Unraveling multifaceted contributions of small regulatory RNAs to photomorphogenic development in Arabidopsis.

BACKGROUND: Post-transcriptional control of gene expression mediated by small regulatory RNAs (sRNAs) is vital for growth and development of diverse organisms. The biogenesis of sRNAs is regulated by both positive and negative regulators known to regulate photomorphogenic development. Two microRNAs (miRNAs), miR157 and miR319, also regulate photomorphogenesis. However, genome-wide profiling of sRNAs and their regulation of target genes during photomorphogenesis has been missing. We provide a comprehensive view of sRNA-controlled gene expression in this developmental process. RESULTS: By profiling sRNAs and the 5' ends of degraded mRNAs during the first 24 h of photomorphogenic development in Arabidopsis, we identified 335 sRNA-mediated mRNA cleavage events in de-etiolating seedlings. These cleavage events are primarily resulted from actions of highly expressed miRNAs and irrelevant to the abundance of target mRNAs. In the light, the expression of the slicer protein gene ARGONAUTE1 in the miRNA functioning pathway could be fine-tuned by miRNA168a/b. We also found that miR396a/b positively regulates de-etiolation by suppressing GROWTH REGULATING FACTORs. Our results suggest that the miRNAs are required to tune down the target mRNAs and regulate photomorphogenesis. CONCLUSION: sRNAs may have a broad impact on gene expression regulation for optimized photomorphogenic development. With both positive and negative regulators under the control of sRNAs, young Arabidopsis seedlings can have a timely but not exaggerated developmental adaptation to light.

HUA ENHANCER1 is involved in posttranscriptional regulation of positive and negative regulators in Arabidopsis photomorphogenesis.

Light regulates growth and developmental processes in plants via global transcriptome adjustment, translational control, and multilayered posttranslational modification of proteins. The transcriptional activation and repression of light-responsive genes has been well documented; however, the impact of posttranscriptional regulation on conveying light signals has been less addressed. Here, we examined whether optimal photomorphogenesis in Arabidopsis thaliana requires the proper biogenesis of small regulatory RNAs that play pivotal roles in the posttranscriptional regulation of gene expression. Arabidopsis carrying a mutation in HUA ENHANCER1 (HEN1), required for stabilization of small regulatory RNAs, showed defects in multiple aspects of photomorphogenic and skotomorphogenic development. HEN1 negatively regulated Arabidopsis photomorphogenesis. Light-activated HEN1 expression depended on the photoreceptors phytochrome A (phyA), phyB, cryptochrome 1 (cry1), and cry2 and key transcriptional regulators ELONGATED HYPOCOTYL5 (HY5) and HY5-HOMOLOG. We also demonstrate the involvement of the small regulatory RNAs miR157d and miR319 in modulating the expression of a positive regulator, HY5, and negative regulators TEOSINTE BRANCHED1, CYCLOIDEA AND PCF family proteins, respectively, for optimal photomorphogenic development in Arabidopsis.

Translational landscape of photomorphogenic Arabidopsis.

Translational control plays a vital role in regulating gene expression. To decipher the molecular basis of translational regulation in photomorphogenic Arabidopsis thaliana, we adopted a ribosome profiling method to map the genome-wide positions of translating ribosomes in Arabidopsis etiolated seedlings in the dark and after light exposure. We found that, in Arabidopsis, a translating ribosome protects an ~30-nucleotide region and moves in three-nucleotide periodicity, characteristics also observed in Saccharomyces cerevisiae and mammals. Light enhanced the translation of genes involved in the organization and function of chloroplasts. Upstream open reading frames initiated by ATG but not CTG mediated translational repression of the downstream main open reading frame. Also, we observed widespread translational repression of microRNA target genes in both light- and dark-grown Arabidopsis seedlings. This genome-wide characterization of transcripts undergoing translation at the nucleotide-resolution level reveals that a combination of multiple translational mechanisms orchestrates and fine-tunes the translation of diverse transcripts in plants with environmental responsiveness.

Family Members Additively Repress the Ectopic Expression of BASIC PENTACYSTEINE3 to Prevent Disorders in Arabidopsis Circadian Vegetative Development.

BARLEY B-RECOMBINANT/BASIC PENTACYSTEINE (BBR/BPC) family members are plant-specific GAGA-motif binding factors (GAFs) controlling multiple developmental processes of growth and propagation. BPCs recruit histone remodeling factors for transcriptional repression of downstream targets. It has been revealed that BPCs have an overlapping and antagonistic relationship in regulating development. In this study, we showed disturbances interfering with the homeostasis of BPC expressions impede growth and development. The ectopic expression of BPC3 results in the daily growth defect shown by higher-order bpc mutants. Oscillations of multiple circadian clock genes are phase-delayed in the quadruple mutant of bpc1 bpc2 bpc4 bpc6 (bpc1,2,4,6). By introducing the overexpression of BPC3 into wild-type Arabidopsis, we found that BPC3 is a repressor participating in its repression and repressing multiple regulators essential to the circadian clock. However, the induction of BPC3 overexpression did not fully replicate clock defects shown by the quadruple mutant, indicating that in addition to the BPC3 antagonization, BPC members also cofunction in the circadian clock regulation. A leaf edge defect similar to that shown by bpc1,2,4,6 is also observed under BPC3 induction, accompanied by repression of a subset of TCPs required for the edge formation. This proves that BPC3 is a repressor that must be confined during the vegetative phase. Our findings demonstrate that BPCs form a meticulous repressor network for restricting their repressive functions to molecular mechanisms controlling plant growth and development.

