Specific binding of a 14-3-3 protein to autophosphorylated WPK4, an SNF1-related wheat protein kinase, and to WPK4-phosphorylated nitrate reductase

WPK4 is a wheat protein kinase related to the yeast protein kinase SNF1, which plays a role in catabolite repression. To identify proteins involved in signal transduction through WPK4, we performed yeast two-hybrid screens and isolated two cDNA clones designated as TaWIN1 and TaWIN2. Both encode 14-3-3 proteins that, upon autophosphorylation, bind the C-terminal regulatory domain of WPK4. Mutational analysis through amino acid substitution revealed that TaWIN1 and TaWIN2 primarily bind WPK4 through phosphoserines at the positions 388 and 418, both located in the C-terminal region. Mutations in the conserved residues of the TaWIN1 amphipathic groove impaired the ability of TaWIN1 to bind to WPK4. A screen for in vitro phosphorylation of proteins involved in nutrient metabolism revealed a putative WPK4 substrate, nitrate reductase; its hinge 1 region was efficiently phosphorylated by WPK4. Subsequent far Western blots showed that it specifically bound TaWIN1. Since nitrate reductase has been shown to be inactivated by phosphorylation upon 14-3-3 binding, the present findings strongly suggest that WPK4 is the protein kinase responsible for controlling the nitrogen metabolic pathway, assembling the nitrate reductase and 14-3-3 complex through its phosphorylation specificity.     

Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm

In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the α-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting α-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds.     

Characterization of a maize beta-amylase cDNA clone and its expression during seed germination.

A maize (Zea mays L.) cDNA clone (pZMB2) encoding beta-amylase was isolated from a cDNA library prepared from the aleurone RNA of germinating kernels. The cDNA encodes a predicted product of 488 amino acids with significant similarity to known beta-amylases from barley (Hordeum vulgare), rye (Secale cereale), and rice (Oryza sativa). Glycine-rich repeats found in the carboxyl terminus of the endosperm-specific beta-amylase of barley and rye are absent from the maize gene product. The N-terminal sequence of the first 20 amino acids of a beta-amylase peptide derived from purified protein is identical to the 5th through 24th amino acids of the predicted cDNA product, indicating the absence of a conventional signal peptide in the maize protein. Recombinant inbred mapping data indicate that the cDNA clone is single-copy gene that maps to chromosome 7L at position 83 centimorgans. Northern blot analysis and in vitro translation-immunoprecipitation data indicate that the maize beta-amylase is synthesized de novo in the aleurone cells but not in the scutellum during seed germination.     

Identification and molecular characterization of ZAG1, the maize homolog of the Arabidopsis floral homeotic gene AGAMOUS.

Recent genetic and molecular studies in Arabidopsis and Antirrhinum suggest that mechanisms controlling floral development are well conserved among dicotyledonous species. To assess whether similar mechanisms also operate in more distantly related monocotyledonous species, we have begun to clone homologs of Arabidopsis floral genes from maize. Here we report the characterization of two genes, designated ZAG1 and ZAG2 (for Zea AG), that were cloned from a maize inflorescence cDNA library by low stringency hybridization with the AGAMOUS (AG) cDNA from Arabidopsis. ZAG1 encodes a putative polypeptide of 286 amino acids having 61% identity with the AGAMOUS (AG) protein. Through a stretch of 56 amino acids, constituting the MADS domain, the two proteins are identical except for two conservative amino acid substitutions. The ZAG2 protein is less similar to AG, with 49% identity overall and substantially less similarity than ZAG1 outside the well-conserved MADS domain. Like AG, ZAG1 RNA accumulates early in stamen and carpel primordia. In contrast, ZAG2 expression begins later and is restricted to developing carpels. Hybridization to genomic DNA with the full-length ZAG1 cDNA under moderately stringent conditions indicated the presence of a large family of related genes. Mapping data using maize recombinant inbreds placed ZAG1 and ZAG2 near two loci that are known to affect maize flower development, Polytypic ear (Pt) and Tassel seed4 (Ts4), respectively. The ZAG1 protein from in vitro translations binds to a consensus target site that is recognized by the AG protein. These data suggest that maize contains a homolog of the Arabidopsis floral identity gene AG and that this gene is conserved in sequence and function.     

