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The target of rapamycin (TOR) kinase pathway regulates various biological processes, including translation, synthesis of ribosomal proteins, and transcription of rRNA. The ribosomal protein S6 (RPS6) is one of the well known downstream components of the TOR pathway. Ribosomal proteins have been known to have diverse functions in regulating cellular metabolism as well as protein synthesis. So far, however, little is known about other possible role(s) of RPS6 in plants, besides being a component of the 40 S ribosomal subunit and acting as a target of TOR. Here, we report that RPS6 may have a novel function via interaction with histone deacetylase 2B (AtHD2B) that belongs to the plant-specific histone deacetylase HD2 family. RPS6 and AtHD2B were localized to the nucleolus. Co-expression of RPS6 and AtHD2B caused a change in the location of both RPS6 and AtHD2B to one or several nucleolar spots. ChIP analysis suggests that RPS6 directly interacts with the rRNA gene promoter. Protoplasts overexpressing both AtHD2B and RPS6 exhibited down-regulation of pre-18 S rRNA synthesis with a concomitant decrease in transcription of some of the ribosomal proteins, suggesting their direct role in ribosome biogenesis and plant development. This is consistent with the mutation in rps6b that results in reduction in 18 S rRNA transcription and decreased root growth. We propose that the interaction between RPS6 and AtHD2B brings about a change in the chromatin structure of rDNA and thus plays an important role in linking TOR signaling to rDNA transcription and ribosome biogenesis in plants.

Symbiotic nodule formation on legume roots is characterized with a series of developmental reprograming in root tissues, including extensive proliferation of cortical cells. We examined a possible involvement of the target of rapamycin (TOR) pathway, a central regulator of cell growth and proliferation in animals and yeasts, during soybean nodule development. Our results show that transcription of both GmTOR and its key downstream effector, GmS6K1, are activated during nodulation, which is paralleled with higher kinase activities of these gene products as well. RNAi-mediated knockdown of GmS6K1 impaired the nodule development with severely reduced nodule weight and numbers. In addition, expression of a few nodulins including leghemoglobin was also decreased, and consequently nitrogen fixation was found to be reduced by half. Proteomic analysis of the GmS6K1-RNAi nodules identified glutamine synthetase (GS), an essential enzyme for nitrogen assimilation in nodules, as one of the proteins that are significantly down regulated. These results appear to provide solid evidence for a functional link between GmS6K1 and nodule development.

1, * The role of Arabidopsis S6 Kinase 1 (AtS6K1), a down- stream target of TOR kinase, in controlling plant growth and ribosome biogenesis was characterized after generat- ing transgenic plants expressing AtS6K1 under auxin- inducible promoter. Down-regulation of selected cell cycle regulatory genes upon auxin treatment was observed in the transgenic plants, confirming the negative regulatory role of AtS6K1 in the plant cell cycle progression reported earlier. Callus tissues established from these transgenic plants grew to larger cell masses with more number of enlarged cells than untransformed control, demonstrating functional implication of AtS6K1 in the control of plant cell size. The observed negative correlation between the ex- pression of AtS6K1 and the cell cycle regulatory genes, however, was completely reversed in protoplasts gener- ated from the transgenic plants expressing AtS6K1, sug- gesting a possible existence of dual regulatory mechanism of the plant cell cycle regulation mediated by AtS6K1. An alternative method of kinase assay, termed "substrate- mediated kinase pull down", was employed to examine the additional phosphorylation on other domains of AtS6K1 and verified the phosphorylation of both amino- and car- boxy-terminal domains, which is a novel finding regarding the phosphorylation target sites on plant S6Ks by up- stream regulatory kinases. In addition, this kinase assay under the stress conditions revealed the salt- and sugar- dependencies of AtS6K1 phosphorylations.

The cell wall is a critical extracellular structure that provides protection and structural support in plant cells. To study the biological function of the cell wall and the regulation of cell wall resynthesis, we examined cellular responses to enzymatic removal of the cell wall in rice (Oryza sativa) suspension cells using proteomic approaches. We find that removal of cell wall stimulates cell wall synthesis from multiple sites in protoplasts instead of from a single site as in cytokinesis. Nucleus DAPI stain and MNase digestion further show that removal of the cell wall is concomitant with substantial chromatin reorganization. Histone post-translational modification studies using both Western blots and isotope labeling assisted quantitative mass spectrometry analyses reveal that substantial histone modification changes, particularly H3K18(AC) and H3K23(AC), are associated with the removal and regeneration of the cell wall. Label-free quantitative proteome analyses further reveal that chromatin associated proteins undergo dramatic changes upon removal of the cell wall, along with cytoskeleton, cell wall metabolism, and stress-response proteins. This study demonstrates that cell wall removal is associated with substantial chromatin change and may lead to stimulation of cell wall synthesis using a novel mechanism.

A leucine-rich repeat receptor-like kinase (LRR-RLK) encoded by one of the genes highly expressed in a specific stage of soybean seed development, referred to as GmLRK1, was identified and characterized. Examination of its kinase domain indicated that GmLRK1 may be a catalytically inactive atypical receptor kinase. An autophosphorylation assay confirmed that GmLRK1 is incapable of autophosphorylation in vitro. However, the phosphorylation of GmRLK1 could be induced after incubation with plant protein extracts, suggesting that some plant proteins may interact with GmLRK1 and phosphorylate the protein in vivo. Analyses of the expression profiles of GmLRK1 and its Arabidopsis ortholog At2g36570 revealed that they may be involved in regulation of more fundamental metabolic and/or developmental pathways, rather than a specialized developmental program such as seed development. Our results further indicate that the GmLRK1 and At2g36570 may play a role in the regulation of certain cellular processes that lead to cell elongation and expansion.

