The blue light signaling inhibitor 3-bromo-7-nitroindazole affects gene translation at the initial reception of blue light in young Arabidopsis seedlings.

Initial light reception after germination is a dramatic life event when a seedling starts proper morphogenesis. Blue light contains a range of light wavelengths that plants can perceive. A previous report suggested that the chemical compound 3-bromo-7-nitroindazole (3B7N) inhibits blue light-mediated suppression of hypocotyl elongation by physically interacting with the blue light receptor Cryptochrome 1 (CRY1). We previously examined changes of genome-wide gene expression in Arabidopsis seedlings germinated in the dark and then exposed to blue light by RNA-seq and Ribo-seq analyses. The expression of ribosome-related genes was translationally upregulated in response to the initial blue light exposure, depending on signals from both the nucleus and chloroplasts. Here, we re-analyzed our previous data and examined the effect of 3B7N treatment on changes in gene expression upon blue light exposure. The results showed that 3B7N negatively affected translation of ribosome-related genes and, interestingly, the effects were similar to not only those in cry1cry2 mutants but also plants under suppression of photosynthesis. We propose an apparent crosstalk between chloroplast function and blue light signaling.

BRZ-INSENSITIVE-LONG HYPOCOTYL8 inhibits kinase-mediated phosphorylation to regulate brassinosteroid signaling.

Glycogen synthase kinase 3 (GSK3) is an evolutionarily conserved serine/threonine protein kinase in eukaryotes. In plants, the GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) functions as a central signaling node through which hormonal and environmental signals are integrated to regulate plant development and stress adaptation. BIN2 plays a major regulatory role in brassinosteroid (BR) signaling and is critical for phosphorylating/inactivating BRASSINAZOLE-RESISTANT 1 (BZR1), also known as BRZ-INSENSITIVE-LONG HYPOCOTYL 1 (BIL1), a master transcription factor of BR signaling, but the detailed regulatory mechanism of BIN2 action has not been fully revealed. In this study, we identified BIL8 as a positive regulator of BR signaling and plant growth in Arabidopsis (Arabidopsis thaliana). Genetic and biochemical analyses showed that BIL8 is downstream of the BR receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and promotes the dephosphorylation of BIL1/BZR1. BIL8 interacts with and inhibits the activity of the BIN2 kinase, leading to the accumulation of dephosphorylated BIL1/BZR1. BIL8 suppresses the cytoplasmic localization of BIL1/BZR1, which is induced via BIN2-mediated phosphorylation. Our study reveals a regulatory factor, BIL8, that positively regulates BR signaling by inhibiting BIN2 activity.

ARGONAUTE1-binding Tudor domain proteins function in small interfering RNA production for RNA-directed DNA methylation.

Transposable elements (TEs) contribute to plant evolution, development, and adaptation to environmental changes, but the regulatory mechanisms are largely unknown. RNA-directed DNA methylation (RdDM) is 1 TE regulatory mechanism in plants. Here, we identified that novel ARGONAUTE 1 (AGO1)-binding Tudor domain proteins Precocious dissociation of sisters C/E (PDS5C/E) are involved in 24-nt siRNA production to establish RdDM on TEs in Arabidopsis thaliana. PDS5 family proteins are subunits of the eukaryote-conserved cohesin complex. However, the double mutant lacking angiosperm-specific subfamily PDS5C and PDS5E (pds5c/e) exhibited different developmental phenotypes and transcriptome compared with those of the double mutant lacking eukaryote-conserved subfamily PDS5A and PDS5B (pds5a/b), suggesting that the angiosperm-specific PDS5C/E subfamily has a unique function in angiosperm plants. Proteome and imaging analyses revealed that PDS5C/E interact with AGO1. The pds5c/e double mutant had defects in 24-nt siRNA accumulation and CHH DNA methylation on TEs. In addition, some lncRNAs that accumulated in the pds5c/e mutant were targeted by AGO1-loading 21-nt miRNAs and 21-nt siRNAs. These results indicate that PDS5C/E and AGO1 participate in 24-nt siRNA production for RdDM in the cytoplasm. These findings indicate that angiosperm plants evolved a new regulator, the PDS5C/E subfamily, to control the increase in TEs during angiosperm evolution.

DNA- and Selectable-Marker-Free Genome-Editing System Using Zygotes from Recalcitrant Maize Inbred B73.

