Frontiers in plant RNA research in ICAR2023: from lab to innovative agriculture.

The recent growth in global warming, soil contamination, and climate instability have widely disturbed ecosystems, and will have a significant negative impact on the growth of plants that produce grains, fruits and woody biomass. To conquer this difficult situation, we need to understand the molecular bias of plant environmental responses and promote development of new technologies for sustainable maintenance of crop production. Accumulated molecular biological data have highlighted the importance of RNA-based mechanisms for plant stress responses. Here, we report the most advanced plant RNA research presented in the 33rd International Conference on Arabidopsis Research (ICAR2023), held as a hybrid event on June 5-9, 2023 in Chiba, Japan, and focused on "Arabidopsis for Sustainable Development Goals". Six workshops/concurrent sessions in ICAR2023 targeted plant RNA biology, and many RNA-related topics could be found in other sessions. In this meeting report, we focus on the workshops/concurrent sessions targeting RNA biology, to share what is happening now at the forefront of plant RNA research.

Modeling alternative translation initiation sites in plants reveals evolutionarily conserved cis-regulatory codes in eukaryotes.

mRNA translation relies on identifying translation initiation sites (TISs) in mRNAs. Alternative TISs are prevalent across plant transcriptomes, but the mechanisms for their recognition are unclear. Using ribosome profiling and machine learning, we developed models for predicting alternative TISs in the tomato (Solanum lycopersicum). Distinct feature sets were predictive of AUG and nonAUG TISs in 5' untranslated regions and coding sequences, including a novel CU-rich sequence that promoted plant TIS activity, a translational enhancer found across dicots and monocots, and humans and viruses. Our results elucidate the mechanistic and evolutionary basis of TIS recognition, whereby cis-regulatory RNA signatures affect start site selection. The TIS prediction model provides global estimates of TISs to discover neglected protein-coding genes across plant genomes. The prevalence of cis-regulatory signatures across plant species, humans, and viruses suggests their broad and critical roles in reprogramming the translational landscape.

Translation initiation at AUG and non-AUG triplets in plants.

In plants and other eukaryotes, precise selection of translation initiation site (TIS) on mRNAs shapes the proteome in response to cellular events or environmental cues. The canonical translation of mRNAs initiates at a 5' proximal AUG codon in a favorable context. However, the coding and non-coding regions of plant genomes contain numerous unannotated alternative AUG and non-AUG TISs. Determining how and why these unexpected and prevalent TISs are activated in plants has emerged as an exciting research area. In this review, we focus on the selection of plant TISs and highlight studies that revealed previously unannotated TISs used in vivo via comparative genomics and genome-wide profiling of ribosome positioning and protein N-terminal ends. The biological signatures of non-AUG TIS-initiated open reading frames (ORFs) in plants are also discussed. We describe what is understood about cis-regulatory RNA elements and trans-acting eukaryotic initiation factors (eIFs) in the site selection for translation initiation by featuring the findings in plants along with supporting findings in non-plant species. The prevalent, unannotated TISs provide a hidden reservoir of ORFs that likely help reshape plant proteomes in response to developmental or environmental cues. These findings underscore the importance of understanding the mechanistic basis of TIS selection to functionally annotate plant genomes, especially for crops with large genomes.

Translation initiation landscape profiling reveals hidden open-reading frames required for the pathogenesis of tomato yellow leaf curl Thailand virus.

Plant viruses with densely packed genomes employ noncanonical translational strategies to increase the coding capacity for viral function. However, the diverse translational strategies used make it challenging to define the full set of viral genes. Here, using tomato yellow leaf curl Thailand virus (TYLCTHV, genus Begomovirus) as a model system, we identified genes beyond the annotated gene sets by experimentally profiling in vivo translation initiation sites (TISs). We found that unanticipated AUG TISs were prevalent and determined that their usage involves alternative transcriptional and/or translational start sites and is associated with flanking mRNA sequences. Specifically, two downstream in-frame TISs were identified in the viral gene AV2. These TISs were conserved in the begomovirus lineage and led to the translation of different protein isoforms localized to cytoplasmic puncta and at the cell periphery, respectively. In addition, we found translational evidence of an unexplored gene, BV2. BV2 is conserved among TYLCTHV isolates and localizes to the endoplasmic reticulum and plasmodesmata. Mutations of AV2 isoforms and BV2 significantly attenuated disease symptoms in tomato (Solanum lycopersicum). In conclusion, our study pinpointing in vivo TISs untangles the coding complexity of a plant viral genome and, more importantly, illustrates the biological significance of the hidden open-reading frames encoding viral factors for pathogenicity.

