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Main conclusion: This is the first report on the involvement of abscisic acid signaling in regulating post-germination growth under Cs stress, not related to potassium deficiency. Cesium (Cs) is known to exert toxicity in plants by competition and interference with the transport of potassium (K). However, the precise mechanism of how Cs mediates its damaging effect is still unclear. This fact is mainly attributed to the large effects of lower K uptake in the presence of Cs that shadow other crucial effects by Cs that were not related to K. RNA-seq was conducted on Arabidopsis roots grown to identify putative genes that are functionally involved to investigate the difference between Cs stress and low K stress. Our transcriptome data demonstrated Cs-regulated genes only partially overlap to low K-regulated genes. In addition, the divergent expression trend of High-affinity K+Transporter (HAK5) from D4 to D7 growth stage suggested participation of other molecular events besides low K uptake under Cs stress. Potassium deficiency triggers expression level change of the extracellular matrix, transfer/carrier, cell adhesion, calcium-binding, and DNA metabolism genes. Under Cs stress, genes encoding translational proteins, chromatin regulatory proteins, membrane trafficking proteins and defense immunity proteins were found to be primarily regulated. Pathway enrichment and protein network analyses of transcriptome data exhibit that Cs availability are associated with alteration of abscisic acid (ABA) signaling, photosynthesis activities and nitrogen metabolism. The phenotype response of ABA signaling mutants supported the observation and revealed Cs inhibition of root growth involved in ABA signaling pathway. The rather contrary response of loss-of-function mutant of Late Embryogenesis Abundant 7 (LEA7) and Translocator Protein (TSPO) further suggested low K stress and Cs stress may activate different salt tolerance responses. Further investigation on the crosstalk between K transport, signaling, and salt stress-responsive signal transduction will provide a deeper understanding of the mechanisms and molecular regulation underlying Cs toxicity. [ABSTRACT FROM AUTHOR]

Chloroplast development adapts to the environment for performing suitable photosynthesis. Brassinosteroids (BRs), plant steroid hormones, have crucial effects on not only plant growth but also chloroplast development. However, the detailed molecular mechanisms of BR signaling in chloroplast development remain unclear. Here, we identify a regulator of chloroplast development, BPG4, involved in light and BR signaling. BPG4 interacts with GOLDEN2-LIKE (GLK) transcription factors that promote the expression of photosynthesis-associated nuclear genes (PhANGs), and suppresses their activities, thereby causing a decrease in the amounts of chlorophylls and the size of light-harvesting complexes. BPG4 expression is induced by BR deficiency and light, and is regulated by the circadian rhythm. BPG4 deficiency causes increased reactive oxygen species (ROS) generation and damage to photosynthetic activity under excessive high-light conditions. Our findings suggest that BPG4 acts as a chloroplast homeostasis factor by fine-tuning the expression of PhANGs, optimizing chloroplast development, and avoiding ROS generation. The authors identify BPG4, a novel regulator of chloroplast development. BPG4 directly suppresses transcriptional activity of GLK to fine-tune photosynthesis associated nuclear gene expression, and regulates chloroplast development and homeostasis. [ABSTRACT FROM AUTHOR]

Light is one of the indispensable elements that plants need in order to grow and develop. In particular, it is essential for inducing morphogenesis, such as suppression of hypocotyl elongation and cotyledon expansion, that plants undergo when they first emerge after germination. However, there is a lack of knowledge about the gene expression and, in particular, the translational levels that induce a response upon light exposure. We have investigated the translational expression of nuclear genes in Arabidopsis thaliana seedlings germinated in the dark and then exposed to blue monochromatic light. In this study, ribosome profiling analysis was performed in the blue-light-receptor mutant cry1cry2 and the light-signaling mutant hy5 to understand which signaling pathways are responsible for the changes in gene expression at the translational level after blue-light exposure. The analysis showed that the expression of certain chloroplast- and ribosome-related genes was up-regulated at the translational level in the wild type. However, in both mutants the translational up-regulation of ribosome-related genes was apparently compromised. This suggests that light signaling through photoreceptors and the HY5 transcription factor are responsible for translation of ribosome-related genes. To further understand the effect of photoreception by chloroplasts on nuclear gene expression, chloroplast function was inhibited by adding a photosynthesis inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and a carotenoid synthesis inhibitor, norflurazon. The results show that inhibition of chloroplast function did not lead to an increase in the expression of ribosome-related genes at the translational level. These results suggest that signals from both the nucleus and chloroplasts are required to activate translation of ribosome-related genes during blue-light reception. [ABSTRACT FROM AUTHOR]