Summary Callose (β-1,3-glucan) is produced at different locations in response to biotic and abiotic cues. Arabidopsis contains 12 genes encoding callose synthase (CalS). We demonstrate that one of these genes, CalS5, encodes a callose synthase which is responsible for the synthesis of callose deposited at the primary cell wall of meiocytes, tetrads and microspores, and the expression of this gene is essential for exine formation in pollen wall. CalS5 encodes a transmembrane protein of 1923 amino acid residues with a molecular mass of 220 kDa. Knockout mutations of the CalS5 gene by T-DNA insertion resulted in a severe reduction in fertility. The reduced fertility in the cals5 mutants is attributed to the degeneration of microspores. However, megagametogenesis is not affected and the female gametes are completely fertile in cals5 mutants. The CalS5 gene is also expressed in other organs with the highest expression in meiocytes, tetrads, microspores and mature pollen. Callose deposition in the cals5 mutant was nearly completely lacking, suggesting that this gene is essential for the synthesis of callose in these tissues. As a result, the pollen exine wall was not formed properly, affecting the baculae and tectum structure and tryphine was deposited randomly as globular structures. These data suggest that callose synthesis has a vital function in building a properly sculpted exine, the integrity of which is essential for pollen viability.

Symbiotic nitrogen fixation requires the internalization of rhizobia into root cells of legumes and subsequent formation of symbiosomes, rhizobia-containing organelles inside the plant cells. A small GTP-binding protein from soybean, sRab7, was shown to be essential for nodule development. When yeast ypt7 (Rab7) null mutant was transformed with the srab7 gene, highly fragmented vacuoles seen in the mutant were replaced with a prominent central vacuole, as in the wild-type cells. These results confirm that sRab7 is an authentic homolog of Ypt7p. When nodules obtained at different stages of development were hybridized with srab7 cDNA, 7-day-old nodules showed the most intense hybridization signal. Hybridization was only detected in the infection zone of forming nodules, implying that sRab7 may be involved in endocytosis of rhizobia. SCaM-1 and SCaM-4, soybean calmodulin genes, were also expressed in the same region at this stage of nodulation as shown by in situ hybridization. RT-PCR analysis confirmed the expression of these genes. These results indicate that both the srab7 and SCaM genes were expressed at an early stage of nodulation. Since membrane fusion of vesicles has been shown to be dependent on calmodulin and Ypt7p, the similarity of the expression patterns between the two genes in soybean nodules may indicate that sRab7 and SCaM are essential for vesicular fusion during rhizobial endocytosis and symbiosome formation.

We have characterized 4 of the 16 members of the family of dynamin-related proteins (DRP) in Arabidopsis. Three members, DRP1A (previously referred as ADL1), DRP1C and DRP1E, belong to the largest group of phragmoplastin-like proteins. DRP2A (ADL6) is one of the two members that contain a pleckstrin homology (PH) domain and a proline-rich (PR) motif, characteristics of animal dynamins. All four proteins interacted in yeast two-hybrid assays with phragmoplastin, and showed different patterns of localization at the forming cell plate during cytokinesis. GFP-tagged DRP1A and DRP1C proteins were found to be associated with the cytoskeleton in G1 phase of the cell cycle. The distribution pattern of DRP1A was sensitive to propyzamid and insensitive to cytochalasin D, suggesting that DRP1A is associated with microtubules and not actin filaments. The association of DRP1A with microtubules was confirmed in vitro by spin-down assays. A GTPase-defective phragmoplastin acted as a dominant negative mutant, reduced transport of vesicles to the cell plate and formed dense tubule-like structures in the cell plate. We propose that DRP1 proteins may provide an anchor for Golgi-derived vesicles to attach to microtubules, which in turn direct the vesicles to the forming cell plate during cytokinesis. Whereas the DRP1 subfamily members are involved in tubulization of membranes, DRP2 may be involved in endocytosis and membrane recycling via clathrin-coated vesicles.

Phragmoplastin, a dynamin-like GTPase, is associated with cell plate formation in plants. We expressed a phragmoplastin-GFP construct (Phr G ) under the control of a 35S promoter in transgenic tobacco plants. High levels of expression of this chimeric protein in transgenic plants were confirmed by Western blot analysis. T1 seedlings formed morphologically normal cotyledons, but most were unable to develop beyond the first pair of true leaves. Only 5–10% of the seedlings could develop into small plants whose progenies continued to exhibit severe growth defects. The primary root growth was arrested after 7 days and no lateral roots were formed. The mature primary root meristem consisted of disorganized isodiametrically enlarged cells and no recognizable quiescent center or collumela. Adventitious root primordia were initiated in the hypocotyl, but completely arrested at an early stage and contained differentiated treachery elements. The orientation of cell plates was abnormal, suggesting a possible defect in its positioning. However, unlike other cytokinesis mutants, no binucleate or polyploid cells were found. Fluorescence image cytometry studies indicated that Phr G root meristem cells had 2C levels of DNA and appear to be arrested in G1 stage of the cell cycle. Heavy callose deposition was observed at the nascent cell plate and callose accumulation persisted in new cell walls. Electron microscopy revealed unusual electron dense substances in the maturing cell plate and accumulation of multivesicular bodies around the cell plate. Since phragmoplastin interacts with callose synthase complex, its overexpression may affect accumulation of callose arresting plant growth due to perturbation in cell division progression.

Callose is synthesized on the forming cell plate and several other locations in the plant. We cloned an Arabidopsis cDNA encoding a callose synthase (CalS1) catalytic subunit. The CalS1 gene comprises 42 exons with 41 introns and is transcribed into a 6.0-kb mRNA. The deduced peptide, with an approximate molecular mass of 226 kD, showed sequence homology with the yeast 1,3-β-glucan synthases and is distinct from plant cellulose synthases. CalS1 contains 16 predicted transmembrane helices with the N-terminal region and a large central loop facing the cytoplasm. CalS1 interacts with two cell plate–associated proteins, phragmoplastin and a novel UDP-glucose transferase that copurifies with the CalS complex. That CalS1 is a cell plate–specific enzyme is demonstrated by the observations that the green fluorescent protein–CalS1 fusion protein was localized at the growing cell plate, that expression of CalS1 in transgenic tobacco cells enhanced callose synthesis on the forming cell plate, and that these cell lines exhibited higher levels of CalS activity. These data also suggest that plant CalS may form a complex with UDP-glucose transferase to facilitate the transfer of substrate for callose synthesis.