Variable interactions between sucrose non-fermented 1-related protein kinases and regulatory proteins in higher plants

WPK4 is a sucrose non-fermented 1 (SNF1)-related wheat protein kinase, and was previously reported to interact with 14-3-3 proteins. We identified four Arabidopsis thaliana WPK4-like genes, and designated them AtWL1 through AtWL4. Yeast two-hybrid analysis, however, indicated that none of the AtWLs interacted with any of A. thaliana 14-3-3 (At14-3-3) proteins, although WPK4 itself interacted with six of them. Structurally, AtWLs were classified into a subfamiliy of AtCIPK, which generally interacts with calucineurin B-like proteins (CBL). This was also the case for AtWL1 and AtWL2, showing an efficient interaction with AtCBL2. In contrast, WPK4 interacted with none of the CBLs. In addition, to ascertain the possible interaction in vivo, expression of those genes was examined with a promoter-GUS assay. These results suggested that the interacting partner of SNF1-related protein kinases varies among plant species, and that, in the case of A. thaliana, it was CBLs, some of which were predicted to broadly regulate multiple CIPKs.     

Expression of the immunity protein of plantaricin 423, produced by Lactobacillus plantarum 423, and analysis of the plasmid encoding the bacteriocin

Plantaricin 423 is a class IIa bacteriocin produced by Lactobacillus plantarum isolated from sorghum beer. It has been previously determined that plantaricin 423 is encoded by a plasmid designated pPLA4, which is now completely sequenced. The plantaricin 423 operon shares high sequence similarity with the operons of coagulin, pediocin PA-1, and pediocin AcH, with small differences in the DNA sequence encoding the mature bacteriocin peptide and the immunity protein. Apart from the bacteriocin operon, no significant sequence similarity could be detected between the DNA or translated sequence of pPLA4 and the available DNA or translated sequences of the plasmids encoding pediocin AcH, pediocin PA-1, and coagulin, possibly indicating a different origin. In addition to the bacteriocin operon, sequence analysis of pPLA4 revealed the presence of two open reading frames (ORFs). ORF1 encodes a putative mobilization (Mob) protein that is homologous to the pMV158 superfamily of mobilization proteins. Highest sequence similarity occurred between this protein and the Mob protein of L. plantarum NCDO 1088. ORF2 encodes a putative replication protein that revealed low sequence similarity to replication proteins of plasmids pLME300 from Lactobacillus fermentum and pYIT356 from Lactobacillus casei. The immunity protein of plantaricin 423 contains 109 amino acids. Although plantaricin 423 shares high sequence similarity with the pediocin PA-1 operon, no cross-reactivity was recorded between the immunity proteins of plantaricin 423 and pediocin PA-1.     

水稻OsTB1基因的结构及其表达分析

TCP基因是一类植物中新发现的、可能具有转录因子活性的基因家族,成员包括金鱼草的Cyclodiea (Cyc)、玉米的Teosinte Branched1 (TB1)以及水稻中的PCF1、PCF2等.玉米的TB1基因有维持玉米顶端优势的作用,与分蘖的发生密切相关;水稻和玉米同属禾本科,在发育的过程中都有分蘖的发生.通过筛选水稻的基因组文库,得到了水稻中的一个TB1同源基因Oryza sativa Teosinte Branched1 (OsTB1).该基因不含内含子,基因编码一个长度为388个氨基酸的蛋白,在氨基酸水平上与TB1的同源性为70%,含有保守的TCP区和R区,是属于TCP基因家族的一个成员.RT-PCR和mRNA原位杂交分析结果表明,OsTB1在水稻的侧芽中有很强的表达,在花序中有较弱的表达.以上结果显示该基因可能在水稻侧芽和花序的起始和发育过程中起重要作用.