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The majority of plant viral disease is transmitted and spread by insect vectors in the field. The small brown planthopper, Laodelphax striatellus (Fallén), is the only efficient vector for rice black-streaked dwarf virus (RBSDV), a devastating plant virus that infects multiple grain crops, including rice, maize, and wheat. Adenosine triphosphate (ATP)-binding cassette (ABC) transporters participate in various biological processes. However, little is known about whether ABC transporters affect virus infection in insects. In this study, RBSDV accumulation was significantly reduced in L. striatellus after treatment with verapamil, an effective inhibitor of ABC transporters. Thirty-four ABC transporter genes were identified in L. striatellus and expression analysis showed that LsABCF2 and LsABCG9 were significantly upregulated and downregulated, respectively, after RBSDV infection. LsABCF2 and LsABCG9 were expressed during all developmental stages, and LsABCG9 was highly expressed in the midgut of L. striatellus. Knockdown of LsABCF2 promoted RBSDV accumulation, while knockdown of LsABCG9 suppressed RBSDV accumulation in L. striatellus. Our data showed that L. striatellus might upregulate the expression of LsABCF2 and downregulate LsABCG9 expression to suppress RBSDV infection. These results will contribute to understanding the effects of ABC transporters on virus transmission and provide theoretical basis for virus management in the field.

Aphis craccivora (Koch) is an economically important pest that affects legumes in worldwide. Chemical control is still the primary efficient method for A. craccivora management. However, the mechanism underlying insecticide resistance in A. craccivora has not been elucidated. A previous study observed that piperonyl butoxide (PBO) and diethyl maleate (DEM) significantly synergized imidacloprid in A. craccivora field populations, indicating that cytochrome P450 (P450) and glutathione S-transferase (GST) genes may play pivotal roles in imidacloprid resistance. In this study, 38 P450 genes and 10 GST genes were identified in A. craccivora through transcriptomic analysis. The expression levels of these P450 and GST genes were measured in susceptible (SUS) strains of A. craccivora under imidacloprid treatment with LC15, LC50, and LC85 doses. The expression levels of CYP18A1, CYP6CY21, CYP6DA1, CYP6DA2, CYP4CJ1, CYP4CJ2, and CYP380C6 were up-regulated in the three treatments. Most of these genes belong to CYP3 and CYP4 Clans. In addition, the expression levels of all P450 and GST genes in A. craccivora were also measured in the Juye (JY) and Linqing (LQ) field populations. The expression levels of CYP6DA2, CYP4CJ1, and CYP380C6 were up-regulated in the SUS strain after imidacloprid treatment at three doses, and these genes were overexpressed in the JY population. Furthermore, the sensitivity of A. craccivora to imidacloprid was significantly increased after knockdown of CYP380C6 and CYP6DA2 through RNA interference. These results may help to elucidate the mechanisms underlying of imidacloprid resistance in A. craccivora.

Aphis craccivora (Koch) is an economically important pest that affects legumes in worldwide. Chemical control is still the primary efficient method for A. craccivora management. However, the mechanism underlying insecticide resistance in A. craccivora has not been elucidated. A previous study observed that piperonyl butoxide (PBO) and diethyl maleate (DEM) significantly synergized imidacloprid in A. craccivora field populations, indicating that cytochrome P450 (P450) and glutathione S-transferase (GST) genes may play pivotal roles in imidacloprid resistance. In this study, 38 P450 genes and 10 GST genes were identified in A. craccivora through transcriptomic analysis. The expression levels of these P450 and GST genes were measured in susceptible (SUS) strains of A. craccivora under imidacloprid treatment with LC15, LC50, and LC85 doses. The expression levels of CYP18A1, CYP6CY21, CYP6DA1, CYP6DA2, CYP4CJ1, CYP4CJ2, and CYP380C6 were up-regulated in the three treatments. Most of these genes belong to CYP3 and CYP4 Clans. In addition, the expression levels of all P450 and GST genes in A. craccivora were also measured in the Juye (JY) and Linqing (LQ) field populations. The expression levels of CYP6DA2, CYP4CJ1, and CYP380C6 were up-regulated in the SUS strain after imidacloprid treatment at three doses, and these genes were overexpressed in the JY population. Furthermore, the sensitivity of A. craccivora to imidacloprid was significantly increased after knockdown of CYP380C6 and CYP6DA2 through RNA interference. These results may help to elucidate the mechanisms underlying of imidacloprid resistance in A. craccivora.

