Maho Tanaka

The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression

The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single‐copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory‐chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ‘two out of three’ and ‘U:N wobble’ mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

Positive regulatory role of strigolactone in plant responses to drought and salt stress

Significance Environmental stresses, such as drought and high salinity, adversely affect plant growth and productivity. Although various phytohormones are known to be involved in regulation of plant stress responses, the role of strigolactone (SL) in this important process remains elusive. By using different molecular and physiological approaches, we provide compelling evidence that, in Arabidopsis, SL acts as positive regulator of plant responses to drought and salt stress, which was associated with shoot- rather than root-related traits. Comparative transcriptome analysis suggests that plants integrate multiple hormone-response pathways—at least SL, abscisic acid, and cytokinin pathways—for adaptation to environmental stress. Our findings demonstrate that genetic modulation of SL content/response could provide a new approach for development of crops with improved stress tolerance. This report provides direct evidence that strigolactone (SL) positively regulates drought and high salinity responses in Arabidopsis. Both SL-deficient and SL-response [more axillary growth (max)] mutants exhibited hypersensitivity to drought and salt stress, which was associated with shoot- rather than root-related traits. Exogenous SL treatment rescued the drought-sensitive phenotype of the SL-deficient mutants but not of the SL-response mutant, and enhanced drought tolerance of WT plants, confirming the role of SL as a positive regulator in stress response. In agreement with the drought-sensitive phenotype, max mutants exhibited increased leaf stomatal density relative to WT and slower abscisic acid (ABA)-induced stomatal closure. Compared with WT, the max mutants exhibited increased leaf water loss rate during dehydration and decreased ABA responsiveness during germination and postgermination. Collectively, these results indicate that cross-talk between SL and ABA plays an important role in integrating stress signals to regulate stomatal development and function. Additionally, a comparative microarray analysis of the leaves of the SL-response max2 mutant and WT plants under normal and dehydrative conditions revealed an SL-mediated network controlling plant responses to stress via many stress- and/or ABA-responsive and cytokinin metabolism-related genes. Our results demonstrate that plants integrate multiple hormone-response pathways for adaptation to environmental stress. Based on our results, genetic modulation of SL content/response could be applied as a potential approach to reduce the negative impact of abiotic stress on crop productivity.

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.

Small open reading frames associated with morphogenesis are hidden in plant genomes

It is likely that many small ORFs (sORFs; 30–100 amino acids) are missed when genomes are annotated. To overcome this limitation, we identified ∼8,000 sORFs with high coding potential in intergenic regions of the Arabidopsis thaliana genome. However, the question remains as to whether these coding sORFs play functional roles. Using a designed array, we generated an expression atlas for 16 organs and 17 environmental conditions among 7,901 identified coding sORFs. A total of 2,099 coding sORFs were highly expressed under at least one experimental condition, and 571 were significantly conserved in other land plants. A total of 473 coding sORFs were overexpressed; ∼10% (49/473) induced visible phenotypic effects, a proportion that is approximately seven times higher than that of randomly chosen known genes. These results indicate that many coding sORFs hidden in plant genomes are associated with morphogenesis. We believe that the expression atlas will contribute to further study of the roles of sORFs in plants.

Differential Gene Expression in Soybean Leaf Tissues at Late Developmental Stages under Drought Stress Revealed by Genome-Wide Transcriptome Analysis

The availability of complete genome sequence of soybean has allowed research community to design the 66 K Affymetrix Soybean Array GeneChip for genome-wide expression profiling of soybean. In this study, we carried out microarray analysis of leaf tissues of soybean plants, which were subjected to drought stress from late vegetative V6 and from full bloom reproductive R2 stages. Our data analyses showed that out of 46093 soybean genes, which were predicted with high confidence among approximately 66000 putative genes, 41059 genes could be assigned with a known function. Using the criteria of a ratio change > = 2 and a q-value<0.05, we identified 1458 and 1818 upregulated and 1582 and 1688 downregulated genes in drought-stressed V6 and R2 leaves, respectively. These datasets were classified into 19 most abundant biological categories with similar proportions. There were only 612 and 463 genes that were overlapped among the upregulated and downregulated genes, respectively, in both stages, suggesting that both conserved and unconserved pathways might be involved in regulation of drought response in different stages of plant development. A comparative expression analysis using our datasets and that of drought stressed Arabidopsis leaves revealed the existence of both conserved and species-specific mechanisms that regulate drought responses. Many upregulated genes encode either regulatory proteins, such as transcription factors, including those with high homology to Arabidopsis DREB, NAC, AREB and ZAT/STZ transcription factors, kinases and two-component system members, or functional proteins, e.g. late embryogenesis-abundant proteins, glycosyltransferases, glycoside hydrolases, defensins and glyoxalase I family proteins. A detailed analysis of the GmNAC family and the hormone-related gene category showed that expression of many GmNAC and hormone-related genes was altered by drought in V6 and/or R2 leaves. Additionally, the downregulation of many photosynthesis-related genes, which contribute to growth retardation under drought stress, may serve as an adaptive mechanism for plant survival. This study has identified excellent drought-responsive candidate genes for in-depth characterization and future development of improved drought-tolerant transgenic soybeans.

Genome-wide analysis of endogenous abscisic acid-mediated transcription in dry and imbibed seeds of Arabidopsis using tiling arrays.