Evolutionarily conserved hierarchical gene regulatory networks for plant salt stress response.

Plant cells constantly alter their gene expression profiles to respond to environmental fluctuations. These continuous adjustments are regulated by multi-hierarchical networks of transcription factors. To understand how such gene regulatory networks (GRNs) have stabilized evolutionarily while allowing for species-specific responses, we compare the GRNs underlying salt response in the early-diverging and late-diverging plants Marchantia polymorpha and Arabidopsis thaliana. Salt-responsive GRNs, constructed on the basis of the temporal transcriptional patterns in the two species, share common trans-regulators but exhibit an evolutionary divergence in cis-regulatory sequences and in the overall network sizes. In both species, WRKY-family transcription factors and their feedback loops serve as central nodes in salt-responsive GRNs. The divergent cis-regulatory sequences of WRKY-target genes are probably associated with the expansion in network size, linking salt stress to tissue-specific developmental and physiological responses. The WRKY modules and highly linked WRKY feedback loops have been preserved widely in other plants, including rice, while keeping their binding-motif sequences mutable. Together, the conserved trans-regulators and the quickly evolving cis-regulatory sequences allow salt-responsive GRNs to adapt over a long evolutionary timescale while maintaining some consistent regulatory structure. This strategy may benefit plants as they adapt to changing environments.

Prevalence of alternative AUG and non-AUG translation initiators and their regulatory effects across plants.

Translation initiation is a key step determining protein synthesis. Studies have uncovered a number of alternative translation initiation sites (TISs) in mammalian mRNAs and showed their roles in reshaping the proteome. However, the extent to which alternative TISs affect gene expression across plants remains largely unclear. Here, by profiling initiating ribosome positions, we globally identified in vivo TISs in tomato and Arabidopsis and found thousands of genes with more than one TIS. Of the identified TISs, >19% and >20% were located at unannotated AUG and non-AUG sites, respectively. CUG and ACG were the most frequently observed codons at non-AUG TISs, a phenomenon also found in mammals. In addition, although alternative TISs were usually found in both orthologous genes, the TIS sequences were not conserved, suggesting the conservation of alternative initiation mechanisms but flexibility in using TISs. Unlike upstream AUG TISs, the presence of upstream non-AUG TISs was not correlated with the translational repression of main open reading frames, a pattern observed across plants. Also, the generation of proteins with diverse N-terminal regions through the use of alternative TISs contributes to differential subcellular localization, as mutating alternative TISs resulted in the loss of organelle localization. Our findings uncovered the hidden coding potential of plant genomes and, importantly, the constraint and flexibility of translational initiation mechanisms in the regulation of gene expression across plant species.

A HemK class glutamine-methyltransferase is involved in the termination of translation and essential for iron homeostasis in Arabidopsis.