An ethylene receptor gene named BoERS1 was cloned from a bamboo (Bambusa oldhamii) cDNA library. The open reading frame of BoERS1 was 1,899 bp and encoded a 632-amino acid protein, which contains the five conserved motifs (H, N, G1, F, and G2 boxes) of the bacterial two-component system histidine kinases and shows high sequence similarity with other ethylene receptors in plants, such as rice and maize. Expression of BoERS1 in bamboo shoots increased with the growth of the emerging shoots. In an in vitro kinase assay, the expressed histidine kinase domain of BoERS1 (BHK) was phosphorylated in the presence of Mn2+, and LC-ESI-MS/MS analysis showed that four amino acids, namely T442, S444, S489, and S503, were phosphorylated. It is interesting to note that S489 and S503 are located in a loop region (L1) that is found only in plant histidine kinase-containing enzymes. The identification of multiple phosphorylation sites on BoERS1 provides a new avenue for future structure–function studies of the ethylene receptor protein family.

To gain the sensitive fault information, proper sampling point configuration is essential and important. A method based on ensemble empirical mode decomposition and variable precision rough set is proposed to optimize the monitoring points in the gearbox fault diagnosis system. First, the vibration signal was processed by ensemble empirical mode decomposition, the energetic characteristic vector can be got by the intrinsic mode functions. Samples in different conditions constructed the decision table, each monitoring point corresponded to a decision table. Second, the approximate dependence values of condition attributes to decision attributes of every monitoring point were got by variable precision rough set. Finally, the importance of the sampling points was achieved by the approximate dependence value. The experimental results show the method is effective and feasible for the monitoring points configuration, and it can minimize the impact of noise as well as improve the efficiency and accuracy of the fault diagnosis system.

In order to obtain the characteristic parameters reflecting fault state of high-speed automaton (HSA), the fault feature extraction method based on motion morphology decomposition and wavelet packet transform (WPT) was presented. According to the movement law of the automaton, the vibration signal generated in three bursts of fire was decomposed into three separate signals, then the response signal in each shooting is a separate signal. Then using WPT to respectively extract wavelet packet energy from three separate signals as the fault characteristic parameters of HSA. By the example, the results show that the extracted fault features can well reflect the working conditions of automaton. Thus the proposed method could be used to extract the fault feature of automaton for monitoring the condition and diagnosing the fault of automaton.

The promoter regions of two cell wall invertase genes, Boβfruct1 and Boβfruct2, and a vacuolar invertase gene, Boβfruct3, in Bambusa oldhamii were cloned, and putative regulatory cis-elements were identified. The expression of these three genes in multiple shoots of bamboo that were cultured in vitro under different conditions was analyzed by real-time PCR. The two cell wall invertase genes were upregulated by indole-3-acetic acid and cytokinins but responded differently to other phytohormones and different temperatures. Boβfruct1 was also upregulated by sucrose and glucose. In contrast, the Boβfruct2 expression was induced by the depletion of sucrose, and this induction could be suppressed by glucose and sucrose. The expression of Boβfruct3 was light-dependent; however, abscisic acid (ABA) could induce its expression in the dark. ABA and light exhibited an additive effect on the expression of Boβfruct3. Our results suggest that these three Boβfruct genes have individual roles in the adaption of the plant to environmental changes. Boβfruct2 might also have an essential role in the immediate response of cells to sucrose availability and in the maintenance of sink activity. Moreover, Boβfruct3 might be one of the interacting nodes of the light and ABA signaling pathways.

