers and pollen and the central leaf vein. Quantitative measurements in extracts prepared from different organs suggested that expression is higher in roots. The analysis of progressive upstream deletions of the promoter suggested the presence of negative regulatory elements, preferentially active in leaves, between nucleotides -609 and -387 from the translation start site. A further deletion down to nucleotide -195 completely abolished expression. The inclusion of sucrose or the cytokinin 6-benzylaminopurine in the culture medium induced COX5b-1 promoter-dependent beta-glucuronidase expression. This induction was observed with all constructs that produced beta-glucuronidase activity. Putative regulatory elements involved in the regulation of other genes were detected in the promoter fragment required for expression. A detailed analysis of these elements will help to elucidate the molecular mechanisms that participate in the expression of this and, possibly, other components of the cytochrome c-dependent respiratory pathway.

The effect of supplying either nitrate or ammonium on nitrate reductase activity (NRA) was investigated in Brassica napus seedlings. In roots, nitrate reductase activity (NRA) increased as a function of nitrate content in tissues and decreased when ammonium was the sole nitrogen source. Conversely, in the shoots (comprising the cotyledons and hypocotyl), NRA was shown to be independent of nitrate content. Moreover, when ammonium was supplied as the sole nitrogen source, NRA in the shoots was surprisingly higher than under nitrate supply and increased as a function of the tissue ammonium content. Under 15 mM of exogenous ammonium, the NRA was up to 2.5-fold higher than under nitrate supply after 6 d of culture. The NR mRNA accumulation under ammonium nutrition was 2-fold higher than under nitrate supply. The activation state of NR in shoots was especially high compared with roots: from nearly 80% under nitrate supply it reached 94% under ammonium. This high NR activation state under ammonium supply could be the consequence of the slight acidification observed in the shoot tissue. The effect of ammonium on NRA was only observed in cotyledons and when more than 3 mM ammonium was supplied. No such NRA increase was evident in the roots or in foliar discs. Addition of 1 mM nitrate under ammonium nutrition halved NRA and decreased the ammonium content in shoots. Thus, this unusual NRA was restricted to seedling cotyledons when nitrate was lacking in the nitrogen source.

The non dormant-1 (nd-1) mutant of sunflower (Helianthus annuus L.) is characterized by an albino and viviparous phenotype. Pigment analysis by spectrophotometer and HPLC demonstrated in nd-1 cotyledons the absence of beta-carotene, lutein and violaxanthin. Additionally, we found a strong accumulation of zeta-carotene and, to a lesser extent, of phytofluene and cis-phytoene in nd-1 seedlings grown in very dim light (1 micro mol m(-2) s(-1)). These results suggested that zeta-carotene desaturation was impaired in the mutant plants. To understand the molecular basis of the nd-1 mutation, we cloned and characterized the zeta-carotene desaturase (Zds) gene from sunflower. A reconstructed full-length sequence (1,916 bp) of the Zds cDNA was obtained from homozygous Nd-1/Nd-1 wild-type plants. It contains a 1,761-bp CDS, 62 nucleotides of 5'-untranslated region (UTR), and 77 nucleotides of 3'-UTR. The predicted protein (64.9 kDa) consists of 587 amino acid residues with a putative transit sequence for plastid targeting in the N-terminal region and a typical amino oxidase domain that includes the flavin adenosine dinucleotide (FAD) binding motif. The phylogenetic analysis demonstrated that the sunflower Zds was clustered to marigold (Tagetes) Zds gene, for which it showed an overall aminoacidic identity of 96.6% and resulted strictly correlated with other Zds sequences of higher plants. Interestingly, RT-PCR analyses showed that nd-1 plants were unable to accumulate Zds transcripts. Sequence information from the Zds cDNA was used to design specific primers and to isolate the full-length exons/introns region of the gene. The sunflower Zds gene (HaZds) comprises 14 exons and 13 introns scattered in a ca. 5.0-kb region. Also, HaZds showed a high conservation of the distribution and size of the exons with rice Zds gene. Based on genomic Southern analysis, the nd-1 genome disclosed a large deficiency at the Zds locus.