Recent emerging evidence has shown that readthrough transcripts (RTs), including polycistronic mRNAs, are also transcribed in eukaryotes. However, the post-transcriptional regulation for these remains to be elucidated. Here, we identify 271 polycistronic RT-producing loci in Arabidopsis. Increased accumulation of RTs is detected in the nonsense-mediated mRNA decay (NMD)-deficient mutants compared with wild type, and the second open reading frames (ORFs) of bicistronic mRNAs are rarely translated in contrast to the first ORFs. Intergenic splicing (IS) events which occur between first and second genes are seen in 158 RTs. Splicing inhibition assays suggest that IS eliminates the chance of transcription termination at the polyadenylation sites of the first gene and promotes accumulation of RTs. These results indicate that RTs arise from genes whose transcription termination is relatively weak or attenuated by IS, but NMD selectively degrades them. Ultimately, this report presents a eukaryotic strategy for RNA metabolism. Readthrough transcripts (RTs) are identified in the genome of Arabidopsis using a long-read sequencing technology and structural and fate examination reveals that RTs are post-transcriptionally regulated by splicing, transcription termination and NMD. [ABSTRACT FROM AUTHOR]

Photoreceptors are conserved in green algae to land plants and regulate various developmental stages. In the ocean, blue light penetrates deeper than red light, and blue-light sensing is key to adapting to marine environments. Here, a search for blue-light photoreceptors in the marine metagenome uncover a chimeric gene composed of a phytochrome and a cryptochrome (Dualchrome1, DUC1) in a prasinophyte, Pycnococcus provasolii. DUC1 detects light within the orange/far-red and blue spectra, and acts as a dual photoreceptor. Analyses of its genome reveal the possible mechanisms of light adaptation. Genes for the light-harvesting complex (LHC) are duplicated and transcriptionally regulated under monochromatic orange/blue light, suggesting P. provasolii has acquired environmental adaptability to a wide range of light spectra and intensities. Blue light penetrates deeper than red light in ocean, thus blue light sensing is critical for adaptation to marine environments. Here, the authors report the genome of Pyconococcus provasolii and identify a chimeric dual orange/far-red and blue light receptor composed of a phytochrome and a cryptochrome by analyzing the marine metagenomes. [ABSTRACT FROM AUTHOR]

Plants have established different mechanisms to cope with environmental fluctuations and accordingly fine-tune their growth and development through the regulation of complex molecular networks. It is largely unknown how the network architectures change and what the key regulators in stress responses and plant growth are. Here, we investigated a complex, highly interconnected network of 20 Arabidopsis transcription factors (TFs) at the basis of leaf growth inhibition upon mild osmotic stress. We tracked the dynamic behavior of the stress-responsive TFs over time, showing the rapid induction following stress treatment, specifically in growing leaves. The connections between the TFs were uncovered using inducible overexpression lines and were validated with transient expression assays. This study resulted in the identification of a core network, composed of ERF6, ERF8, ERF9, ERF59, and ERF98, which is responsible for most transcriptional connections. The analyses highlight the biological function of this core network in environmental adaptation and its redundancy. Finally, a phenotypic analysis of loss-of-function and gain-of-function lines of the transcription factors established multiple connections between the stress-responsive network and leaf growth. [ABSTRACT FROM AUTHOR]