Bamboos are ecologically and economically important grasses, and are distinguished by their rapid growth. To identify genes associated with bamboo growth, PCR-based mRNA differential display was used to clone genes that were differentially expressed in various tissues of bamboo (Bambusa oldhamii) shoots at different growth stages. In total, 260 different cDNA sequences were obtained. These genes displayed complex expression profiles across the different tissues and growth stages as revealed by a cDNA microarray analysis. Notable among them were genes that were temporally up-regulated or down-regulated in the internode-containing region of rapidly elongating shoots. These genes might participate in the rapid elongation of the bamboo culm. Of the 36 up-regulated and 46 down-regulated genes, 16 genes and 8 genes, respectively, were predicted to encode hypothetical proteins or were unknown sequences. Aside from these, genes involved in hormonal signaling and homeostasis, stress responses, peptide processing and signaling and lignin biosynthesis composed most of the up-regulated genes; genes involved in DNA replication, nucleic acid binding and signal transduction were highly represented among the down-regulated genes. These results suggested that genes associated with plant hormonal signaling and homeostasis, peptide signaling, reactive oxygen species signaling and homeostasis, several stress-related genes and a monocot-specific unknown gene, BoMSP41, play important roles in the elongation of bamboo internodes. Multiple signaling pathways might form a complex interconnected network that controls the rapid growth of this giant grass.

Plant acid invertases, which are either associated with the cell wall or present in vacuoles, belong to family 32 of glycoside hydrolases (GH32). Homology modeling of bamboo vacuolar invertase Bobetafruct3 using Arabidopsis cell-wall invertase AtcwINV1 as a template showed that its overall structure is similar to GH32 enzymes, and that the three highly conserved motifs, NDPNG, RDP and EC, are located in the active site. This study also used site-directed mutagenesis to examine the roles of the conserved amino acid residues in these three motifs, which include Asp135, Arg259, Asp260, Glu316 and Cys317, and a conserved Trp residue (Trp159) that resides between the NDPNG and RDP motifs. The mutants W159F, W159L, E316Q and C317A retained acid invertase activity, but no invertase activity was observed for the mutant E316A or mutants with changes at Asp135, Arg259, or Asp260. The apparent K(m) values of the four mutants with invertase activity were all higher than that of the wild-type enzyme. The mutants W159L and E316Q exhibited lower k(cat) values than the wild-type enzyme, but an increase in the k(cat) value was observed for the mutants W159F and C317A. The results of this study demonstrate that these residues have individual functions in catalyzing sucrose hydrolysis.

In order to explore the genetic basis for chlorophyll content in maize (Zea mays L.), a total of 189 F2 individuals derived from the single cross between the inbred lines A150-3-2 and Mo17 were used as mapping population. Four traits associated with chlorophyll content were measured at the trumpet stage and at the flowering stage. Total 32 QTLs were investigated on all the chromosomes except for chromosomes 6 and 10. There were 24 QTLs located on chromosomes 1, 2, 3, 5, 7, 8, and 9 at the trumpet stage. Six QTLs were investigated for chlorophyll-a content (chla), chlorophyll-b content (chlb), other chlorophyll content (chlc), and total chlorophyll content (chlz), respectively. QTLs for four traits were located in the same marker intervals in many cases. The distance among different QTLs of the four traits in the same marker intervals ranged from 0 to 2 cM. The four major QTLs for chla, chlb, chlc, and chlz at the trumpet stage, which explained 11.63%, 10.3%, 10.77%, and 11.51% of the phenotypic variance, respectively, were investigated between umc1098 and bnlg557 on chromosome 5. There were 8 QTLs located on chromosomes 4 and 5 at the flowering stage, with 2 QTLs for chla, chlb, chlc, and chlz, respectively. QTLs were investigated between umc1098 and bnlg557, which controlled the four chlorophyll content traits (chla, chlb, chlc, and chlz), at the trumpet stage and two chlorophyll content traits (chla and chlb) at the flowering stage. QTLs between umc2308 and bnlg386 for the four traits related to chlorophyll content were investigated only at the flowering stage.