Synthesis of callose (β-1,3-glucan) in plants has been a topic of much debate over the past several decades. Callose synthase could not be purified to homogeneity and most partially purified cellulose synthase preparations yielded β-1,3-glucan in vitro, leading to the interpretation that cellulose synthase might be able to synthesize callose. While a rapid progress has been made on the genes involved in cellulose synthesis in the past five years, identification of genes for callose synthases has proven difficult because cognate genes had not been identified in other organisms. An Arabidopsis gene encoding a putative cell plate-specific callose synthase catalytic subunit (CalS1) was recently cloned. CalS1 shares high sequence homology with the well-characterized yeast β-1,3-glucan synthase and transgenic plant cells over-expressing CalS1 display higher callose synthase activity and accumulate more callose. The callose synthase complex exists in at least two distinct forms in different tissues and interacts with phragmoplastin, UDP-glucose transferase, Rop1 and, possibly, annexin. There are 12 CalS isozymes in Arabidopsis, and each may be tissue-specific and/or regulated under different physiological conditions responding to biotic and abiotic stresses.

The Δ1-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned) is the rate-limiting enzyme in proline (Pro) biosynthesis in plants and is subject to feedback inhibition by Pro. It has been suggested that the feedback regulation of P5CS is lost in plants under stress conditions. We compared Pro levels in transgenic tobacco (Nicotiana tabacum) plants expressing a wild-type form of Vigna aconitifolia P5CS and a mutated form of the enzyme (P5CSF129A) whose feedback inhibition by Pro was removed by site-directed mutagenesis. Transgenic plants expressing P5CSF129A accumulated about 2-fold more Pro than the plants expressing V. aconitifolia wild-type P5CS. This difference was further increased in plants treated with 200 mm NaCl. These results demonstrated that the feedback regulation of P5CS plays a role in controlling the level of Pro in plants under both normal and stress conditions. The elevated Pro also reduced free radical levels in response to osmotic stress, as measured by malondialdehyde production, and significantly improved the ability of the transgenic seedlings to grow in medium containing up to 200 mm NaCl. These findings shed new light on the regulation of Pro biosynthesis in plants and the role of Pro in reducing oxidative stress induced by osmotic stress, in addition to its accepted role as an osmolyte.

Phragmoplastin, a high molecular weight GTPase belonging to the dynamin superfamily of proteins, becomes associated with the cell plate during cytokinesis in plants. Growth of the cell plate requires continuous fusion of vesicles, and phragmoplastin appears to play a role in the formation of vesicle-tubule-vesicle structures at the cell plate. In this study, we have demonstrated that two self-assembly domains (SA1 and SA2) are involved in polymerization of phragmoplastin. SA1 is about 42 amino acids long and is located near the N terminus overlapping with the GTP-binding region. SA2, containing at least 24 amino acids, is located in the middle of the molecule outside the GTP-binding domain. Peptides containing either SA1 or SA2 interact efficiently with the full-length phragmoplastin. The SA1 domain of one phragmoplastin molecule also binds to SA2 of another as confirmed in vitro by using radiolabeled peptides. This interaction leads to the formation of polymers with a staggered contoured spiral structure. Electron microscopy studies revealed that helical arrays of phragmoplastin can be induced by reducing salt concentration. Our results suggest that phragmoplastin may assemble into helical arrays that wrap around and squeeze vesicles into vesicle-tubule-vesicle structures observed on the forming cell plate.

A Δ 1 -pyrroline-5-carboxylate synthetase (P5CS) cDNA from mothbean ( Vigna aconitifolia L.) was introduced into rice ( Oryza sativa L.) genome by the biolistic method. Expression of this P5CS transgene under the control of a stress-inducible promoter led to stress-induced overproduction of the P5CS enzyme and proline accumulation in transgenic rice plants. Second-generation (R 1 ) transgenic rice plants showed an increase in biomass under salt-stress and water-stress conditions as compared to the non-transformed control plants.

Many plants accumulate proline in response to osmotic stress. Δ1-pyrroline-5-carboxylate synthetase (P5CS) is the rate limiting enzyme in proline biosynthesis in plants. The promoter of an Arabidopsis P5CS gene was isolated and analyzed using a β-glucuronidase (GUS) reporter gene in transgenic plants. NaCl stress induced GUS activity to the maximum level within 3 h while dehydration continued to enhance the GUS activity up to 24 h. The P5CS gene promoter was found to contain two transcription start sites, and dehydration stimulated the transcription predominantly from the downstream site. Abscisic acid (ABA) failed to induce the P5CS gene expression although ABA is known to increase during water stress. These observations suggest that induction of the P5CS gene is a specific response of plants to salt and water stresses.

Legume-Rhizobium spp. symbiosis requires signaling between the symbiotic partners and differential expression of plant genes during nodule development. Previously, we cloned a gene encoding a putative β-carotene hydroxylase (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In this work, we extended our study to three GmBCHs to examine their possible role(s) in nodule development, as they were additionally identified as nodule specific, along with the completion of the soybean genome. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in Escherichia coli. Localization of GmBCHs by transfecting Arabidopsis (Arabidopsis thaliana) protoplasts with green fluorescent protein fusions and by electron microscopic immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids, while GmBCH1 appeared to be cytosolic. RNA interference of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids in nodules. Therefore, we examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings, we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation.