Nondestructive circadian profiling of starch content in fresh intact Arabidopsis leaf with two-photon fluorescence and second-harmonic generation imaging.

Plant chloroplasts conduct photosynthesis to convert solar energy into sugars for the carbon source essential for cell living and growth during the day. One fraction of photosynthetic products is stored in chloroplasts by forming starch granules to continue the provision of carbon energy during the night. Currently, profiling the starch temporal pattern requires either: (i) sacrificing the leaves, or (ii) generating transgenic plants at the risk of changing the metabolisms by incorporating a genetically modified granule-bound starch synthase (GBSS). In this paper, we demonstrated a nondestructive method using two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging to quantify starch granules within chloroplasts of fresh intact leaves across a day-night cycle. We did so using two Arabidopsis lines having normal and excess starch contents: wild-type (Columbia-0) and starch excess 1 (sex1). The starch granules were visualized by SHG imaging, while the chloroplasts in mesophyll cells were visualized by TPF imaging. Our results provided micron scale spatial resolution of starch distribution within leaves and showed starch circadian patterns consistent with those profiled by enzymatic assays in previous studies. We demonstrated that TPF-SHG imaging is a potential tool for revealing the real-time heterogeneity of starch circadian rhythm in leaf cells, without the need for destructive sample preparation.

Investigating the Viral Suppressor HC-Pro Inhibiting Small RNA Methylation through Functional Comparison of HEN1 in Angiosperm and Bryophyte.

In plants, HEN1-facilitated methylation at 3' end ribose is a critical step of small-RNA (sRNA) biogenesis. A mutant of well-studied Arabidopsis HEN1 (AtHEN1), hen1-1, showed a defective developmental phenotype, indicating the importance of sRNA methylation. Moreover, Marchantia polymorpha has been identified to have a HEN1 ortholog gene (MpHEN1); however, its function remained unfathomed. Our in vivo and in vitro data have shown MpHEN1 activity being comparable with AtHEN1, and their substrate specificity towards duplex microRNA (miRNA) remained consistent. Furthermore, the phylogenetic tree and multiple alignment highlighted the conserved molecular evolution of the HEN1 family in plants. The P1/HC-Pro of the turnip mosaic virus (TuMV) is a known RNA silencing suppressor and inhibits HEN1 methylation of sRNAs. Here, we report that the HC-Pro physically binds with AtHEN1 through FRNK motif, inhibiting HEN1's methylation activity. Moreover, the in vitro EMSA data indicates GST-HC-Pro of TuMV lacks sRNA duplex-binding ability. Surprisingly, the HC-Pro also inhibits MpHEN1 activity in a dosage-dependent manner, suggesting the possibility of interaction between HC-Pro and MpHEN1 as well. Further investigations on understanding interaction mechanisms of HEN1 and various HC-Pros can advance the knowledge of viral suppressors.

Starch synthesis in Arabidopsis is achieved by spatial cotranscription of core starch metabolism genes.

Starch synthesis and degradation require the participation of many enzymes, occur in both photosynthetic and nonphotosynthetic tissues, and are subject to environmental and developmental regulation. We examine the distribution of starch in vegetative tissues of Arabidopsis (Arabidopsis thaliana) and the expression of genes encoding core enzymes for starch synthesis. Starch is accumulated in plastids of epidermal, mesophyll, vascular, and root cap cells but not in root proper cells. We also identify cells that can synthesize starch heterotrophically in albino mutants. Starch synthesis in leaves is regulated by developmental stage and light. Expression of gene promoter-beta-glucuronidase fusion constructs in transgenic seedlings shows that starch synthesis genes are transcriptionally active in cells with starch synthesis and are inactive in root proper cells except the plastidial phosphoglucose isomerase. In addition, ADG2 (for ADPG PYROPHOSPHORYLASE2) is not required for starch synthesis in root cap cells. Expression profile analysis reveals that starch metabolism genes can be clustered into two sets based on their tissue-specific expression patterns. Starch distribution and expression pattern of core starch synthesis genes are common in Arabidopsis and rice (Oryza sativa), suggesting that the regulatory mechanism for starch metabolism genes may be conserved evolutionarily. We conclude that starch synthesis in Arabidopsis is achieved by spatial coexpression of core starch metabolism genes regulated by their promoter activities and is fine-tuned by cell-specific endogenous and environmental controls.

LWD-TCP complex activates the morning gene CCA1 in Arabidopsis.

A double-negative feedback loop formed by the morning genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) and the evening gene TIMING OF CAB EXPRESSION1 (TOC1) contributes to regulation of the circadian clock in Arabidopsis. A 24-h circadian cycle starts with the peak expression of CCA1 at dawn. Although CCA1 is targeted by multiple transcriptional repressors, including PSEUDO-RESPONSE REGULATOR9 (PRR9), PRR7, PRR5 and CCA1 HIKING EXPEDITION (CHE), activators of CCA1 remain elusive. Here we use mathematical modelling to infer a co-activator role for LIGHT-REGULATED WD1 (LWD1) in CCA1 expression. We show that the TEOSINTE BRANCHED 1-CYCLOIDEA-PCF20 (TCP20) and TCP22 proteins act as LWD-interacting transcriptional activators. The concomitant binding of LWD1 and TCP20/TCP22 to the TCP-binding site in the CCA1 promoter activates CCA1. Our study reveals activators of the morning gene CCA1 and provides an action mechanism that ensures elevated expression of CCA1 at dawn to sustain a robust clock.