Sucrose synthase (SuS), which catalyzes the reversible conversion of sucrose and uridine diphosphate (UDP) into fructose and UDP-glucose, is a key enzyme in sucrose metabolism in higher plants. SuS belongs to family 4 of the glycosyltransferases (GT4) and contains an E-X7-E motif that is conserved in members of GT4 and two other GT families. To gain insight into the roles of this motif in rice sucrose synthase 3 (RSuS3), the two conserved glutamate residues (E678 and E686) in this motif and a phenylalanine residue (F680) that resides between the two glutamate residues were changed by site-directed mutagenesis. All mutant proteins maintained their tetrameric conformation. The mutants E686D and F680Y retained partial enzymatic activity and the mutants E678D, E678Q, F680S, and E686Q were inactive. Substrate binding assays indicated that UDP and fructose, respectively, were the leading substrates in the sucrose degradation and synthesis reactions of RSuS3. Mutations on E678, F680, and E686 affected the binding of fructose, but not of UDP. The results indicated that E678, F680, and E686 in the E-X7-E motif of RSuS3 are essential for the activity of the enzyme and the sequential binding of substrates. The sequential binding of the substrates implied that the reaction catalyzed by RSuS can be controlled by the availability of fructose and UDP, depending on the metabolic status of a tissue.

Two cDNAs (Ib beta fruct2 and Ib beta fruct3) encoding vacuolar invertases were cloned from sweet potato leaves, expressed in Pichia pastoris, and the recombinant proteins were purified by ammonium sulfate fractionation and chromatography on Ni-NTA agarose. The deduced amino acid sequences encoded by the cDNAs contained characteristic conserved elements of vacuolar invertases, including the sequence R[G/A/P]xxxGVS[E/D/M]K[S/T/A/R], located in the prepeptide region, Wxxx[M/I/V]LxWQ, located around the starting site of the mature protein, and an intact beta-fructosidase motif. The pH optimum, the substrate specificity, and the apparent K(m) values for sucrose exhibited by the recombinant proteins were similar to those of vacuolar invertases purified from sweet potato leaves and cell suspensions, thus confirming that the proteins encoded by Ib beta fruct2 and Ib beta fruct3 are vacuolar invertases. Moreover, northern analysis revealed that the expression of the two genes was differentially regulated. With the exception of mature leaves and sprouting storage roots, Ib beta fruct2 mRNA is widely expressed among the tissues of the sweet potato and is more abundant in young sink tissues. By contrast, Ib beta fruct3 mRNA was only detected in shoots and in young and mature leaves. It appears, therefore, that these two vacuolar invertases play different physiological roles during the development of the sweet potato plant.

In suspension-cultured cells of rice (Oryza sativa L. cv. Tainung 67), two sucrose synthase genes, RSus1 and RSus2, were found to be differentially regulated by sugars. Exogenously provided sugars triggered marked accumulation of the RSusl transcripts and this required de novo synthesized proteins. In addition to being regulated at the transcriptional level, other regulatory mechanisms might be involved in the sugar-modulated expression of RSusl since the RSuS1 proteins were extremely stable in sucrose-depleted cells. Glucose analogues, 3-O-methyl glucose, 6-deoxyglucose and mannose, did not trigger the induction of RSus1 expression, suggesting that hexose uptake per se and hexokinase are not involved in the sugar-sensing pathway controlling RSus1 expression. The accumulation of RSus2 mRNA was observed regardless of the presence or absence of sugars. However, newly synthesized proteins induced by sucrose and/or starvation were involved in the regulation of RSus2 expression at transcriptional and/or post-transcriptional levels. The protein level of RSuS2 was elevated under prolonged starvation condition but a reciprocal change was observed in the sucrose-fed cell. The constant presence of RSuS1 and RSuS2 proteins in both sucrose-starved and sucrose-provided cells may allow the cells to respond to sugar availability immediately.

An invertase cDNA (Ibbetafruct1) was cloned from sweet potato leaves and characterized. The deduced amino acid sequence of the Ibbetafruct1-encoded protein was closely related to vacuolar invertases and included the WECVD catalytic domain characteristic of them. An expression plasmid containing the coding region of Ibbetafruct1 under the control of the alcohol oxidase promoter was used to transform the methylotrophic yeast Pichia pastoris. The biochemical properties for the expressed recombinant enzyme, which was determined to be the acid beta-fructofuranosidase with an acidic pI value (5.1), were similar to those of vacuolar invertases purified from sweet potato. Periodic acid/Schiff staining and Con A-Sepharose gel-binding experiments revealed the recombinant invertase to be a glycoprotein containing glucose and/or mannose residues. Furthermore, the carbohydrate moiety appears to be a key determinant of the enzyme's sucrose hydrolysis activity, substrate affinity, and thermal stability.