Genome-editing tools such as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system have become essential tools for increasing the efficiency and accuracy of plant breeding. Using such genome-editing tools on maize, one of the most important cereal crops of the world, will greatly benefit the agriculture and the mankind. Conventional genome-editing methods typically used for maize involve insertion of a Cas9-guide RNA expression cassette and a selectable marker in the genome DNA; however, using such methods, it is essential to eliminate the inserted DNA cassettes to avoid legislative concerns on gene-modified organisms. Another major hurdle for establishing an efficient and broadly applicable DNA-free genome-editing system for maize is presented by recalcitrant genotypes/cultivars, since cell/tissue culture and its subsequent regeneration into plantlets are crucial for producing transgenic and/or genome-edited maize. In this study, to establish a DNA-free genome-editing system for recalcitrant maize genotypes/cultivars, Cas9-gRNA ribonucleoproteins were directly delivered into zygotes isolated from the pollinated flowers of the maize-B73 cultivar. The zygotes successfully developed and were regenerated into genome-edited plantlets by co-culture with phytosulfokine, a peptide phytohormone. The method developed herein made it possible to obtain DNA- and selectable-marker-free genome-edited recalcitrant maize genotypes/cultivars with high efficiency. This method can advance the molecular breeding of maize and other important cereals, regardless of their recalcitrant characteristics.

Arabidopsis transcriptomic analysis reveals cesium inhibition of root growth involves abscisic acid signaling.

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.

BPG4 regulates chloroplast development and homeostasis by suppressing GLK transcription factors and involving light and brassinosteroid signaling.

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.

Advances in single-cell metabolomics to unravel cellular heterogeneity in plant biology.

Single-cell metabolomics is a powerful tool that can reveal cellular heterogeneity and can elucidate the mechanisms of biological phenomena in detail. It is a promising approach in studying plants, especially when cellular heterogeneity has an impact on different biological processes. In addition, metabolomics, which can be regarded as a detailed phenotypic analysis, is expected to answer previously unrequited questions which will lead to expansion of crop production, increased understanding of resistance to diseases, and in other applications as well. In this review, we will introduce the flow of sample acquisition and single-cell techniques to facilitate the adoption of single-cell metabolomics. Furthermore, the applications of single-cell metabolomics will be summarized and reviewed.

Translational activation of ribosome-related genes at initial photoreception is dependent on signals derived from both the nucleus and the chloroplasts in Arabidopsis thaliana.

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.

Intergenic splicing-stimulated transcriptional readthrough is suppressed by nonsense-mediated mRNA decay in Arabidopsis.

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.

BRZ-INSENSITIVE-PALE GREEN 1 is encoded by chlorophyll biosynthesis enzyme gene that functions in the downstream of brassinosteroid signaling.

Brassinosteroids (BRs), a kind of phytohormone, have various biological activities such as promoting plant growth, increasing stress resistance, and chloroplast development. Though BRs have been known to have physiological effects on chloroplast, the detailed mechanism of chloroplast development and chlorophyll biosynthesis in BR signaling remains unknown. Here we identified a recessive pale green Arabidopsis mutant, Brz-insensitive-pale green1 (bpg1), which was insensitive to promoting of greening by BR biosynthesis-specific inhibitor Brz in the light. BPG1 gene encoded chlorophyll biosynthesis enzyme, 3, 8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), and bpg1 accumulated divinyl chlorophylls. Chloroplast development was suppressed in bpg1. Brz dramatically increased the expression of chlorophyll biosynthesis enzyme genes, including BPG1. These results suggest that chlorophyll biosynthesis enzymes are regulated by BR signaling in the aspect of gene expression and BPG1 plays an important role in regulating chloroplast development.

A collection of inducible transcription factor-glucocorticoid receptor fusion lines for functional analyses in Arabidopsis thaliana.