Membrane trafficking plays pivotal roles in many cellular processes including plant immunity. Here, we report the characterization of OsVAMP714, an intracellular SNARE protein, focusing on its role in resistance to rice blast disease caused by the fungal pathogen Magnaporthe oryzae. Disease resistance tests using OsVAMP714 knockdown and overexpressing rice plants demonstrated the involvement of OsVAMP714 in blast resistance. The overexpression of OsVAMP7111, whose product is highly homologous to OsVAMP714, did not enhance blast resistance to rice, implying a potential specificity of OsVAMP714 to blast resistance. OsVAMP714 was localized to the chloroplast in mesophyll cells and to the cellular periphery in epidermal cells of transgenic rice plant leaves. We showed that chloroplast localization is critical for the normal OsVAMP714 functioning in blast resistance by analyzing the rice plants overexpressing OsVAMP714 mutants whose products did not localize in the chloroplast. We also found that OsVAMP714 was located in the vacuolar membrane surrounding the invasive hyphae of M. oryzae. Furthermore, we showed that OsVAMP714 overexpression promotes leaf sheath elongation and that the first 19 amino acids, which are highly conserved between animal and plant VAMP7 proteins, are crucial for normal rice plant growths. Our studies imply that the OsVAMP714-mediated trafficking pathway plays an important role in rice blast resistance as well as in the vegetative growth of rice. [ABSTRACT FROM AUTHOR]

Screening of 40,000 Arabidopsis FOX (Full-length cDNA Over-eXpressor gene hunting system) lines expressing rice full-length cDNAs brings us to identify four cadmium (Cd)-tolerant lines, one of which carried OsREX1- S as a transgene. OsREX1- S shows the highest levels of identity to Chlamydomonas reinhardtii REX1- S (referred to as CrREX1- S, in which REX denotes Required for Excision) and to yeast and human TFB5s (RNA polymerase II transcription factor B5), both of which are components of the general transcription and DNA repair factor, TFIIH. Transient expression of OsREX1-S consistently localized the protein to the nucleus of onion cells. The newly generated transgenic Arabidopsis plants expressing OsREX1- S reproducibly displayed enhanced Cd tolerance, confirming that the Cd-tolerance of the initial identified line was conferred solely by OsREX1- S expression. Furthermore, transgenic Arabidopsis plants expressing OsREX1- S exhibited ultraviolet-B (UVB) tolerance by reducing the amounts of cyclobutane pyrimidine dimers produced by UVB radiation. Moreover, those transgenic OsREX1- S Arabidopsis plants became resistant to bleomycin (an inducer of DNA strand break) and mitomycin C (DNA intercalating activity), compared to wild type. Our results indicate that OsREX1-S renders host plants tolerant to Cd, UVB radiation, bleomycin and mitomycin C through the enhanced DNA excision repair. [ABSTRACT FROM AUTHOR]

Plant steroid hormones, brassinosteroids, are essential for growth, development and responses to environmental stresses in plants. Although BR signaling proteins are localized in many organelles, i.e., the plasma membrane, nuclei, endoplasmic reticulum and vacuole, the details regarding the BR signaling pathway from perception at the cellular membrane receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) to nuclear events include several steps. Brz (Brz220) is a specific inhibitor of BR biosynthesis. In this study, we used Brz-mediated chemical genetics to identify Brz- insensitive- long hypocotyls 2- 1D ( bil2- 1D). The BIL2 gene encodes a mitochondrial-localized DnaJ/Heat shock protein 40 (DnaJ/Hsp40) family, which is involved in protein folding. BIL2-overexpression plants ( BIL2- OX) showed cell elongation under Brz treatment, increasing the growth of plant inflorescence and roots, the regulation of BR-responsive gene expression and suppression against the dwarfed BRI1-deficient mutant. BIL2- OX also showed resistance against the mitochondrial ATPase inhibitor oligomycin and higher levels of exogenous ATP compared with wild-type plants. BIL2 participates in resistance against salinity stress and strong light stress. Our results indicate that BIL2 induces cell elongation during BR signaling through the promotion of ATP synthesis in mitochondria. [ABSTRACT FROM AUTHOR]

