Chung Sun An

The Regulation of DWARF4 Expression Is Likely a Critical Mechanism in Maintaining the Homeostasis of Bioactive Brassinosteroids in Arabidopsis

Mutants that are defective in brassinosteroid (BR) biosynthesis or signaling display severely retarded growth patterns due to absence of growth-promoting effects by BRs. Arabidopsis (Arabidopsis thaliana) DWARF4 (DWF4) catalyzes a flux-determining step in the BR biosynthetic pathways. Thus, it is hypothesized that the tissues of DWF4 expression may represent the sites of BR biosynthesis in Arabidopsis. Here we show that DWF4 transcripts accumulate in the actively growing tissues, such as root, shoot apices with floral clusters, joint tissues of root and shoot, and dark-grown seedlings. Conforming to the RNA gel-blot analysis, DWF4:β-glucuronidase (GUS) histochemical analyses more precisely define the tissues that express the DWF4 gene. Examination of the endogenous levels of BRs in six and seven different tissues of wild type and brassinosteroid insensitive1-5 mutant, respectively, revealed that BRs are significantly enriched in roots, shoot tips, and joint tissues of roots and shoots. In addition, DWF4:GUS expression was negatively regulated by BRs. DWF4:GUS activity was increased by treatment with brassinazole, a BR biosynthetic inhibitor, and decreased by exogenous application of bioactive BRs. When DWF4:GUS was expressed in a different genetic background, its level was down-regulated in brassinazole resistant1-D, confirming that BRASSINAZOLE RESISTANT1 acts as a negative regulator of DWF4. Interestingly, in the brassinosteroid insensitive2/dwf12-1D background, DWF4:GUS expression was intensified and delocalized to elongating zones of root, suggesting that BRASSINOSTEROID INSENSITIVE2 is an important factor that limits DWF4 expression. Thus, it is likely that the DWF4 promoter serves as a focal point in maintaining homeostasis of endogenous bioactive BR pools in specific tissues of Arabidopsis.

Arabidopsis cyp51 Mutant Shows Postembryonic Seedling Lethality Associated with Lack of Membrane Integrity

CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14α-demethylase involved in the postsqualene sterol biosynthetic pathway. According to the current gene annotation, the Arabidopsis (Arabidopsis thaliana) genome contains two putative CYP51 genes, CYP51A1 and CYP51A2. Our studies revealed that CYP51A1 should be considered an expressed pseudogene. To study the functional importance of the CYP51A2 gene in plant growth and development, we isolated T-DNA knockout alleles for CYP51A2. Loss-of-function mutants for CYP51A2 showed multiple defects, such as stunted hypocotyls, short roots, reduced cell elongation, and seedling lethality. In contrast to other sterol mutants, such as fk/hydra2 and hydra1, the cyp51A2 mutant has only minor defects in early embryogenesis. Measurements of endogenous sterol levels in the cyp51A2 mutant revealed that it accumulates obtusifoliol, the substrate of CYP51, and a high proportion of 14α-methyl-Δ8-sterols, at the expense of campesterol and sitosterol. The cyp51A2 mutants have defects in membrane integrity and hypocotyl elongation. The defect in hypocotyl elongation was not rescued by the exogenous application of brassinolide, although the brassinosteroid-signaling cascade is apparently not affected in the mutants. Developmental defects in the cyp51A2 mutant were completely rescued by the ectopic expression of CYP51A2. Taken together, our results demonstrate that the Arabidopsis CYP51A2 gene encodes a functional obtusifoliol 14α-demethylase enzyme and plays an essential role in controlling plant growth and development by a sterol-specific pathway.

Postembryonic Seedling Lethality in the Sterol-Deficient Arabidopsis cyp51A2 Mutant Is Partially Mediated by the Composite Action of Ethylene and Reactive Oxygen Species

Seedling-lethal phenotypes of Arabidopsis (Arabidopsis thaliana) mutants that are defective in early steps in the sterol biosynthetic pathway are not rescued by the exogenous application of brassinosteroids. The detailed molecular and physiological mechanisms of seedling lethality have yet to be understood. Thus, to elucidate the underlying mechanism of lethality, we analyzed transcriptome and proteome profiles of the cyp51A2 mutant that is defective in sterol 14α-demethylation. Results revealed that the expression levels of genes involved in ethylene biosynthesis/signaling and detoxification of reactive oxygen species (ROS) increased in the mutant compared with the wild type and, thereby, that the endogenous ethylene level also increased in the mutant. Consistently, the seedling-lethal phenotype of the cyp51A2 mutant was partly attenuated by the inhibition of ethylene biosynthesis or signaling. However, photosynthesis-related genes including Rubisco large subunit, chlorophyll a/b-binding protein, and components of photosystems were transcriptionally and/or translationally down-regulated in the mutant, accompanied by the transformation of chloroplasts into gerontoplasts and a reduction in both chlorophyll contents and photosynthetic activity. These characteristics observed in the cyp51A2 mutant resemble those of leaf senescence. Nitroblue tetrazolium staining data revealed that the mutant was under oxidative stress due to the accumulation of ROS, a key factor controlling both programmed cell death and ethylene production. Our results suggest that changes in membrane sterol contents and composition in the cyp51A2 mutant trigger the generation of ROS and ethylene and eventually induce premature seedling senescence.