Arabidopsis possesses approximately 2000 transcription factors (TFs) in its genome. They play pivotal roles in various biological processes but analysis of their function has been hampered by the overlapping nature of their activities. To uncover clues to their function, we generated inducible TF lines using glucocorticoid receptor (GR) fusion techniques in Arabidopsis. These TF-GR lines each express one of 1255 TFs as a fusion with the GR gene. An average 14 lines of T2 transgenic TF-GR lines were generated for each TF to monitor their function. To evaluate these transcription lines, we induced the TF-GR lines of phytochrome-interacting factor 4, which controls photomorphogenesis, with synthetic glucocorticoid dexamethasone. These phytochrome-interacting factor 4-GR lines showed the phenotype described in a previous report. We performed screening of the other TF-GR lines for TFs involved in light signaling under blue and far-red light conditions and identified 13 novel TF candidates. Among these, we found two lines showing higher anthocyanin accumulation under light conditions and we examined the regulating genes. These results indicate that the TF-GR lines can be used to dissect functionally redundant genes in plants and demonstrate that the TF-GR line collection can be used as an effective tool for functional analysis of TFs.

Tracking metabolites at single-cell resolution reveals metabolic dynamics during plant mitosis.

Analyzing only one cell allows the changes and characteristics of intracellular metabolites during the chromosome segregation process to be precisely captured and mitotic sub-phases to be dissected at the metabolite level.

Transformation of Jatropha curcas L. for production of larger seeds and increased amount of biodiesel.

The development of green energy is important to mitigate global warming. Jatropha (Jatropha curcas L.) is a promising candidate for the production of alternative biofuel, which could reduce the burden on the Earth's resources. Jatropha seeds contain a large quantity of lipids that can be used to produce biofuel, and the rest of the plant has many other uses. Currently, techniques for plant genetic transformation are extensively employed to study, create, and improve the specific characteristics of the target plant. Successful transformation involves the alteration of plants and their genetic materials. The aim of this study was to generate Jatropha plants that can support biofuel production by increasing their seed size using genes found via the rice FOX-hunting system. The present study improved previous protocols, enabling the production of transgenic Jatropha in two steps: the first step involved using auxins and dark incubation to promote root formation in excised shoots and the second step involved delaying the timing of antibiotic selection in the cultivation medium. Transgenic plants were subjected to PCR analysis; the transferred gene expression was confirmed via RT-PCR and the ploidy level was investigated. The results suggest that the genes associated with larger seed size in Arabidopsis thaliana, which were found using the rice FOX-hunting system, produce larger seeds in Jatropha.

Cryptic promoter activation occurs by at least two different mechanisms in the Arabidopsis genome.

In gene-trap screening of plant genomes, promoterless reporter constructs are often expressed without trapping of annotated gene promoters. The molecular basis of this phenomenon, which has been interpreted as the trapping of cryptic promoters, is poorly understood. Here, we found that cryptic promoter activation occurs by at least two different mechanisms using Arabidopsis gene-trap lines in which a firefly luciferase (LUC) open reading frame (ORF) without an apparent promoter sequence was expressed from intergenic regions: one mechanism is 'cryptic promoter capturing', in which the LUC ORF captured pre-existing promoter-like chromatin marked by H3K4me3 and H2A.Z, and the other is 'promoter de novo origination', in which the promoter chromatin was newly formed near the 5' end of the inserted LUC ORF. The latter finding raises a question as to how the inserted LUC ORF sequence is involved in this phenomenon. To examine this, we performed a model experiment with chimeric LUC genes in transgenic plants. Using Arabidopsis psaH1 promoter-LUC constructs, we found that the functional core promoter region, where transcription start sites (TSSs) occur, cannot simply be determined by the upstream nor core promoter sequences; rather, its positioning proximal to the inserted LUC ORF sequence was more critical. This result suggests that the insertion of the coding sequence alters the local distribution of TSSs in the plant genome. The possible impact of the two types of cryptic promoter activation mechanisms on plant genome evolution and endosymbiotic gene transfer is discussed.

Identification of a dual orange/far-red and blue light photoreceptor from an oceanic green picoplankton.

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.

Two types of bHLH transcription factor determine the competence of the pericycle for lateral root initiation.

The molecular basis of the competence of the pericycle cell to initiate lateral root primordium formation is totally unknown. Here, we report that in Arabidopsis, two types of basic helix-loop-helix (bHLH) transcription factors, named PERICYCLE FACTOR TYPE-A (PFA) proteins and PERICYCLE FACTOR TYPE-B (PFB) proteins, govern the competence of pericycle cells to initiate lateral root primordium formation. Overexpression of PFA genes confers hallmark pericycle characteristics, including specific marker gene expression and auxin-induced cell division, and multiple loss-of-function mutations in PFA genes or the repression of PFB target genes results in the loss of this specific pericycle function. PFA and PFB proteins physically interact and are under mutual- and self-regulation, forming a positive feedback loop. This study unveils the transcriptional regulatory system that determines pericycle participation in lateral root initiation.