Plants have developed certain adaptive responses to environmental stresses that cause adverse effects on growth. To identify genes involved in the adaptive mechanisms, we constructed a large population of transgenic Arabidopsis expressing rice full-length cDNAs, and performed gain-of-function screening under high-salinity stress. In this study, we identified a rice R2R3-type MYB transcription factor gene, JAmyb, as a gene whose overexpression causes tolerance to high salinity. JAmyb overexpression in transgenic Arabidopsis improved tolerance to high-salinity stress during seed germination, seedling growth, and root elongation. In rice seedlings, JAmyb expression was induced by high-salinity and high-osmotic stresses and reactive oxygen species (ROS), suggesting that JAmyb is responsible for abiotic stress response. Microarray analysis showed that the overexpression of JAmyb stimulates the expression of several defense-associated genes, some of which have been predicted to be involved in osmotic adjustment, ROS removal, and ion homeostasis. Several transcription factors involved in the jasmonate (JA)-mediated stress response are also regulated by JAmyb. JAmyb has been reported to be associated with disease response. Our observations suggest that JAmyb plays a role in JA-mediated abiotic stress response in addition to biotic stress response in rice. [ABSTRACT FROM AUTHOR]

Cassava ( Manihot esculenta Crantz) is an important tropical root crop that provides food security and income generation for many farmers. Cassava ranks as the third most important source of calories in the tropics and provides sustenance to 800 million people worldwide. Cassava is also an efficient source of modified starch and bioethanol. In spite of the many advantages and high potential of cassava, the research community lacks functional genomic resources. Therefore, RIKEN has been working to establish a cassava functional genomics platform, which includes full-length cDNAs, DNA microarrays, transformation capabilities, and a searchable database in collaboration with the International Center for Tropical Agriculture (CIAT) of Colombia and Mahidol University of Thailand. In this review, we summarize the present status and future perspectives of our cassava functional genomic approaches. [ABSTRACT FROM AUTHOR]

The full-length cDNA over-expressing (FOX) gene hunting system is useful for genome-wide gain-of-function analysis. The screening of FOX lines requires a high-throughput metabolomic method that can detect a wide range of metabolites. Fourier transform-near-infrared (FT-NIR) spectroscopy in combination with the chemometric approach has been used to analyze metabolite fingerprints. Since FT-NIR spectroscopy can be used to analyze a solid sample without destructive extraction, this technique enables untargeted analysis and high-throughput screening focusing on the alteration of metabolite composition. We performed non-destructive FT-NIR-based fingerprinting to screen seed samples of 3000 rice- Arabidopsis FOX lines; the samples were obtained from transgenic Arabidopsis thaliana lines that overexpressed rice full-length cDNA. Subsequently, the candidate lines exhibiting alteration in their metabolite fingerprints were analyzed by gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) in order to assess their metabolite profiles. Finally, multivariate regression using orthogonal projections to latent structures (O2PLS) was used to elucidate the predictive metabolites obtained in FT-NIR analysis by integration of the datasets obtained from FT-NIR and GC-TOF/MS analyses. FT-NIR-based fingerprinting is a technically efficient method in that it facilitates non-destructive analysis in a high-throughput manner. Furthermore, with the integrated analysis used here, we were able to discover unique metabotypes in rice- Arabidopsis FOX lines; thus, this approach is beneficial for investigating the function of rice genes related to metabolism. [ABSTRACT FROM AUTHOR]

Plants have evolved intricate mechanisms to respond and adapt to a wide variety of biotic and abiotic stresses in their environment. The Arabidopsis DEAR1 (DREB and EAR motif protein 1; At3g50260) gene encodes a protein containing significant homology to the DREB1/CBF (dehydration-responsive element binding protein 1/C-repeat binding factor) domain and the EAR (ethylene response factor-associated amphiphilic repression) motif. We show here that DEAR1 mRNA accumulates in response to both pathogen infection and cold treatment. Transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) showed a dwarf phenotype and lesion-like cell death, together with constitutive expression of PR genes and accumulation of salicylic acid. DEAR1ox also showed more limited P. syringae pathogen growth compared to wild-type, consistent with an activated defense phenotype. In addition, transient expression experiments revealed that the DEAR1 protein represses DRE/CRT (dehydration-responsive element/C-repeat)-dependent transcription, which is regulated by low temperature. Furthermore, the induction of DREB1/CBF family genes by cold treatment was suppressed in DEAR1ox, leading to a reduction in freezing tolerance. These results suggest that DEAR1 has an upstream regulatory role in mediating crosstalk between signaling pathways for biotic and abiotic stress responses. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is one of the most important technologies currently available for the analysis of gene function. However, despite the development of various methods, it is still difficult to construct RNAi vectors for plants with the appropriate inverted repeat fragments to produce double-stranded RNA for knockdown experiments. To solve this problem we have developed an easy and simple method to make RNAi constructs using two long oligonucleotides consisting of partially complementary sequences without the need for PCR amplification and multiple cloning steps. CHS RNAi plants generated using this method showed yellow seed color and a decrease in antocyanin content—phenotypes typically observed in CHS loss-of-function mutants. Moreover, we demonstrated specific knockdown of both the PHYA and PHYB genes using a tandem RNAi construct. This method thus represents a powerful tool for gene knockdown in plants. [ABSTRACT FROM AUTHOR]