An infected root nodule cell may contain several thousand rhizobial symbionts, each enclosed in a membrane envelope, the peribacteroid membrane (PBM). The PBM is derived from the host plasma membrane, but shares properties with the vacuolar membrane and contains several nodule-specific proteins (nodulins) that perform unique functions for symbiosis.

Plant cells undergoing cytokinesis (cytoplasmic division) form a cell plate in the center of the phragmoplast. The phragmoplast consists of both cytoskeletal and membranous elements. The cytoskeletal elements are required for the delivery of vesicles to the equatorial region of the cell and provide guidance to the cell plate. Vesicles, originating from the Golgi apparatus, travel along microtubules and fuse in a unique manner at the center of the phragmoplast to give rise to the new plasma membranes of the two daughter cells. The cargo they carry contributes to the cell-plate matrix. The production, delivery and fusion of these vesicles at an appropriate time and place are critical for the correct positioning of the forming cell-plate. Recent evidence suggests that a dynamin-like protein — phragmoplastin — may be involved in ‘squeezing' the cell-plate vesicles into specialized tubular structures, facilitating membrane fusion and the release of the vesicle contents. We describe the dynamics of this complex pathway and our progress in understanding it at the molecular level.

Phosphatidylinositol 3-kinase (PI3K) is an important component of various receptor tyrosine kinase complexes in mammalian cells and a key enzyme required for cell division and vacuolar protein sorting in yeast. To our knowledge, this enzyme has not been characterized in plants. We report the cloning and characterization of soybean PI3K cDNAs and present evidence for the induction of a distinctive form of this enzyme specific to nodule organogenesis. Expression of the root form of PI3K is repressed during nodule organogenesis and is reinduced in mature nodules. Primer-extension results showed that the gene encoding the nodule form of PI3K is highly expressed in young (12-15 day old) root nodules in parallel with membrane proliferation but is repressed in mature nodules. The root form of the PI3K cDNA (SPI3K-5) encodes a peptide of 814 amino acids and the nodule form (SPI3K-1) encodes a peptide of 812 amino acids. Both cDNAs share 98% sequence identity in the coding region but differ in the noncoding regions. The polypeptides encoded by soybean PI3K cDNAs show significant sequence homology (50-60% similarity and 20-40% identity) to both PI3Ks and phosphatidylinositol 4-kinases from mammalian and yeast cells. Escherichia coli expressed soybean PI3K phosphorylated phosphatidylinositol specifically at the D-3 position of the inositol ring to generate phosphatidylinositol 3-phosphate. The temporal increase of a specific PI3K activity during membrane proliferation in young nodules suggests that PI3K plays a pivotal role in development of the peribacteroid membrane forming a subcellular compartment.

Nodulin-24 is a nodule-specific protein of the peribacteroid membrane (PBM) in soybean. It has an apparent molecular mass of 33 kDa while its full-length cDNA encodes a polypeptide of only 24 kDa. In vitro transcription of nodulin-24 cDNA followed by translation resulted in a peptide translocated into microsomal membranes with cleavage of a signal sequence. The cleavage site of the signal sequence in nodulin-24 was determined to be between Ala (A25) and Arg (R26) by microsequencing of the [3H]leucine-labeled processed peptide. Fusion of the signal sequence of nodulin-24 with the beta-glucuronidase peptide prevented co-translational cleavage of the signal sequence although the translocation of the fused protein into microsomes occurred co-translationally. Trypsin treatment of membrane-translocated nodulin-24 did not result in any alteration in size suggesting that the newly synthesized peptide is fully protected in the membrane vesicle. Fusion of nodulin-24 with beta-glucuronidase also showed no change in size following trypsin treatment, suggesting that nodulin-24 has no membrane-spanning region. In addition, in vitro synthesized nodulin-24 was present in the supernatant fraction after sonication of microsomal membranes. Mature nodulin-24, on the other hand, is not solubilized from PBM by sodium carbonate (pH 11) or EGTA and is soluble only in detergent. These data suggest that nodulin-24 is synthesized as a lumenal protein in the endoplasmic reticulum and post-translationally attached to the membranes en route to the PBM. This processing results in a significant increase in the apparent molecular mass of nodulin-24 which may be due to the attachment of membrane lipids as this protein shares characteristics with membrane lipoproteins of many pathogenic bacteria.

The expression of Delta(1)-pyrroline-5-carboxylate reductase (P5CR) gene was found to be higher in soybean root nodules than in leaves and roots, and its expression in roots appeared to be osmoregulated (AJ Delauney, DPS Verma [1990] Mol Gen Genet 221: 299-305). P5CR was purified to homogeneity as a monomeric protein of 29 kilodaltons by overexpression of a soybean P5CR cDNA clone in Escherichia coli. The pH optimum of the purified P5CR was altered by increasing the salt concentration, and maximum enzyme activity was attainable at a lower pH under high salt (0.2-1 molar NaCl). Kinetic studies of the purified enzyme suggested that nicotinamide adenine dinucleotide phosphate(+) inhibited P5CR activity, whereas nicotinamide adenine dinucleotide(+) did not. Subcellular fractionation and antibodies raised against purified soybean P5CR were used to investigate location of the enzyme in different parts of soybean as well as in leaves of transgenic tobacco plants synthesizing soybean P5CR. P5CR activity was present in cytoplasm of soybean roots and nodules as well as in leaves, but in leaves, about 15% of the activity was detected in the plastid fraction. The location of P5CR was further confirmed by western blot assay of the proteins from cytosol and plastid fractions of different parts of the plant. Expression of soybean nodule cytosolic P5CR in transgenic tobacco under the control of cauliflower mosaic virus 35S promoter led to the accumulation of this protein exclusively in the cytoplasm, suggesting that the chloroplastic activity may be due to the presence of a plastid form of the enzyme. The different locations of P5CR in root and leaf suggested that proline may be synthesized in different subcellular compartments in root and leaf. Proline concentration was not significantly increased in transgenic plants exhibiting high level P5CR activity, indicating that reduction of P5C is not a rate-limiting step in proline production.