Method for Phenotypic Chemical Screening to Identify Cryptochrome Inhibitors.

After germination, plants determine their morphogenesis, such as hypocotyl elongation and cotyledon opening, by responding to various wavelengths of light (photomorphogenesis). Cryptochrome is a blue-light photoreceptor that controls de-etiolation, stomatal opening and closing, flowering time, and shade avoidance. Successful incorporation of these phenotypes as indicators into a chemical screening system results in faster selection of candidate compounds. Here, we describe phenotypic screening for the blue-light response of Arabidopsis thaliana seedling and the resulting process that clarifies that the compound obtained in the screening is an inhibitor of cryptochromes.

Regulatory Potential of bHLH-Type Transcription Factors on the Road to Rubber Biosynthesis in Hevea brasiliensis.

Natural rubber is the main component of latex obtained from laticifer cells of Hevea brasiliensis. For improving rubber yield, it is essential to understand the genetic molecular mechanisms responsible for laticifer differentiation and rubber biosynthesis. Jasmonate enhances both secondary laticifer differentiation and rubber biosynthesis. Here, we carried out time-course RNA-seq analysis in suspension-cultured cells treated with methyljasmonic acid (MeJA) to characterize the gene expression profile. Gene Ontology (GO) analysis showed that the term "cell differentiation" was enriched in upregulated genes at 24 hours after treatment, but inversely, the term was enriched in downregulated genes at 5 days, indicating that MeJA could induce cell differentiation at an early stage of the response. Jasmonate signaling is activated by MYC2, a basic helix-loop-helix (bHLH)-type transcription factor (TF). The aim of this work was to find any links between transcriptomic changes after MeJA application and regulation by TFs. Using an in vitro binding assay, we traced candidate genes throughout the whole genome that were targeted by four bHLH TFs: Hb_MYC2-1, Hb_MYC2-2, Hb_bHLH1, and Hb_bHLH2. The latter two are highly expressed in laticifer cells. Their physical binding sites were found in the promoter regions of a variety of other TF genes, which are differentially expressed upon MeJA exposure, and rubber biogenesis-related genes including SRPP1 and REF3. These studies suggest the possibilities that Hb_MYC2-1 and Hb_MYC2-2 regulate cell differentiation and that Hb_bHLH1 and Hb_bHLH2 promote rubber biosynthesis. We expect that our findings will help to increase natural rubber yield through genetic control in the future.

Light Activates Brassinosteroid Biosynthesis to Promote Hook Opening and Petiole Development in Arabidopsis thaliana.

Although brassinosteroids (BRs) have been proposed to be negative regulators of photomorphogenesis, their physiological role therein has remained elusive. We studied light-induced photomorphogenic development in the presence of the BR biosynthesis inhibitor, brassinazole (Brz). Hook opening was inhibited in the presence of Brz; this inhibition was reversed in the presence of brassinolide (BL). Hook opening was accompanied by cell expansion on the inner (concave) side of the hook. This cell expansion was inhibited in the presence of Brz but was restored upon the addition of BL. We then evaluated light-induced organ-specific expression of three BR biosynthesis genes, DWF4, BR6ox1 and BR6ox2, and a BR-responsive gene, SAUR-AC1, during the photomorphogenesis of Arabidopsis. Expression of these genes was induced, particularly in the hook region, in response to illumination. The induction peaked after 3 h of light exposure and preceded hook opening. Phytochrome-deficient mutants, hy1, hy2 and phyAphyB, and a light-signaling mutant, hy5, were defective in light-induced expression of BR6ox1, BR6ox2 and SAUR-AC1. Light induced both expression of BR6ox genes and petiole development. Petiole development was inhibited in the presence of Brz. Our results largely contradict the early view that BRs are negative regulators of photomorphogenesis. Our data collectively suggest that light activates the expression of BR biosynthesis genes in the hook region via a phytochrome-signaling pathway and HY5 and that BR biosynthesis is essential for hook opening and petiole development during photomorphogenesis.