Yukio Kurihara

‘Omics’ analyses of regulatory networks in plant abiotic stress responses

Plants must respond and adapt to abiotic stresses to survive in various environmental conditions. Plants have acquired various stress tolerance mechanisms, which are different processes involving physiological and biochemical changes that result in adaptive or morphological changes. Recent advances in genome-wide analyses have revealed complex regulatory networks that control global gene expression, protein modification, and metabolite composition. Genetic regulation and epigenetic regulation, including changes in nucleosome distribution, histone modification, DNA methylation, and npcRNAs (non-protein-coding RNA) play important roles in abiotic stress gene networks. Transcriptomics, metabolomics, bioinformatics, and high-through-put DNA sequencing have enabled active analyses of regulatory networks that control abiotic stress responses. Such analyses have markedly increased our understanding of global plant systems in responses and adaptation to stress conditions.

Genome-wide suppression of aberrant mRNA-like noncoding RNAs by NMD in Arabidopsis

The nonsense-mediated mRNA decay (NMD) pathway is a well-known eukaryotic surveillance mechanism that eliminates aberrant mRNAs that contain a premature termination codon (PTC). The UP-Frameshift (UPF) proteins, UPF1, UPF2, and UPF3, are essential for normal NMD function. Several NMD substrates have been identified, but detailed information on NMD substrates is lacking. Here, we noticed that, in Arabidopsis, most of the mRNA-like nonprotein-coding RNAs (ncRNAs) have the features of an NMD substrate. We examined the expression profiles of 2 Arabidopsis mutants, upf1-1 and upf3-1, using a whole-genome tiling array. The results showed that expression of not only protein-coding transcripts but also many mRNA-like ncRNAs (mlncRNAs), including natural antisense transcript RNAs (nat-RNAs) transcribed from the opposite strands of the coding strands, were up-regulated in both mutants. The percentage of the up-regulated mlncRNAs to all expressed mlncRNAs was much higher than that of the up-regulated protein-coding transcripts to all expressed protein- coding transcripts. This finding demonstrates that one of the most important roles of NMD is the genome-wide suppression of the aberrant mlncRNAs including nat-RNAs.

Arabidopsis HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1.

Heterochromatin silencing is pivotal for genome stability in eukaryotes. In Arabidopsis, a plant-specific mechanism called RNA–directed DNA methylation (RdDM) is involved in heterochromatin silencing. Histone deacetylase HDA6 has been identified as a component of such machineries; however, its endogenous targets and the silencing mechanisms have not been analyzed globally. In this study, we investigated the silencing mechanism mediated by HDA6. Genome-wide transcript profiling revealed that the loci silenced by HDA6 carried sequences corresponding to the RDR2-dependent 24-nt siRNAs, however their transcript levels were mostly unaffected in the rdr2 mutant. Strikingly, we observed significant overlap of genes silenced by HDA6 to those by the CG DNA methyltransferase MET1. Furthermore, regardless of dependence on RdDM pathway, HDA6 deficiency resulted in loss of heterochromatic epigenetic marks and aberrant enrichment for euchromatic marks at HDA6 direct targets, along with ectopic expression of these loci. Acetylation levels increased significantly in the hda6 mutant at all of the lysine residues in the H3 and H4 N-tails, except H4K16. Interestingly, we observed two different CG methylation statuses in the hda6 mutant. CG methylation was sustained in the hda6 mutant at some HDA6 target loci that were surrounded by flanking DNA–methylated regions. In contrast, complete loss of CG methylation occurred in the hda6 mutant at the HDA6 target loci that were isolated from flanking DNA methylation. Regardless of CG methylation status, CHG and CHH methylation were lost and transcriptional derepression occurred in the hda6 mutant. Furthermore, we show that HDA6 binds only to its target loci, not the flanking methylated DNA, indicating the profound target specificity of HDA6. We propose that HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1, possibly recruiting MET1 to specific loci, thus forming the foundation of silent chromatin structure for subsequent non-CG methylation.

Specific Enrichment of miRNAs in Arabidopsis thaliana Infected with Tobacco mosaic virus

Abstract RNA silencing is a broadly conserved machinery and is involved in many biological events. Small RNAs are key molecules in RNA silencing pathway that guide sequence-specific gene regulations and chromatin modifications. The silencing machinery works as an anti-viral defense in virus-infected plants. It is generally accepted that virus-specific small interfering (si) RNAs bind to the viral genome and trigger its cleavage. Previously, we have cloned and obtained sequences of small RNAs from Arabidopsis thaliana infected or uninfected with crucifer Tobacco mosaic virus. MicroRNAs (miRNAs) accumulated to a higher percentage of total small RNAs in the virus-infected plants. This was partly because the viral replication protein binds to the miRNA/miRNA* duplexes. In the present study, we mapped the sequences of small RNAs other than virus-derived siRNAs to the Arabidopsis genome and assigned each small RNA. It was demonstrated that only miRNAs increased as a result of viral infection. Furthermore, some newly identified miRNAs and miRNA candidates were found from the virus-infected plants despite a limited number of examined sequences. We propose that it is advantageous to use virus-infected plants as a source for cloning and identifying new miRNAs.

Identification of the candidate genes regulated by RNA-directed DNA methylation in Arabidopsis.