Soybean nodulin-26, a homologue of bovine eye lens major intrinsic protein (MIP-26), is an integral protein of the peribacteroid membrane in symbiotic root nodules. It comprises 271 amino acids with six potential transmembrane domains and lacks an amino-terminal signal sequence. A full-length nodulin-26 cDNA and its various deletion derivatives were transcribed in vitro after linking them to bacteriophage T3 promoter. In vitro translation of these transcripts in a rabbit reticulocyte lysate, in the presence or absence of canine pancreatic microsomal membranes, suggested that nodulin-26 is cotranslationally inserted into the microsomes without a cleavable signal peptide. The first two transmembrane domains (103 amino acids) of the protein are sufficient for microsomal membrane insertion. Membrane-translocated nodulin-26 binds to Con-A and is sensitive to endoglycosidase-H treatment, suggesting that it is glycosylated. Native nodulin-26 from root nodules retains its sugar moiety as it, too, binds to Con-A. Chemical cleavage mapping at cysteine residues, a trypsin protection assay, and the Con-A binding affinity of nodulin-26 suggested that both the NH2 and COOH termini of this protein are on the cytoplasmic surface of the peribacteroid membrane, while the glycosidic residue is on the surface of the membrane facing the bacteroids. In vitro phosphorylation experiments showed that nodulin-26 is a major phosphorylated protein in the peribacteroid membrane. This phosphorylation is mediated by a Ca(2+)-dependent, calmodulin-independent protein kinase located in the peribacteriod membrane. Externally supplied acid phosphatase dephosphorylates this protein, but alkaline phosphatase does not. Based on its homology with several eukaryotic and prokaryotic channel-type membrane proteins, nodulin-26 may form a channel translocating specific molecules to the bacteroids during endosymbiosis in legume plants.

Starch content and activities of some enzymes of starch metabolism were determined in wild-type, N2-fixing (fix+) nodules and in two non-N2-fixing (fix−) nodules induced by Bradyrhizobium japonicum mutant strains, T5-95 and T8-1, on soybean (Glycine max L.) roots. The T5-95 nodules are similar to wild type in ultrastructure, but the T8-1 nodules are different in that the bacteroids are not released from the infection thread. After initial accumulation to relatively high concentration, starch was depleted during nitrogen fixation in fix+ nodules. However, in fix− nodules, the accumulated starch was not metabolized. The activity of starch-bound starch synthase (EC 2.4.1.21) declined in fix+ nodules but remained high in fix− nodules. The activity of α-amylase (EC 3.2.1.1) was only slightly higher than wild type in T5-95 but was four times higher than wild type in T8-1 nodules. The activity of starch phosphorylase (EC 2.4.1.1) increased in all nodule types from 14 to 21 days postinfection. A positive correlation was observed between the capacity of nodules to fix N2 and their capacity to degrade starch. Collectively, these results support the concept that starch accumulated during early stages of nodule development is metabolized to supply energy for nitrogen fixation and to meet the metabolic demands of bacteroids. Key words: nitrogen fixation, starch content, effective and ineffective nodules, starch synthase, starch phosphorylase, α-amylase.

A complete nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3) was isolated from a soybean (Glycine max L. var. Prize) nodule cDNA expression library using a previously isolated partial cDNA clone. The N-35 cDNA was expressed in Escherichia coli driven by the lacZ promoter and was found to be functionally active. The uricase activity was detected in the cytoplasmic fraction of E. coli with the same pH optimum and apparent K(m) values as that in the nodules. Because a stop codon is located 15 base pairs upstream of the N-35 initiation codon, it appears that a fusion protein with LacZ was not made, but reinitiation occurred due to the presence of a putative Shine-Dalgarno sequence in the appropriate region. The size of the N-35 polypeptide made in E. coli is identical to that present in soybean nodules and is able to assemble into a tetrameric holoenzyme with the same molecular weight as the native uricase. Thus, the presence of peroxisomes does not appear to be essential for the proper assembly of the holoenzyme in E. coli. These data also indicate that posttranslational modifications or membrane transport are not essential either for the assembly of N-35 into a holoenzyme or for the activity of uricase.

A full-length cDNA clone encoding cytosolic glutamine synthetase (GS), expressed in roots and root nodules of soybean, was isolated by direct complementation of an Escherichia coli gln A- mutant. This sequence is induced in roots by the availability of ammonia. A 3.5-kilobase promoter fragment of a genomic clone (lambda GS15) corresponding to this cDNA was isolated and fused with a reporter [beta-glucuronidase (GUS)] gene. The GS-GUS fusion was introduced into a legume (Lotus corniculatus) and a nonlegume (tobacco) plant by way of Agrobacterium-mediated transformations. This chimeric gene was found to be expressed in a root-specific manner in both tobacco and L. corniculatus, the expression being restricted to the growing root apices and the vascular bundles of the mature root. Treatment with ammonia increased the expression of this chimeric gene in the legume background (i.e., L. corniculatus); however, no induction was observed in tobacco roots. Histochemical localization of GUS activity in ammonia-treated transgenic L. corniculatus roots showed a uniform distribution across all cell types. These data suggest that the tissue specificity of the soybean cytosolic GS gene is conserved in both tobacco and L. corniculatus; however, in the latter case, this gene is ammonia inducible. Furthermore, the ammonia-enhanced GS gene expression in L. corniculatus is due to an increase in transcription. That this gene is directly regulated by externally supplied or symbiotically fixed nitrogen is also evident from the expression of GS-GUS in the infection zone, including the uninfected cells, and the inner cortex of transgenic L. corniculatus nodules, where a flux of ammonia is encountered by this tissue. The lack of expression of GS-GUS in the outer cortex of the nodules suggests that ammonia may not be able to diffuse outside the endodermis.