The phytohormone abscisic acid (ABA) plays important roles in the induction and maintenance of seed dormancy. Although application of exogenous ABA inhibits germination, the effects of exogenous ABA on ABA-mediated gene transcription differ from those of endogenous ABA. To understand how endogenous ABA regulates the transcriptomes in seeds, we performed comprehensive expression analyses using whole-genome Affymetrix tiling arrays in two ABA metabolism mutants - an ABA-deficient mutant (aba2) and an ABA over-accumulation mutant (cyp707a1a2a3 triple mutant). Hierarchical clustering and principal components analyses showed that differences in endogenous ABA levels do not influence global expression of stored mRNA in dry seeds. However, the transcriptome after seed imbibition was related to endogenous ABA levels in both types of mutant. Endogenous ABA-regulated genes expressed in imbibed seeds included those encoding key ABA signaling factors and gibberellin-related components. In addition, cohorts of ABA-upregulated genes partially resembled those of dormant genes, whereas ABA-downregulated genes were partially overlapped with after-ripening-regulated genes. Bioinformatic analyses revealed that 6105 novel genes [non-Arabidopsis Genome Initiative (AGI) transcriptional units (TUs)] were expressed from unannotated regions. Interestingly, approximately 97% of non-AGI TUs possibly encoded hypothetical non-protein-coding RNAs, including a large number of antisense RNAs. In dry and imbibed seeds, global expression profiles of non-AGI TUs were similar to those of AGI genes. For both non-AGI TUs and AGI code genes, we identified those that were regulated differently in embryo and endosperm tissues. Our results suggest that transcription in Arabidopsis seeds is more complex and dynamic than previously thought.

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.

Transcriptome Analyses of a Salt-Tolerant Cytokinin-Deficient Mutant Reveal Differential Regulation of Salt Stress Response by Cytokinin Deficiency

Soil destruction by abiotic environmental conditions, such as high salinity, has resulted in dramatic losses of arable land, giving rise to the need of studying mechanisms of plant adaptation to salt stress aimed at creating salt-tolerant plants. Recently, it has been reported that cytokinins (CKs) regulate plant environmental stress responses through two-component systems. A decrease in endogenous CK levels could enhance salt and drought stress tolerance. Here, we have investigated the global transcriptional change caused by a reduction in endogenous CK content under both normal and salt stress conditions. Ten-day-old Arabidopsis thaliana wild-type (WT) and CK-deficient ipt1,3,5,7 plants were transferred to agar plates containing either 0 mM (control) or 200 mM NaCl and maintained at normal growth conditions for 24 h. Our experimental design allowed us to compare transcriptome changes under four conditions: WT-200 mM vs. WT-0 mM, ipt1,3,5,7-0 mM vs. WT-0 mM, ipt1,3,5,7-200 mM vs. ipt1,3,5,7-0 mM and ipt1,3,5,7-200 mM vs. WT-200 mM NaCl. Our results indicated that the expression of more than 10% of all of the annotated Arabidopsis genes was altered by CK deficiency under either normal or salt stress conditions when compared to WT. We found that upregulated expression of many genes encoding either regulatory proteins, such as NAC, DREB and ZFHD transcription factors and the calcium sensor SOS3, or functional proteins, such as late embryogenesis-abundant proteins, xyloglucan endo-transglycosylases, glycosyltransferases, glycoside hydrolases, defensins and glyoxalase I family proteins, may contribute to improved salt tolerance of CK-deficient plants. We also demonstrated that the downregulation of photosynthesis-related genes and the upregulation of several NAC genes may cause the altered morphological phenotype of CK-deficient plants. This study highlights the impact of CK regulation on the well-known stress-responsive signaling pathways, which regulate plant adaptation to high salinity as well as other environmental stresses.

Arabidopsis AHP2, AHP3, and AHP5 histidine phosphotransfer proteins function as redundant negative regulators of drought stress response

Cytokinin is an essential phytohormone controlling various biological processes, including environmental stress responses. In Arabidopsis, although the cytokinin (CK)-related phosphorelay—consisting of three histidine kinases, five histidine phosphotransfer proteins (AHPs), and a number of response regulators—has been known to be important for stress responses, the AHPs required for CK signaling during drought stress remain elusive. Here, we report that three Arabidopsis AHPs, namely AHP2, AHP3, and AHP5, control responses to drought stress in negative and redundant manner. Loss of function of these three AHP genes resulted in a strong drought-tolerant phenotype that was associated with the stimulation of protective mechanisms. Specifically, cell membrane integrity was improved as well as an increased sensitivity to abscisic acid (ABA) was observed rather than an alteration in ABA-mediated stomatal closure and density. Consistent with their negative regulatory functions, all three AHP genes’ expression was down-regulated by dehydration, which most likely resulted from a stress-induced reduction of endogenous CK levels. Furthermore, global transcriptional analysis of ahp2,3,5 leaves revealed down-regulation of many well-known stress- and/or ABA-responsive genes, suggesting that these three AHPs may control drought response in both ABA-dependent and ABA-independent manners. The discovery of mechanisms of activation and the targets of the downstream components of CK signaling involved in stress responses is an important and challenging goal for the study of plant stress regulatory network responses and plant growth. The knowledge gained from this study also has broad potential for biotechnological applications to increase abiotic stress tolerance in plants.

Arabidopsis HDA6 is required for freezing tolerance

Many plants exhibit altered gene expression patterns in response to low nonfreezing temperatures and an increase in freezing tolerance in a phenomenon known as cold acclimation. Here we show, for the first time, that the histone deacetylase gene HDA6 is required for cold acclimation and freezing tolerance in Arabidopsis. HDA6 is transcriptionally upregulated during long-term cold treatment. Cold-treated hda6 mutants showed reduced freezing tolerance compared with the cold-treated wild-type plants. Freezing-caused electrolyte leakage increased in the cold-treated hda6 mutant. In contrast, the non-cold-treated hda6 mutants showed no significant difference in survivability and electrolyte leakage compared to wild-type plants. Transcriptome analysis identified the genes that showed aberrant expression in the hda6 mutant after cold acclimation. We conclude that HDA6 plays a critical role in regulating cold acclimation process that confers freezing resistance on Arabidopsis.