Environmental stresses limit plant growth and crop production worldwide. We attempted to isolate rice genes involved in conferring tolerance to environmental stresses by using a transgenic Arabidopsis population expressing full-length cDNAs of rice. Among these lines, a thermotolerant line, R08946, was detected. The rice cDNA inserted in R08946 encoded a NAC transcription factor, ONAC063. This protein was localized in the nucleus and showed transactivation activity at the C-terminus. ONAC063 expression was not induced by high-temperature but highly induced by high-salinity in rice roots. High-osmotic pressure and reactive oxygen species levels also induced ONAC063 expression. The seeds of ONAC063-expressing transgenic Arabidopsis showed enhanced tolerance to high-salinity and osmotic pressure. Microarray and real-time reverse transcription-polymerase chain reaction analyses showed upregulated expression of some salinity-inducible genes, including the amylase gene AMY1, in ONAC063-expressing transgenic Arabidopsis. Thus, ONAC063 may play an important role in eliciting responses to high-salinity stress. [ABSTRACT FROM AUTHOR]

Plant growth and crop yields are limited by high-temperature stresses. In this study, we attempted to isolate the rice genes responsible for high-temperature stress tolerance using a transformed Arabidopsis population expressing a full-length cDNA library of rice. From approximately 20,000 lines of transgenic Arabidopsis, we isolated a thermotolerant line, R04333, that could survive transient heat stress at the cotyledon stage. The rice cDNA inserted in R04333 encodes OsHsfA2e, a member of the heat stress transcription factors. The thermotolerant phenotype was observed in newly constructed transgenic Arabidopsis plants expressing OsHsfA2e. Among 5 A2-type HSF genes encoded in the rice genome, four genes, including OsHsfA2e, are induced by high temperatures in rice seedlings. The OsHsfA2e protein was localized to the nuclear region and exhibited transcription activation activity in the C-terminal region. Microarray analysis demonstrated that under unstressed conditions transgenic Arabidopsis overexpressing OsHsfA2e highly expressed certain stress-associated genes, including several classes of heat-shock proteins. The thermotolerant phenotype was observed not only in the cotyledons but also in rosette leaves, inflorescence stems and seeds. In addition, transgenic Arabidopsis exhibited tolerance to high-salinity stress. These observations suggest that the OsHsfA2e may be useful in molecular breeding designed to improve the environmental stress tolerance of crops. [ABSTRACT FROM AUTHOR]

In the original version of this Article, the label of the domain structure that has 612 amino acids presented in Fig. These authors contributed equally: Yuko Makita, Shigekatsu Suzuki, Keiji Fushimi, Setsuko Shimada. [Extracted from the article]