A vacuolar type β-d-fructofuranosidase (Osβfruct3) was cloned from etiolated rice seedlings cDNA library. It encodes an open reading frame of 688 residues. The deduced amino acid sequence had 58% identity to the vacuolar type β-d-fructofuranosidase of maize (Ivr1). Osβfruct3 exists as a single copy per genome. Northern analyses showed that Osβfruct3 undergoes organ-specific expression and is involved in the adjustment of plant responses to environmental signals and metabolizable sugars. Osβfruct3 was also heterologously expressed in Pichia pastoris. The recombinant proteins were confirmed to be a vacuolar type β-d-fructofuranosidase.

One alkaline invertase and two acid invertase activities were detected in the shoots of etiolated rice (Oryza sativa) seedlings. The alkaline invertase (AIT) was purified to homogeneity through steps of ammonium sulphate fractionation, concanavalin A-Sepharose affinity chromatography (non-retained), DEAE-Sephacel chromatography and preparative electrophoresis. The pH optimum of AIT was 7.0 and the molecular mass, determined by gel filtration, was 240 kDa. It is apparently a homotetrameric enzyme (subunit molecular mass 60 kDa). The isoelectric point was 4.4 by isoelectric focusing. The best substrate of the enzyme was sucrose, with a Km of 2.53 mM. The enzyme also hydrolysed raffinose, but not maltose or lactose, so it is a β-D-fructofuranosidase. It gave negative glycoprotein staining. Of the hydrolysis products, fructose was a competitive inhibitor and glucose was a non-competitive inhibitor. Treatment with an alkaline phosphatase could activate AIT, whereas other proteins such as BSA, concanavalin A and urease had no effect on the enzyme activity. The enzyme activity was inhibited by Tris, thiol reagents and heavy metal ions.

The spatial and temporal distribution of sucrose synthase (RSuS) in rice (Oryza sativa L.) was studied by Western and immunohistochemical analyses using the monospecific antibodies for three RSuS isoforms. In leaf tissues, RSuS1 was localized in the mesophyll while RSuS2 was in the phloem in addition to the mesophyll. In the roots, only RSuS1 was found in the phloem. No RSuS3 could be detected in any parts of etiolated seedlings. The expression of each RSus gene is closely linked to the seed development. RSuS1 was present in the aleurone layers of developing seeds, and at a low level in endosperm cells. RSuS2 was evenly distributed in seed tissues other than the endosperm. RSuS3 was localized predominantly in the endosperm cells. The tissue specific localizations of the three gene products suggest that RSuS1 plays a role in sugar transport into endosperm cells where the reaction catalyzed by RSuS3 provides the precursor of starch synthesis. RSus2, which is ubiquitously expressed, may play a housekeeping role.

To study the preventive effects of jinghua weikang capsule (JWC) on nonsteroidal anti-inflammatory drugs (NSAIDs) induced injury to the mucosa of the small intestine.Thirty-two Wistar rats were randomly divided into four groups, i.e., the blank control group, the model group, the JWC group, and the esomeprazole group. Diclofenac was administered to rats in the model group, the JWC group, and the esomeprazole group at the daily dose of 15 mg/kg. JWC and esomeprazole was respectively given to those in the JWC group, and the esomeprazole group one day ahead. Normal saline was given to rats in the blank control group. Rats were killed 3 days later. The pathological changes of the small intestine were observed by hematoxylin and eosin stain.Compared with the blank control group, the general score for the small intestine (4.63 +/-0.52 vs 0.00 +/-0. 00) and the pathological score (4.00 +/-0.90 vs 0.00 +/-0. 00) obviously increased in the model group, showing statistical difference (P0.05). Compared with the model group, the general score for the small intestine (1.88 +/-0.99) and the pathological score (2.11 +/-1.11) obviously decreased in the JWG group, showing statistical difference (P0.05). Compared with the model group, the general score for the small intestine (2.75 +/-1.28) and the pathological score (2. 30 +/-0.94) obviously decreased in the esomeprazole group, showing statistical difference (P0.05).JWC could prevent NSAIDs induced injury to the mucosa of the small intestine.