Building a cell plate during cytokinesis in plant cells requires the participation of a number of proteins in a multistep process. We previously identified phragmoplastin as a cell plate-specific protein involved in creating a tubulovesicular network at the cell plate. We report here the identification and characterization of a phragmoplastin-interacting protein, PHIP1, in Arabidopsis (Arabidopsis thaliana). It contains multiple functional motifs, including a lysine-rich domain, two RNA recognition motifs, and three CCHC-type zinc fingers. Polypeptides with similar motif structures were found only in plant protein databases, but not in the sequenced prokaryotic, fungal, and animal genomes, suggesting that PHIP1 represents a plant-specific RNA-binding protein. In addition to phragmoplastin, two Arabidopsis small GTP-binding proteins, Rop1 and Ran2, are also found to interact with PHIP1. The zinc fingers of PHIP1 were not required for its interaction with Rop1 and phragmoplastin, but they may participate in its binding with the Ran2 mRNA. Immunofluorescence, in situ RNA hybridization, and green fluorescent protein tagging experiments showed the association of PHIP1 with the forming cell plate during cytokinesis. Taken together, our data suggest that PHIP1 is a novel RNA-binding protein and may play a unique role in the polarized mRNA transport to the vicinity of the cell plate.

Commitment for Cell Division and Cell Cycle Control.- Circadian Regulation of Cell Division.- Transcriptional Control of the Plant Cell Cycle.- Division Plane Orientation in Plant Cells.- G1/S Transition and the Rb-E2F Pathway.- The Endoreduplication Cell Cycle: Regulation and Function.- Dynamics of Chromosomes, Cytoskeleton and Organelles During Cell Division.- Chromosome Dynamics in Meiosis.- Cytoskeletal and Vacuolar Dynamics During Plant Cell Division: Approaches Using Structure-Visualized Cells.- Mitotic Spindle Assembly and Function.- Cytoskeletal Motor Proteins in Plant Cell Division.- Organelle Dynamics During Cell Division.- Open Mitosis: Nuclear Envelope Dynamics.- Cell Plate Formation and Cytokinesis.- MAP Kinase Signaling During M Phase Progression.- Plant Cytokinesis - Insights Gained from Electron Tomography Studies.- Vesicle Traffic at Cytokinesis.- Molecular Analysis of the Cell Plate Forming Machinery.- Cell Division and Differentiation.- Asymmetric Cell Divisions: Zygotes of Fucoid Algae as a Model System.- Stomatal Patterning and Guard Cell Differentiation.- Genetic Control of Anther Cell Division and Differentiation.- Coordination of Cell Division and Differentiation.

We have isolated several cDNA clones encoding delta 1-pyrroline-5-carboxylate reductase (P5CR, L-proline: NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) which catalyzes the terminal step in proline biosynthesis, by direct complementation of a proC mutation in Escherichia coli with an expression library of soybean root nodule cDNA. The library was constructed in the lambda ZapII vector, converted to a plasmid library by in vivo excision of recombinant pBluescript phagemids, and used for transformation of the E. coli mutant. Complementing plasmids contained inserts of about 1.2 kb which hybridized to a 1.3 kb RNA transcript in nodules, uninfected roots and leaves. DNA sequence analysis of one full length cDNA clone showed that it encodes a 28 586 Mr polypeptide with 39% amino acid identity to the E. coli P5CR sequence. Genomic analysis showed that there are two to three copies of the P5CR gene in the soybean genome. The steady-state level of P5CR mRNA in root nodules was twice as high as in uninfected roots and about five times higher than in leaves. Subjecting young seedlings to osmotic stress by watering with 400 mM NaCl resulted in an almost six-fold increase in the level of root P5CR mRNA, suggesting that this gene may be osmoregulated.

Doubly transformed tobacco plants containing both sense and antisense chloramphenicol aceltyltransferase (CAT) genes and showing > 90% inhibition of CAT gene expression were previously produced. One such plant was back-crossed with untransformed tobacco, and progeny were selected in which the CAT and anti-CAT genes were segregated. Progency containing the CAT gene but lacking the antisense construct showed fully restored CAT activity whereas those containing only the antisense genes expressed no CAT activity. The latter were stably maintained through two generations. Recombination of the sense and antisense genes by sexual crossing resulted in efficient antisense RNA-mediated inactivation of the CAT gene.

The cell plate is built using Golgi-derived vesicles carrying various proteins, sugars and lipids,which are required for the de novo synthesis of the cell wall duringcytokinesis. The processes of vesicle fusion, cell plate expansion and maturation are initiated and controlledby a large number of proteins that serve as structural components, transporters, enzymes, and regulatoryelements. Since the identification of phragmoplastin, the first protein marker of the cytokinetic organellephragmoplast, a number of cell plate-associated proteins have been identified and characterized. Someof these proteins appear to be unique to the process of cell plate formation, while others have functionsin different subcellular locations and are recruited to the forming cell plate transiently during cytokinesis.A temporal and spatial orchestration of basic exocytotic and endocytotic processes culminate in toformation of this unique subcellular compartment. Completion of this process in a defined time isessential for a proper cell division.

TARGET OF RAPAMYCIN (TOR) kinase controls many cellular functions in eukaryotic cells in response to stress and nutrient availability and was shown to be essential for embryonic development in Arabidopsis thaliana. We demonstrated that Arabidopsis RAPTOR1 (a TOR regulatory protein) interacts with the HEAT repeats of TOR and that RAPTOR1 regulates the activity of S6 kinase (S6K) in response to osmotic stress. RAPTOR1 also interacts in vivo with Arabidopsis S6K1, a putative substrate for TOR. S6K1 fused to green fluorescent protein and immunoprecipitated from tobacco (Nicotiana tabacum) leaves after transient expression was active in phosphorylating the Arabidopsis ribosomal S6 protein. The catalytic domain of S6K1 could be phosphorylated by Arabidopsis 3-phosphoinositide-dependent protein kinase-1 (PDK1), indicating the involvement of PDK1 in the regulation of S6K. The S6K1 activity was sensitive to osmotic stress, while PDK1 activity was not affected. However, S6K1 sensitivity to osmotic stress was relieved by co-overexpression of RAPTOR1. Overall, these observations demonstrated the existence of a functional TOR kinase pathway in plants. However, Arabidopsis seedlings do not respond to normal physiological levels of rapamycin, which appears to be due its inability to bind to the Arabidopsis homolog of FKBP12, a protein that is essential for the binding of rapamycin with TOR. Replacement of the Arabidopsis FKBP12 with the human FKBP12 allowed rapamycin-dependent interaction with TOR. Since homozygous mutation in TOR is lethal, it suggests that this pathway is essential for integrating the stress signals into the growth regulation.