Extensive studies on the molecular mechanisms of vesicular trafficking have revealed that molecules involved in this cellular function are remarkably well conserved from yeast to higher plants. However, it is not clear at all how a variety of organisms maintain the individual divergent systems using the common machinery of vesicular traffic. We have been attempting to understand the roles and regulatory mechanisms of vesicular traffic in plants through the study of Rab/Ypt GTPases. Ara proteins are Rab/Ypt homologues of Arabidopsis, which are implicated in the regulation of vesicular traffic. Their biochemical properties are similar to those of the Rab/Ypt proteins from animal and yeast cells. The overexpression of ARA2 or ARA4 causes pleiotropic morphological abnormalities in the transgenic tobacco plants. The GTPase cycle of Ara proteins has to be strictly controlled for their proper functions. We have identified two classes of regulator molecules of Ara2 and Ara4. One is the GTPase activating protein (GAP), and the other is the GDP dissociation inhibitor (GDI). GAP has been identified as an activity accelerating the hydrolysis of GTP by Ara2 or Ara4. GDI (AtGDI1) has been isolated as a molecule interacting with Ara4 using a novel method for detecting interactions between foreign molecules in yeast. Further studies on the interacting molecules should unveil the regulatory system of and signal transduction pathway via Ara proteins. [ABSTRACT FROM AUTHOR]

The structure of the gene encoding the apoprotein of phytochrome B1 (PHYB1) in tomato has been determined from genomic and cDNA sequences. In contrast to PHYA, PHYB1 lacks an intron upstream of the first ATG. A single transcription start site was found by 5′ RACE at –116. Tomato PHYB1 spans 7 kb starting from the first ATG. The coding region is organized into four exons as for other angiosperm PHY. The deduced apoprotein consists of 1131 amino acids, with a molecular mass of 125.4 kDa. Tomato phytochrome B1 shares 78% and 74% identity with Arabidopsis phytochromes B and D, respectively. Along with the normally spliced full-length transcripts, sequences of reverse transcriptase-PCR clones revealed five types of alternative transcripts. Each type of alternative transcript was missing a considerable part of the coding region, including the chromophore-binding site. The four putative PHYB1 mutants in tomato, which are temporarily red-light insensitive (tri), were each confirmed to have a mutation in PHYB1. Each mutation arose from a different, single-base substitution. Allele tri1 is presumably a null because the mutation introduces a stop at codon 92. In tri3, val-238 is replaced by Phe. The importance of this valine residue is evidenced by the fact that the tri3 phenotype is as strong as that of tri1. Alleles tri2 and tri4 encode proteins truncated at their C-termini. The former lacks either 170 or 438 amino acids, depending upon which of two types of splicing occurs during transcript maturation, while the latter lacks 225. [ABSTRACT FROM AUTHOR]

Three kinds of cDNAs encoding 26S proteasome subunits have been cloned from spinach (Spinacia oleracea L.). These genes, designated as SOPSC8, SOPSC1 and SOPRS7, encode an α-type and a β-type subunit of the 20S catalytic core, and an ATPase subunit of the 19/22S regulatory complex, respectively. The deduced protein sequences showed high sequence similarities to other proteasome α- and β-type and ATPase subunit proteins. Southern blot analysis indicates that there are additional members of these dispersed proteasome families in the spinach genome. These three subunit genes are expressed simultaneously during germination and reach a maximum one day after sowing followed by a decline. The expression of these genes also increases during cotyledon senescence. [ABSTRACT FROM AUTHOR]

The fungal pathogen Rhizoctonia solani causes devastating diseases in hundreds of plant species. Among these, R. solani causes sheath blight, one of the three major diseases in rice. To date, few genes have been reported that confer resistance to R. solani. Here, rice-FOX Arabidopsis lines identified as having resistance to a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, and a fungal pathogen, Colletotrichum higginsianum were screened for disease resistance to R. solani. BROAD-SPECTRUM RESISTANCE2 (BSR2), a gene encoding an uncharacterized cytochrome P450 protein belonging to the CYP78A family, conferred resistance to R. solani in Arabidopsis. When overexpressed in rice, BSR2 also conferred resistance to two R. solani anastomosis groups. Both Arabidopsis and rice plants overexpressing BSR2 had slower growth and produced longer seeds than wild-type control plants. In contrast, BSR2-knockdown rice plants were more susceptible to R. solani and displayed faster growth and shorter seeds in comparison with the control. These results indicate that BSR2 is associated with disease resistance, growth rate and seed size in rice and suggest that its function is evolutionarily conserved in both monocot rice and dicot Arabidopsis. [ABSTRACT FROM AUTHOR]