RNA-directed DNA methylation (RdDM) is a process in which 24 nucleotide (nt) small interfering RNAs (siRNAs) guide de novo cytosine methylation in the homologous genomic DNA region. Of several factors involving 24 nt siRNA accumulation, RNA-dependent RNA polymerase 2 (RDR2) is a key component, because accumulation of 24 nt siRNA disappears in the Arabidopsis rdr2 mutant. Here, we compared expression profiles among wild-type, rdr2-1 and ddc (drm1drm2cmt3), DNA methyltransferase triple mutant, using a whole genome tiling array to identify the candidate genes directly downregulated by RdDM-related 24 nt siRNAs. Of the transcripts upregulated in the mutants, we searched for those whose coding regions or flanking regions have siRNA-generating loci. We found upregulated expression of 18 transcripts with AGI codes and 19 predicted transcriptional units (TUs) with siRNA loci in both rdr2-1 and ddc. Our study provided important information for understanding the relationship between RdDM and the identified candidate genes.

Arabidopsis Tiling Array Analysis to Identify the Stress-Responsive Genes

Plants respond and adapt to drought, cold, and high-salinity stresses. Stress-inducible gene products function in the stress response and tolerance in plants. Using cDNA microarrays and oligonucleotide microarrays, stress-inducible genes have been identified in various plant species so far. Recently, tiling array technology has become a powerful tool for the whole-genome transcriptome analysis. We applied the Arabidopsis Affymetrix tiling arrays to study the whole-genome transcriptome under drought, cold, and high-salinity stresses and identified a large number of drought, cold, and high-salinity stress-inducible genes and transcriptional units (TUs).

A poly(A)-specific ribonuclease directly regulates the poly(A) status of mitochondrial mRNA in Arabidopsis

Coordination of gene expression in the organelles and the nucleus is important for eukaryotic cell function. Transcriptional and post-transcriptional gene regulation in mitochondria remains incompletely understood in most eukaryotes, including plants. Here we show that poly(A)-specific ribonuclease, which influences the poly(A) status of cytoplasmic mRNA in many eukaryotes, directly regulates the poly(A) tract of mitochondrial mRNA in conjunction with a bacterial-type poly(A) polymerase, AGS1, in Arabidopsis. An Arabidopsis poly(A)-specific ribonuclease-deficient mutant, ahg2-1, accumulates polyadenylated mitochondrial mRNA and shows defects in mitochondrial protein complex levels. Mutations of AGS1 suppress the ahg2-1 phenotype. Mitochondrial localizations of AHG2 and AGS1 are required for their functions in the regulation of the poly(A) tract of mitochondrial mRNA. Our findings suggest that AHG2 and AGS1 constitute a regulatory system that controls mitochondrial mRNA poly(A) status in Arabidopsis.

Cross-protection in Arabidopsis against crucifer tobamovirus Cg by an attenuated strain of the virus.

SUMMARY Cross-protection is a procedure that has been utilized to protect crops against virulent strains of viruses by pre-treatment with closely related attenuated strains of the virus. We constructed a mutant of crucifer tobamovirus Cg, which is analogous to L(11)A, an attenuated strain of Tomato mosaic virus-L (ToMV-L). This mutant, named CgYD, caused few disease symptoms and could spread throughout Arabidopsis thaliana Col-0 plants. Initial infection with CgYD was shown to efficiently cross-protect against a challenge with wild-type Cg. Thus, we have established in Arabidopsis a powerful system for investigating mechanisms of cross-protection. Using this system, we showed that cross-protection was not overcome, even if a higher concentration of the virion, or purified virion RNA, were used in the challenge. We also demonstrated that cross-protection requires that the second virus be very similar in sequence to Cg, which is a characteristic of RNA silencing. However the RNA dependent RNA polymerase SDE1/SGS2 associated with post-transcriptional gene silencing was not required for cross-protection.

Sucrose transporter NtSUT4 from tobacco BY-2 involved in plant cell shape during miniprotoplast culture

Sucrose plays an important role in several cellular processes since it is a general source of metabolic energy, serves as a precursor for starch and cellulose synthesis, and is a metabolic starting point for carboxylate- and amino acid synthesis. While plant vacuole is the main cellular storage pool, where sucrose accumulates to high concentrations, only a small number of vacuolar sugar transporters have been identified and characterized to date. We initially identified a vacuolar sucrose transporter (NtSUT4) from tobacco BY-2 cells and established transgenic tobacco BY-2 cell lines that overexpress NtSUT4-GFP (BY-SUTG cells). Using a model system for synchronous cell elongation in miniprotoplasts (evacuolated cells) prepared from tobacco BY-2 cells, we found that NtSUT4-GFP overexpression inhibited cell growth towards the cell major axis. Moreover, under the same conditions, we found that the cell walls were well stained by calcofluor in BY-SUTG cells than in wild type BY-2 cells. These results suggest that NtSUT4 is involved in cell shape via sucrose homeostasis in plant cells.

Transcriptome Analyses Revealed Diverse Expression Changes in ago1 and hyl1 Arabidopsis Mutants

MicroRNAs (miRNAs) are 20-24 nucleotide endogenous regulatory molecules conserved in higher eukaryotes. In Arabidopsis, miRNAs are produced through step-wise cleavages of primary miRNA precursors (pri-miRNAs) by DICER-LIKE1 (DCL1). This cleavage step is also supported by a double-stranded RNA-binding protein, HYPONASTIC LEAVES1 (HYL1). In many cases, mature miRNA is predominantly incorporated into an endonuclease, ARGONAUTE1 (AGO1), which degrades miRNA-targeted mRNAs. Here, we examined and revealed whole genome transcriptomes in ago1-25 and hyl1-2 mutants using tiling arrays. The data in this paper are valuable for understanding the relationship between the miRNA pathway and its effect on transcriptomes.