Nodules are formed on legume roots as a result of signaling between symbiotic partners and in response to the activities of numerous genes. We cloned fragments of differentially expressed genes in spot-inoculated soybean (Glycine max) roots. Many of the induced clones were similar to known genes related to oxidative stress, such as thioredoxin and β-carotene hydroxylase. The deduced amino acid sequences of full-length soybean cDNAs for thioredoxin and β-carotene hydroxylase were similar to those in other species. In situ RNA hybridization revealed that the thioredoxin gene is expressed on the pericycle of 2-d-old nodules and in the infected cells of mature nodules, suggesting that thioredoxin is involved in nodule development. The thioredoxin promoter was found to contain a sequence resembling an antioxidant responsive element. When a thioredoxin mutant of yeast was transformed with the soybean thioredoxin gene it became hydrogen peroxide tolerant. These observations prompted us to measure reactive oxygen species levels. These were decreased by 3- to 5-fold in 7-d-old and 27-d-old nodules, coincident with increases in the expression of thioredoxin and β-carotene hydroxylase genes. Hydrogen peroxide-producing regions identified with cerium chloride were found in uninoculated roots and 2-d-old nodules, but not in 7-d-old and 27-d-old nodules. RNA interference-mediated repression of the thioredoxin gene severely impaired nodule development. These data indicate that antioxidants such as thioredoxin are essential to lower reactive oxygen species levels during nodule development.

Pro has been shown to play an important role in ameliorating environmental stress in plants and microorganisms, including heavy metal stress. Here, we describe the effects of the expression of a mothbean delta(1)-pyrroline-5-carboxylate synthetase (P5CS) gene in the green microalga Chlamydomonas reinhardtii. We show that transgenic algae expressing the mothbean P5CS gene have 80% higher free-Pro levels than wild-type cells, grow more rapidly in toxic Cd concentrations (100 microM), and bind fourfold more Cd than wild-type cells. In addition, Cd-K edge extended x-ray absorption fine structure studies indicated that Cd does not bind to free Pro in transgenic algae with increased Pro levels but is coordinated tetrahedrally by sulfur of phytochelatin. In contrast to P5CS-expressing cells, Cd is coordinated tetrahedrally by two oxygen and two sulfur atoms in wild-type cells. Measurements of reduced/oxidized GSH ratios and analyses of levels of malondialdehyde, a product of the free radical damage of lipids, indicate that free Pro levels are correlated with the GSH redox state and malondialdehyde levels in heavy metal-treated algae. These results suggest that the free Pro likely acts as an antioxidant in Cd-stressed cells. The resulting increased GSH levels facilitate increased phytochelatin synthesis and sequestration of Cd, because GSH-heavy metal adducts are the substrates for phytochelatin synthase.

Tropical legumes fix nitrogen in association with Bradyrhizobium species. The symbiotically-reduced nitrogen is assimilated by the host plant using a unique pathway that ensures efficient utilization of carbon. Tropical legumes such as soybean, cowpea and bean primarily assimilate and transport fixed-nitrogen as ureides, allantoin and allantoic acid, while temperate legumes such as pea and alfalfa are amide-transporters, producing L-glutamine and L-asparagine. Ureides are produced via de novo biosynthesis and oxidation of purines. The activities of enzymes involved in de novo purine biosynthesis and catabolism vary in effective and ineffective nodules and correlate with the status of the nodules in fixing nitrogen (Atkins, 1991). The ureide-producing nodules are usually determinate in their structure but not all determinate nodules are ureide producers.

Using phragmoplastin as a bait, we isolated an Arabidopsis cDNA encoding a novel UDP-glucose transferase (UGT1). This interaction was confirmed by an in vitro protein--protein interaction assay using purified UGT1 and radiolabeled phragmoplastin. Protein gel blot results revealed that UGT1 is associated with the membrane fraction and copurified with the product-entrapped callose synthase complex. These data suggest that UGT1 may act as a subunit of callose synthase that uses UDP-glucose to synthesize callose, a 1,3-beta-glucan. UGT1 also interacted with Rop1, a Rho-like protein, and this interaction occurred only in its GTP-bound configuration, suggesting that the plant callose synthase may be regulated by Rop1 through the interaction with UGT1. The green fluorescent protein--UGT1 fusion protein was located on the forming cell plate during cytokinesis. We propose that UGT1 may transfer UDP-glucose from sucrose synthase to the callose synthase and thus help form a substrate channel for the synthesis of callose at the forming cell plate.

Root nodule development is a unique process where bacteria reside intracellularly inside a host cell and contribute to the nitrogen requirement of the host plant. A molecular communication between the bacteria and host plants results in the organogenesis of nodules. The host roots exude flavonoids that act as chemotactic agents to attract the rhizobia to move toward the root surface and act as inducers for rhizobial nod genes. The nod genes encode enzymes responsible for biosynthesis and secretion of Nod factors (Carlson et al., 1994). The latter are active in eliciting morphological changes in the host roots and initiate root nodule organogenesis. It has been suggested that such factors may exist in plants to regulate normal morphogenesis programs. The host induces a set of genes encoding the nodulin proteins that are required for organogenesis and function of the root nodules. The metabolism of the host is adapted to meet the requirements of nitrogen fixation by bacteria and their assimilation by the host in root nodules.