By cloning and sequencing cDNA and genomic DNA and transcription initiation site mapping, the total structures including at least 1 kb of putative regulatory sequences upstream of the transcription initiation sites of three genes encoding rice sucrose synthase isoprotomers were either newly established or amended. The third type of SS gene, RSus3, has not been found in other plants. The structures of the three genes and the gene products were compared and their evolutionary sequence was proposed. Specific probes for the three SS mRNA's were developed and used for analyzing their steady state levels at different organs and under some physiological stress conditions. It appears that RSus2 is a house-keeping gene, RSus3 is highly specific to the grain, and the expression of RSus1 shows a tendency to complement that of RSus3. A possible cause of the presence of the third rice SS gene was discussed. We also reported a novel method to synthesize single-stranded DNA for S1 mapping of a transcription initiation site associated with extended secondary structures.

The rice sucrose synthase 1 (RSus1) gene is transcriptionally induced by sucrose, and a region within its promoter, at -1117 to -958 upstream of the transcription initiation site, was found to be essential for enhancing the sucrose-induced expression. Further dissection of this region revealed that a group of nuclear proteins interact with a 39-bp fragment named A-3-2 (-1045 to -1007). A protein that specifically and directly interacted with A-3-2 was isolated from the suspension-cultured cells of rice and was subsequently identified as a purine-rich DNA-binding protein. The amino acid sequence of this protein, OsPurα, exhibited 73% identity with the Arabidopsis Purα-1 protein, and its modeled structure resembled the structure of Pur-α in Drosophila. Recombinant OsPurα expressed and purified from Escherichia coli was demonstrated to have DNA-binding activity and to interact with A-3-2 specifically. Moreover, OsPurα was able to enhance sucrose-induced expression of the β-glucuronidase (GUS) reporter gene, which was transcriptionally fused to two copies of a DNA fragment containing A-3-2 and the cauliflower mosaic virus 35S minimal promoter, in vivo. The level of OsPurα bound to A-3-2 was higher in cells cultured in the presence of sucrose; however, the level of OsPurα mRNA in cells was not affected by sucrose. The results of this study demonstrate that OsPurα participates in the regulation of RSus1 expression in response to sucrose; nevertheless, it may require other partner proteins for full function.

A cDNA, BohLOL1, encoding a protein containing three zf-LSD1 (zinc finger-Lesions Simulating Disease resistance 1) domains was cloned from growing bamboo (Bambusa oldhamii) shoots. A phylogenetic analysis revealed that BohLOL1 is a homolog of Arabidopsis LSD1 and LOL1 (LSD-one-like 1), which have been reported to act antagonistically in controlling cell death via the maintenance of reactive oxygen species homeostasis. The BohLOL1 gene was differentially expressed in various bamboo shoot tissues and was upregulated in shoots with higher rates of culm elongation. The expression level of this gene in multiple shoots of bamboo, which were cultured in vitro, was also upregulated by auxins, cytokinins, pathogen infection, 2,6-dichloroisonicotinic acid (a functional analog of salicylic acid), and hydrogen peroxide. The results suggest that BohLOL1 participates in bamboo growth and in the response to biotic stress. The DNA-binding assays and subcellular localization studies demonstrated that BohLOL1 is a nuclear DNA-binding protein. BohLOL1 might function through protein-DNA interactions and thus affect the expression of its target genes. The results of this study extend the role of plant LSD1 and LOL1 proteins from the regulation of cell death to cell growth. The growth-dependent up-regulation of BohLOL1 expression, which uniquely occurs in growing bamboo, might be one of the critical factors that contribute to the rapid growth of this remarkable plant.