Glyoxysomes are present in etiolated cotyledons and contain enzymes for gluconeogenesis, which constitutes the major function of glyoxysomes. However, 281 genes seemingly related to peroxisomal functions occur in the Arabidopsis genome, implying that many unidentified proteins are present in glyoxysomes. To better understand the functions of glyoxysomes, we performed glyoxysomal proteomic analysis of etiolated Arabidopsis cotyledons. Nineteen proteins were identified as glyoxysomal proteins, including 13 novel proteins, one of which is glyoxysomal protein kinase 1 (GPK1). We cloned GPK1 cDNA by RT-PCR and characterized GPK1. The amino acid sequence deduced from GPK1 cDNA has a hydrophobic region, a putative protein kinase domain, and a possible PTS1 motif. Immunoblot analysis using fractions collected on a Percoll density gradient confirmed that GPK1 is localized in glyoxysomes. Analysis of suborganellar localization and protease sensitivity showed that GPK1 is localized on glyoxysomal membranes as a peripheral membrane protein and that the putative kinase domain is located inside the glyoxysomes. Glyoxysomal proteins are phosphorylated well in the presence of various metal ions and [g-32P]ATP, and one of them is identified as thiolase by immunoprecipitation. Immuno-inhibition of phosphorylation in glyoxysomes suggested that GPK1 phosphorylates a 40-kDa protein. These results show that protein phosphorylation systems are operating in glyoxysomes.

Light signaling has been demonstrated to be an important factor for plant growth and development; however, its role in the regulation of DNA replication and cell cycle has just started to be unraveled. In this work, we have demonstrated that the TOP2 promoter of Pisum sativum (pea) is activated by a broad spectrum of light including far-red light (FR), red light (RL) and blue light (BL). Deletion analyses of the TOP2 promoter in transformed plants, Arabidopsis thaliana and Nicotiana tobaccum (tobacco), define a minimal promoter region that is induced by RL, FR and BL, and is essential and sufficient for light-mediated activation. The minimal promoter of TOP2 follows the phytochrome- mediated low-fluence response similar to complex light regulated promoters. DNA-protein interaction studies reveal the presence of a DNA binding activity specific to a 106 bp region of the minimal promoter that is crucial for light-mediated activation. These results altogether indicate a direct involvement of light signaling in the regulation of expression of TOP2, one of the components of the DNA replication/cell cycle machinery.

Two types of phospholipid degrading enzyme, phospholipase D (PLD; EC 3.1.4.4) and phosphatidyl- inositol-specific phospholipase C (PIP(2)-PLC; PI-PLC 3.1.4.11) were studied during the development of seeds and plants of Brassica napus. PLD exhibits two types of activity; polyphosphoinositide-requiring (PIP(2)-dependent PLD) and polyphosphoinositide-independent requiring millimolar concentrations of calcium (PLDalpha). Significantly different patterns of activity profiles were found for soluble and membrane-associated forms of all three enzymes within both processes. Membrane-associated PIP(2)-dependent PLD activity shows the opposite trend when compared to PLDalpha, while the highest PI-PLC activity appears in the same stages of development of seeds and plants as for PLDalpha. In subcellular fractions of hypocotyls of young plants, phospholipases were localized predominantly on plasma membranes. The biochemical characteristics (Ca(2+), pH) of all three enzymes associated with plasma membrane vesicles, isolated by partitioning in an aqueous dextran- polyethylene glycol two-phase system, are also described. Direct interaction of PLDalpha with G-proteins under in vitro conditions was not confirmed.

Alpha-amylase is expressed at high levels in cotyledons of germinated seeds of Vigna mungo. The mRNA for alpha-amylase appeared in cotyledons of the seeds at 1 d after imbibition started (DAI). Two TGACGT motifs at -445 and at -125 in the promoter region of the gene interacted with nuclear proteins from cotyledons of dry seeds and the activities were detected until 3 DAI. A transient assay with particle bombardment showed that the downstream region from -135 in the promoter was required for high level expression in the cotyledons and the activity was reduced by mutation of the TGACGT motif at -125. The activities to bind the TGACGT motifs were detected in the axes of the seeds at 1 DAI but disappeared at 4 DAI, although the mRNA for alpha-amylase in the axes appeared at 4 DAI and increased in level by 6 DAI. A transient assay experiment showed that a positive regulatory element for the expression in the axes was located in the region from -630 to -453. These results indicated that the TGACGT motif at -125 was required for high level expression of the gene in the cotyledons of the germinated seeds.