The nodulins, nodule-specific host gene products, were first identified from soybean and nodulin-35 was detected to be the second most abundant protein in soybean root nodules 1. Following this, a group of other nodulins were detected using various immunological approaches and 2-D gel analysis2 and nodulin-35 was characterized to be a subunit of the nodule-specific uricase3. These early studies, coupled with DNA sequence hybridization4 and differential screening of nodule cDNA libraries5 established that a group of host genes encoding nodulins is induced following infection of the host plants by rhizobia. This group was later divided into early and late nodulins6 and each group was generally considered to play a role in early root nodule morphogenesis and function, respectively. During the last 10 years almost 200 such genes, in addition to leghemoglobin genes, have been identified from different legume hosts.

Plants generally accumulate free proline under osmotic stress conditions. Upon removal of the osmotic stress, the proline levels return to normal. In order to understand the mechanisms involved in regulating the levels of proline, we cloned and characterized a proline dehydrogenase (PDH) cDNA from Arabidopsis thaliana (AtPDH). The 1745 bp cDNA contains a major open reading frame encoding a peptide of 499 amino acids. The deduced amino acid sequence has high homology with both Saccharomyces cerevisiae and Drosophila melanogaster proline oxidases and contains a putative mitochondrial targeting sequence. When expressed in yeast, the AtPDH cDNA complemented a yeast put1 mutation and exhibited proline oxidase activity. We also determined the free proline contents and the delta 1-pyrroline-5-carboxylate synthetase (P5CS) and PDH mRNA levels under different osmotic stress and recovery conditions. The results demonstrated that the removal of free proline during the recovery from salinity or dehydration stress involves an induction of the PDH gene while the activity of P5CS declines. The reciprocal regulation of P5CS and PDH genes appears to be a key mechanism in the control of the levels of proline during and after osmotic stress. The PDH gene was also significantly induced by exogenously applied proline. The induction of PDH by proline, however, was inhibited by salt stress.

Nodule organogenesis is a highly-programmed developmental process triggered by rhizobial signal molecules and controlled by both Rhizobium and plant (Verma, 1992; Fisher and Long, 1992; Hirsch, 1992). Formation of Rhizobium-legume symbiosis provides a unique experimental system for genetic and molecular studies for communication between bacteria and host plants. Plant flavonoids and rhizobial Nod factors (modified oligosaccharides) are two classes of unique signal molecules that represent direct communication between these two organisms. The host roots exude flavonoids that act as chemotactic agents to attract the rhizobia to move towards the root surface and, more importantly, as strong inducers to initiate rhizobial nod gene expression. The nod genes encode enzymes responsible for biosynthesis and secretion of Nod factors (Carlson et al., 1994). Induction of the nod gene expression results in the production of Nod factors that elicit morphological changes in the host roots. These changes include root hair curling, formation of nodule primodia in the cortical cell and nodule organogenesis.

A plant homolog of yeast HAL2 gene (RHL) was cloned from rice (Orizya sativa L.). The RHL cDNA complemented an Escherichia coli cysteine auxotrophic mutant, cysQ, and the yeast HAL2 mutant, met22. The latter is a methionine auxotroph and cannot use sulfate, sulfite, or sulfide as sulfur sources but exhibits wild-type activities of the enzymes necessary to assimilate sulfate and has normal sulfur uptake system. These results demonstrated that HAL2, cysQ, and RHL genes encode proteins with similar function in sulfur assimilatory pathway. The RHL cDNA expressed a 40-kDa protein that was shown to catalyze the conversion of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to adenosine 5'-phosphosulfate (APS) and 3'(2')-phosphoadenosine 5'-phosphate (PAP) to AMP. The enzyme activity is Mg(2+)-dependent, sensitive to Ca2+, Li+, and Na+ and activated by K+. The inhibition by Ca2+ depends on the Mg2+/Ca2+ ratio and is reversible by high Mg2+ concentration. The substrate specificity and kinetics of RHL enzyme are very similar to the Chlorella 3'(2'),5'-diphosphonucleoside 3'(2')-phosphohydrolase (DPNPase). Our evidence suggests that this enzyme regulates the flux of sulfur in the sulfur-activation pathway by converting PAPS to APS. Several residues that are essential for the activity of this enzyme were identified by site-directed mutagenesis, and the possible role of DPNPase in salt tolerance is discussed.

Soybean (Glycine max) and mothbean (Vigna aconitifolia) cDNA clones encoding glutamine phosphoribosylpyrophosphate amidotransferase (PRAT), the first enzyme of the de novo purine biosynthesis pathway, have been isolated from nodule cDNA libraries. The amino acid sequence deduced from soybean clone showed > 85% homology to the PRAT sequence of mothbean and 33-47% homology to those of bacteria, yeast, chicken, rat and human. The soybean clone encodes a protein with an N-terminal sequence resembling a plastid-targeting peptide. Downstream from this peptide is a sequence similar to the 11 amino acid propeptide found in the Bacillus subtilis, chicken, rat and human PRAT proteins. The mothbean cDNA, although lacking most of the plastid presequence, encodes the putative propeptide and efficiently complements purine auxotrophy in an Escherichia coli purF mutant. Both the soybean and mothbean clones encode characteristic cysteine residues that are known to be involved in the assembly of a [Fe-S] cluster near the C-terminus of this protein. Levels of PRAT mRNA in mothbean nodules were found to increase steadily as the nodules matured from 13 days to 23 days. PRAT mRNA was not detectable in uninfected root tissue but a low level of transcript was detected in leaves. Treatment of uninfected root with L-glutamine induced the PRAT mRNA transcript suggesting that glutamine produced as a result of assimilation of fixed nitrogen is funnelled into the de novo purine biosynthesis and controls the expression of this pathway in root nodules.