Glyoxylate carboligase (Gcl) (EC 4.1.1.47) of Escherichia coli catalyzes the condensation of two molecules of glyoxylate to give tartronic semialdehyde, a key intermediate in glyoxylate catabolism. We report the cloning, genomic location, and DNA sequence of the gene (called gcl) encoding E. coli Gcl and isolation of mutants lacking the enzyme. Gcl is a protein of 593 amino acid residues (64,738 Da) that has a high level (30%) of sequence similarity to the acetohydroxy acid synthases (AHAS) of branched chain amino acid synthetic pathway. Significant sequence identity (26%) was also observed with E. coli pyruvate oxidase, a redox flavoprotein, previously shown to be related to the AHAS enzymes (Chang, Y.-Y., and Cronan, J. E., Jr. (1988) J. Bacteriol. 170, 3937-3945). Consistent with a grouping of Gcl with the AHAS and pyruvate oxidase enzymes. Gcl contains a quinone binding site as well as binding site for thiamine pyrophosphate and FAD. We also found that a gene (orf258) immediately downstream of the gcl gene encoded a protein (Orf258) of 258 residues. Although the gene organization of gcl and orf258 is analogous to that of the ilv gene operons which encode the E. coli AHAS isozyme large and small subunits, Orf258 does not function as a Gcl subunit. Moreover, disruption of the chromosomal copy of orf258 did not affect growth on glyoxylate or glycolate.

The synthesis of cell wall polysaccharides is highly active in rapidly growing bamboo shoots. We cloned a set of BoCesA cDNAs that encode cellulose synthase from bamboo (Bambusa oldhamii) and investigated the expression patterns of the BoCesA2, BoCesA5, BoCesA6 and BoCesA7 genes. The four BoCesA genes were differentially expressed in the different parts of growing bamboo shoots, in various organs, and in multiple shoots that were cultured in vitro. They were down-regulated by alpha-naphthaleneacetic acid and differentially affected by thidiazuron in the multiple shoots. In situ RT-PCR analyses demonstrated that BoCesA2, BoCesA5, BoCesA6, and BoCesA7 mRNAs were present throughout the base and the internode regions of the etiolated shoots that emerged from pseudorhizomes, and in the internode regions of the juvenile branch shoots that emerged from nodes of mature bamboo culms; however, the expression of the four genes in the lignified internode of the branch shoot was predominantly detected in the center of the vascular bundles. Our results for cDNA cloning, expression analyses, and phylogenetic analysis suggest that the 10 BoCesA genes cloned from the etiolated bamboo shoots participate in cellulose synthesis in the primary cell walls of the growing bamboo, and that at least three additional BoCesA genes involved in cellulose synthesis in the secondary walls may be present in the bamboo genome. The expressions of BoCesA genes may be under fine control in response to the various developmental stages and physiological conditions of bamboo.

Pyruvate oxidase of Escherichia coli, an enzyme greatly activated by phospholipids, is a tetramer of a Mr 62,000 subunit. We have utilized the differing electrophoretic mobilities of several mutant oxidases on native polyacrylamide gels to study the role of the quaternary structure of the enzyme in the activation process. We found that when two poxB gene alleles coexisted in cells, heterotetrameric species were formed in addition to homotetramers. The concentration of each tetrameric species varied according to the concentration of the different subunits present, and the distribution seemed virtually identical to those expected from random mixing. We showed that the intrinsic activity of pyruvate oxidase was not affected by interactions among the four subunits. However, binding of the enzyme to lipids, a property required for function in vivo, required that a tetramer contain at least two subunits capable of lipid binding. Our data fit the model proposed previously (Grabau, C., Chang, Y.-Y., and Cronan, J. E., Jr. (1989) J. Biol. Chem. 264, 12510-12519) in which the carboxyl termini of two subunits interact to form a functional lipid-binding domain. We also have detected oxidase activity in a form of oxidase of unusually high electrophoretic mobility. This form seems to be either a monomeric or a dimeric form (more probably the former) of the oxidase subunit.

Three Bo beta fruct cDNAs encoding acid invertases were cloned from shoots of the green bamboo Bambusa oldhamii. On the basis of the amino acid sequences of their products and phylogenetic analyses, Bo beta fruct1 and Bo beta fruct2 were determined to encode cell wall invertases, whereas Bo beta fruct3encodes a vacuolar invertase. The recombinant proteins encoded by Bo beta fruct2 and Bo beta fruct3 were produced in Pichia pastoris and purified to near homogeneity using ammonium sulfate fractionation and immobilized metal affinity chromatography. The pH optima, pI values, and substrate specificities of the isolated enzymes were consistent with those of plant cell wall or vacuolar invertases. The growth-dependent expression of Bo beta fruct1 and Bo beta fruct2 in the base regions of shoots underscores their roles in sucrose unloading and providing substrates for shoot growth. Its high sucrose affinity suggests that the Bo beta fruct2-encoded enzyme is important for maintaining the sucrose gradient between source and sink organs, while the predominant expression of Bo beta fruct3 in regions of active cell differentiation and expansion suggests functions in osmoregulation and cell enlargement.