To investigate the roles of peroxisomal membrane proteins in the reversible conversion of glyoxysomes to leaf peroxisomes, we characterized several membrane proteins of glyoxysomes. One of them was identified as an ascorbate peroxidase (pAPX) that is localized on glyoxysomal membranes. Its cDNA was isolated by immunoscreening. The deduced amino acid sequence encoded by the cDNA insert does not have a peroxisomal targeting signal (PTS), suggesting that pAPX is imported by one or more PTS-independent pathways. Subcellular fractionation of 3- and 5-d-old cotyledons of pumpkin revealed that pAPX was localized not only in the glyoxysomal fraction, but also in the ER fraction. A magnesium shift experiment showed that the density of pAPX in the ER fraction did not increase in the presence of Mg(2+), indicating that pAPX is not localized in the rough ER. Immunocytochemical analysis using a transgenic Arabidopsis which expressed pumpkin pAPX showed that pAPX was localized on peroxisomal membranes, and also on a unknown membranous structure in green cotyledons. The overall results suggested that pAPX is transported to glyoxysomal membranes via this unknown membranous structure.

When auxin stimulates rapid cell elongation growth of cereal coleoptiles, it causes a degradation of 1,3:1,4-beta-glucan in hemicellulosic polysaccharides. We examined gene expressions of endo-1,3:1,4-beta-glucanase (EI) and exo-beta-glucanase (ExoII), of which optimum pH are about 5, and molecular distribution of hemicellulosic polysaccharides in barley (Hordeum vulgare L.) coleoptile segments treated with or without IAA. IAA (10(-5) M) stimulated the gene expression of EI, while it did not affect that of ExoII. IAA induced gene expression of EI after 4 h and increased wall-bound glucanase activity after 8 h. The molecular weight distribution of hemicellulosic polysaccharides from coleoptile cell walls was shifted to lower molecular weight region by 2 h of IAA treatment. Fusicoccin (10(-6) M) mimicked IAA-induced elongation growth and the decrease in molecular weight of hemicellulosic 1,3:1,4-beta-glucan of coleoptiles in the first 4 h, but it did not promote elongation growth thereafter. These facts suggest that acidification of barley cell walls by IAA action enhances pre-existing cell wall-bound glucanase activity in the early first phase of IAA-induced growth and the late second phase involves the gene expression of EI by IAA.

Cotyledons detached from 4-d-old cucumber (Cucumis sativus L.) seedlings were subjected to water stress (air-drying or PEG-treatment) to examine the effects of the stress on carbohydrate metabolism. Amylolytic activity in the cotyledon was increased about 6-fold by water stress within 1 d. The substrate specificity and the action pattern indicated that beta-amylase is responsible for the activity. Activities of azocaseinase, malate dehydrogenase and triose-phosphate isomerase were not affected by water stress, indicating that the effect of the stress on beta-amylase is rather specific. Cycloheximide-treatment strongly reduced the enhancement of beta-amylase activity. The hypocotyl of cucumber seedlings also exhibited an increase in the enzyme activity when subjected to water stress. The major free sugars in cucumber cotyledons were glucose, fructose, maltose, and sucrose; sucrose being the most abundant. Sucrose content in excised, unstressed cotyledons increased markedly during the incubation. Changes in other free sugars were small compared with that of sucrose. Starch also accumulated in unstressed cotyledons. In stressed cotyledons more sucrose and less starch accumulated than in unstressed ones. Such results were discussed in relation to the enhancement of beta-amylase activity.

Seedlings of Brassica napus were cultivated on a slowly rotating clinostat (1 rpm) or in the vertical control for 5 d. The root growth, the cotyledonary reserves and the transport of 14C-labeled sucrose from cotyledons to root system were studied in both cultural conditions. The biomass (fresh weight) of the root system was 35% higher in the horizontally clinorotated seedlings than in the controls. This increase was correlated with a greater degradation of reserve lipids and faster accumulation of sucrose in the cotyledons. The activity of isocitrate lyase, one of the two enzymes necessary to conversion of lipids into glucids, was also greater in the cotyledons of clinorotated seedlings. The labeling distribution of 14C in the cotyledons, the hypocotyl and the root system after 30, 60 and 120 min of application of 14C-labeled sucrose on the cotyledons showed higher translocation of the cotyledonary sucrose to the root system of clinorotated seedlings. In addition, we studied the effects of clinorotation on the biomass of the excised root system (of 10 d old seedlings) cultivated in a medium containing 1% sucrose. The horizontally clinorotated root system grew more than that of the controls. These results showed that the horizontal clinorotation acted on the root system growth and provoked a higher sucrose translocation from source to sink, i.e. from cotyledons to root system.

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.

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.