Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells

The WRKY proteins are a superfamily of regulators that control diverse developmental and physiological processes. This family was believed to be plant specific until the recent identification of WRKY genes in nonphotosynthetic eukaryotes. We have undertaken a comprehensive computational analysis of the rice (Oryza sativa) genomic sequences and predicted the structures of 81 OsWRKY genes, 48 of which are supported by full-length cDNA sequences. Eleven OsWRKY proteins contain two conserved WRKY domains, while the rest have only one. Phylogenetic analyses of the WRKY domain sequences provide support for the hypothesis that gene duplication of single- and two-domain WRKY genes, and loss of the WRKY domain, occurred in the evolutionary history of this gene family in rice. The phylogeny deduced from the WRKY domain peptide sequences is further supported by the position and phase of the intron in the regions encoding the WRKY domains. Analyses for chromosomal distributions reveal that 26% of the predicted OsWRKY genes are located on chromosome 1. Among the dozen genes tested, OsWRKY24, -51, -71, and -72 are induced by abscisic acid (ABA) in aleurone cells. Using a transient expression system, we have demonstrated that OsWRKY24 and -45 repress ABA induction of the HVA22 promoter-beta-glucuronidase construct, while OsWRKY72 and -77 synergistically interact with ABA to activate this reporter construct. This study provides a solid base for functional genomics studies of this important superfamily of regulatory genes in monocotyledonous plants and reveals a novel function for WRKY genes, i.e. mediating plant responses to ABA.     

Defense-related signaling by interaction of arabinogalactan proteins and beta-glucosyl Yariv reagent inhibits gibberellin signaling in barley aleurone cells

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins present at the plasma membrane and in extracellular spaces. A synthetic chemical, beta-glucosyl Yariv reagent (beta-GlcY), binds specifically to AGPs. We previously reported that gibberellin signaling is specifically inhibited by beta-GlcY treatment in barley aleurone protoplasts. In the present study, we found that beta-GlcY also inhibited gibberellin-induced programmed cell death (PCD) in aleurone cells. We examined the universality and specificity of the inhibitory effect of beta-GlcY on gibberellin signaling using microarray analysis and found that beta-GlcY was largely effective in repressing gibberellin-induced gene expression. In addition, >100 genes were up-regulated by beta-GlcY in a gibberellin-independent manner, and many of these were categorized as defense-related genes. Defense signaling triggered by several defense system inducers such as jasmonic acid and a chitin elicitor could inhibit gibberellin-inducible events such as alpha-amylase secretion, PCD and expression of some gibberellin-inducible genes in aleurone cells. Furthermore, beta-GlcY repressed the gibberellin-inducible Ca2+-ATPase gene which is important for gibberellin-dependent gene expression, and induced known repressors of gibberellin signaling, two WRKY genes and a NAK kinase gene. These effects of beta-GlcY were also phenocopied by the chitin elicitor and/or jasmonic acid. These results indicate that gibberellin signaling is under the regulation of defense-related signaling in aleurone cells. It is also probable that AGPs are involved in the perception of stimuli causing defense responses.     

The P450-4 gene of Gibberella fujikuroi encodes ent-kaurene oxidase in the gibberellin biosynthesis pathway

At least five genes of the gibberellin (GA) biosynthesis pathway are clustered on chromosome 4 of Gibberella fujikuroi; these genes encode the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenases. We now describe a fourth cytochrome P450 monooxygenase gene (P450-4). Gas chromatography-mass spectrometry analysis of extracts of mycelia and culture fluid of a P450-4 knockout mutant identified ent-kaurene as the only intermediate of the GA pathway. Incubations with radiolabeled precursors showed that the metabolism of ent-kaurene, ent-kaurenol, and ent-kaurenal was blocked in the transformants, whereas ent-kaurenoic acid was metabolized efficiently to GA(4). The GA-deficient mutant strain SG139, which lacks the 30-kb GA biosynthesis gene cluster, converted ent-kaurene to ent-kaurenoic acid after transformation with P450-4. The B1-41a mutant, described as blocked between ent-kaurenal and ent-kaurenoic acid, was fully complemented by P450-4. There is a single nucleotide difference between the sequence of the B1-41a and wild-type P450-4 alleles at the 3' consensus sequence of intron 2 in the mutant, resulting in reduced levels of active protein due to a splicing defect in the mutant. These data suggest that P450-4 encodes a multifunctional ent-kaurene oxidase catalyzing all three oxidation steps between ent-kaurene and ent-kaurenoic acid.     

Positional cloning of rice semidwarfing gene, sd-1: rice "green revolution gene" encodes a mutant enzyme involved in gibberellin synthesis.

A rice semidwarfing gene, sd-1, known as the "green revolution gene," was isolated by positional cloning and revealed to encode gibberellin 20-oxidase, the key enzyme in the gibberellin biosynthesis pathway. Analysis of 3477 segregants using several PCR-based marker technologies, including cleaved amplified polymorphic sequence, derived-CAPS, and single nucleotide polymorphisms revealed 1 ORF in a 6-kb candidate interval. Normal-type rice cultivars have an identical sequence in this region, consisting of 3 exons (558, 318, and 291 bp) and 2 introns (105 and 1471 bp). Dee-Geo-Woo-Gen-type sd-1 mutants have a 383-bp deletion from the genome (278-bp deletion from the expressed sequence), from the middle of exon 1 to upstream of exon 2, including a 105-bp intron, resulting in a frame-shift that produces a termination codon after the deletion site. The radiation-induced sd-1 mutant Calrose 76 has a 1-bp substitution in exon 2, causing an amino acid substitution (Leu [CTC] to Phe [TTC]). Expression analysis suggests the existence of at least one more locus of gibberellin 20-oxidase which may prevent severe dwarfism from developing in sd-1 mutants.     

The mll6786 gene encodes a repressor protein controlling the degradation pathway for vitamin B6 in Mesorhizobium loti

Pyridoxine is converted to succinic semialdehyde, acetate, ammonia and CO(2) through the actions of eight enzymes. The genes encoding the enzymes occur as a cluster on the chromosomal DNA of Mesorhizobium loti, a symbiotic nitrogen-fixing bacterium. Here, it was found that disruption of the mll6786 gene, which is located between the genes encoding the first and eighth enzymes of the pathway, caused constitutive expression of the eight enzymes. The protein encoded by the mll6786 gene is a member of the GntR family and is designated as PyrR. PyrR comprises 223 amino acid residues and is a dimeric protein with a subunit molecular mass of 25?kDa. The purified PyrR with a C-terminal His(6) -tag could bind to an intergenic 67-bp DNA region, which contains a palindrome sequence and a deduced promoter sequence, between the mll6786 and mlr6787 genes, encoding PyrR and AAMS amidohydrolase, respectively.