Summary • Bamboo is distinguished by its rapid growth. To investigate sucrose metabolism in this plant, we cloned the cDNAs encoding sucrose synthase (SuS) from Bambusa oldhamii and investigated their expression in growing shoots and leaves. • Four cDNA clones, BoSus1, BoSus2, BoSus3 and BoSus4, were isolated by screening a cDNA library from etiolated bamboo shoots. Recombinant BoSuS proteins were produced in Escherichia coli and purified by immobilized metal affinity chromatography and ultrafiltration. Semi-quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) was used to determine the abundance of the transcript of each gene. • BoSus1 and BoSus3 may be duplicate or homeologous genes, the sequences of which show high identity. Similarly, BoSus2 shows high identity with BoSus4. Kinetic analysis showed that the two BoSuS isoforms of each type had similar michaelis constant (Km) values for sucrose, but different values for UDP. The four genes were expressed in various bamboo organs but were differentially regulated. The increase in the abundance of their mRNA paralleled the growth rate of the bamboo. • The results suggest that, in bamboo, SuS is encoded by at least four genes, each with a specific role in providing substrates for the polysaccharide biosynthesis and/or energy production necessary to support the rapid growth of this species.

A vacuolar type beta-D-fructofuranosidase (Os beta fruct3) was cloned from etiolated rice seedlings cDNA library. It encodes an open reading frame of 688 residues. The deduced amino acid sequence had 58% identity to the vacuolar type beta-D-fructofuranosidase of maize (Ivr1). Os beta fruct3 exists as a single copy per genome. Northern analyses showed that Os beta fruct3 undergoes organ-specific expression and is involved in the adjustment of plant responses to environmental signals and metabolizable sugars. Os beta fruct3 was also heterologously expressed in Pichia pastoris. The recombinant proteins were confirmed to be a vacuolar type beta-D-fructofuranosidase.

Presence of homo- and hetero-tetrameric rice sucrose synthase (RSS) isoforms in etiolated rice seedlings was demonstrated by immunoprecipitation using monospecific antibodies. Three RSS isozymes with various pI values were purified by ammonium sulfate fractionation, Sepharose CL-6B gel filtration, DEAE-Sephacel and Mono-Q ion exchange chromatographies. They were characterized as heterotetramers composed of RSS1 and RSS2 subunits. All of them used UDP as the best substrate. The presence of divalent metal ions increased the activities of synthesis but inhibited the cleavage of sucrose.

Cyclopropane fatty acid (CFA) synthase of Escherichia coli catalyzes a modification of the acyl chains of phospholipid bilayers. We report (i) identification of the CFA synthase protein, (ii) overproduction (> 600-fold) and purification to essential homogeneity of the enzyme, and (iii) the amino acid sequence of CFA synthase as deduced from the nucleotide sequence of the cfa gene. CFA synthase was overproduced by use of the T7 promoter/RNA polymerase system under closely defined conditions. The enzyme was readily purified by a two-step procedure requiring only ammonium sulfate fractionation and binding to phospholipid vesicles followed by flotation in sucrose density gradients. The deduced amino acid sequence predicts a protein of 43,913 Da (382 residues) that lacks long hydrophobic segments. The CFA synthase sequence has no significant similarity to known proteins except for sequences found in other enzymes that utilize S-adenosyl-L-methionine. We also report inhibitor studies of the enzyme active site.

By sequencing cDNA clones, we have concluded that three distinct sucrose genes are expressed in rice (Oryza sativa cv. Tainong 67). When the amino acid sequences deduced from these cDNAs as well as those of known sucrose synthase are compared, the highest divergence is found in the C-termini. The most suitable DNA sequences for use as specific for the mRNA derived from these genes have been suggested.