Arabidopsis thaliana Remorins Interact with SnRK1 and Play a Role in Susceptibility to Beet Curly Top Virus and Beet Severe Curly Top Virus.

Remorins, a family of plant-specific proteins containing a variable N-terminal region and conserved C-terminal domain, play a role in various biotic and abiotic stresses, including host-microbe interactions. However, their functions remain to be completely elucidated, especially for the Arabidopsis thaliana remorin group 4 (AtREM4). To elucidate the role of remorins in Arabidopsis, we first showed that AtREM4s have typical molecular characteristics of the remorins, such as induction by various types of biotic and abiotic stresses, localization in plasma membrane and homo- and hetero-oligomeric interaction. Next, we showed that their loss-of-function mutants displayed reduced susceptibility to geminiviruses, Beet Curly Top Virus and Beet Severe Curly Top Virus, while overexpressors enhanced susceptibility. Moreover, we found that they interacted with SnRK1, which phosphorylated AtREM4.1, and were degraded by the 26S proteasome pathway. These results suggest that AtREM4s may be involved in the SnRK1-mediated signaling pathway and play a role as positive regulators of the cell cycle during geminivirus infection.

Sequence analysis and expression patterns of two nifA genes from Frankia EuIK1

Two nifA genes were cloned from Frankia EuIK 1 strain, a symbiont of Elaeagnus umbellata Thunb. and analysed on the basis of their deduced amino acid sequences and expression patterns. The complete nucleotide sequence of 1926 bp of nifA1 and 1524 bp of nifA2 was determined, respectively. A putative NifA-binding site was found -95 to about -80 bp upstream of start codon for nifA 1 ORF as TGT-N 10 -ACA, but the clpB ORF was followed by nifA2 ORF with 15 bp of intergenic sequence. Deduced amino acid sequence showed that two nifA genes encode typical NifA, having three major domains and two linkers, and their central domains of NifAl and NifA2 showed sequence similarity of 70-75% with those from other NifA proteins. However, entire NifA2 ORF is more similar to alternative NifA (60%) than to typical NifA (53%). Conserved amino acid sequence in helix-turn-helix motif of typical NifA was also found in NifA1, but it was not conserved in NifA2, which is also common in alternative NifA proteins. Moreover, the expression of nifA1 during nodule development was similar to that of Rhizobium meliloti in that it was expressed at low level constitutively, while that of nifA2 was similar to the pattern of nifH, structural gene for nitrogenase reductase, in that its transcripts level was changed in accordance with nitrogenase activity. These results indicate that nifA 1 and nifA2 might be classified into typical nifA and alternative nifA, respectively. This is the first report on the presence of two nifA genes in Frankia.

Comprehensive analysis of AHL homologous genes encoding AT-hook motif nuclear Localized protein in rice

The AT-hook motif is a small DNA-binding protein motif that has been found in the high mobility group of non-histone chromosomal proteins. The Arabidopsis genome contains 29 genes encoding the AT-hook motif DNA-binding protein (AHL). Recent studies of Arabidopsis genes (AtAHLs) have revealed that they might play diverse functional roles during plant growth and development. In this report, we mined 20 AHL genes (OsAHLs) from the rice genome database using AtAHL genes as queries and characterized their molecular features. A phylogenetic tree revealed that OsAHL proteins can be classified into 2 evolutionary clades. Tissue expression pattern analysis revealed that all of the OsAHL genes might be functionally expressed genes with 3 distinct expression patterns. Nuclear localization analysis using transgenic Arabidopsis showed that several OsAHL proteins are exclusively localized in the nucleus, indicating that they may act as architectural transcription factors to regulate expression of their target genes during plant growth and development.

Soybean mitogen-activated protein kinase GMK2 is activated with GMK1 in Bradyrhizobium-Soybean interactions

Biological nitrogen fixation in root nodules is the primary nitrogen source for plants. In leguminous plants, nodulation is initiated by the recognition of the nodulation (Nod) factor, the signaling molecule secreted by rhizobia. Our previous study showed that 47-kDa and 44-kDa MAPKs were activated in soybean treated with a genistein-induced culture filtrate (GCF) of Bradyrhizobium japonicum. We investigated the activity and regulation of the 47-kDa MAPK, GMK1; however, the 44-kDa MAPK was not extensively studied. Herein, we identified the 44-kDa MAPK as Glycine max MAP kinase 2 (GMK2) and showed that its transcription was activated by GCF. GMK1 and 44-kDa MAPK were activated by phosphatidic acid (PA) and GCF-mediated induction of GMK1 and GMK2 activities were reduced by treatment with PA generation inhibitors. The activity of GCF-activated GMK2, but not GMK1, was decreased by calcium signaling inhibitors. Our data indicate that GMK2 expression is regulated at the transcriptional level and that GMK2 activity is regulated by phosphatidic acid and calcium during soybean-Bradyrhizobium interactions.