Iron (Fe) transport and utilization are controlled by Fe-dependent transcriptional cascades. Many genes participate in these processes, transcriptionally controlled by Fe-status. Thorough knowledge of the translational check-points is lacking. We identified a non-response to Fe-deficiency1-1 (nrf1-1) mutant of Arabidopsis thaliana, which displayed a hypersensitive phenotype under Fe-deficient conditions. By mapping nrf1-1, we found that the AT3G13440 locus encoding a HemK methyltransferase is responsible for the phenotype. Analyses of ProUBQ10:NRF1CDS overexpression nrf1-1 lines and a T-DNA insertion mutant nrf1-2, confirmed that loss-of-function of NRF1 results in enhanced Fe-starvation-sensitivity. NRF1 is required for the proper expression of the majority of Fe-deficiency-inducible (FDI) genes. The nrf1 mutants accumulated more polysomes in the roots, due to stalled ribosomes on several transcripts. Ribosome-footprint (RF) mapping revealed that ribosomes are stalled at a stop codon that amplified the stalling of trailing ribosomes. We detected higher RF levels in many FDI transcripts in nrf1-2. Our study demonstrates the requirement of NRF1 for an accurate termination of protein synthesis essential not only for a precise iron homeostasis, but also cellular ion balance. NRF1 is also important for normal growth and development. A check-point that fine-tunes peptide release in plants is uncovered.

TOR and RPS6 transmit light signals to enhance protein translation in deetiolating Arabidopsis seedlings.

Deetiolation is an essential developmental process transforming young plant seedlings into the vegetative phase with photosynthetic activities. Light signals initiate this important developmental process by triggering massive reprogramming of the transcriptome and translatome. Compared with the wealth of knowledge of transcriptional regulation, the molecular mechanism underlying this light-triggered translational enhancement remains unclear. Here we show that light-enhanced translation is orchestrated by a light perception and signaling pathway composed of photoreceptors, CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1), the phytohormone auxin, target of rapamycin (TOR), and ribosomal protein S6 (RPS6). In deetiolating Arabidopsis seedlings, photoreceptors, including phytochrome A and cryptochromes, perceive far-red and blue light to inactivate the negative regulator COP1, which leads to activation of the auxin pathway for TOR-dependent phosphorylation of RPS6. Arabidopsis mutants defective in TOR, RPS6A, or RPS6B exhibited delayed cotyledon opening, a characteristic of the deetiolating process to ensure timely vegetative development of a young seedling. This study provides a mechanistic view of light-triggered translational enhancement in deetiolating Arabidopsis.

Photoelastic analysis of stress distributions in the root-bone interface when applying various orthodontic methods to subside lower anterior crowding.

Dental crowding is a prevalent problem in modern society. Various factors, including different bracket systems and geometry of wire, influence the outcome using superelastic wire in the alignment stage of treatment. Currently, the use of light round wire instead of large-sized rectangular wire is emphasized to avoid powerful torsional stiffness. However, these guidelines lack scientific evidence for support. They have been written predominantly based on clinical experience. Therefore, the purpose of this study was to evaluate how factors such as bracket systems and wire geometry affect the stress distribution at the root-bone interface. Models using a photoelastic material (PL-3) to simulate bone tissue were fabricated. The simulated teeth were arranged as in lower anterior crowding. Then, the crowded teeth were subjected to orthodontic treatment with various types of bracket, ligating approaches using ligature wires of different sizes, shapes, and materials. Photoelastic images of the bone area of the models were obtained and compared. The results showed that wire size plays a more significant role than the material or cross-sectional shape of the wire in affecting the stress distribution at the simulated root-bone interface. The teeth ligated with a larger cross-sectional diameter of wire showed the application of excessive torque, which may cause root resorption and slow down tooth movement. These results not only demonstrate the scientific evidence backing clinical experience but also can be a useful reference for further clinical application.

Regulatory Divergence in Wound-Responsive Gene Expression between Domesticated and Wild Tomato.

The evolution of transcriptional regulatory mechanisms is central to how stress response and tolerance differ between species. However, it remains largely unknown how divergence in cis-regulatory sites and, subsequently, transcription factor (TF) binding specificity contribute to stress-responsive expression divergence, particularly between wild and domesticated species. By profiling wound-responsive gene transcriptomes in wild Solanum pennellii and domesticated S. lycopersicum, we found extensive wound response divergence and identified 493 S. lycopersicum and 278 S. pennellii putative cis-regulatory elements (pCREs) that were predictive of wound-responsive gene expression. Only 24-52% of these wound response pCREs (depending on wound response patterns) were consistently enriched in the putative promoter regions of wound-responsive genes across species. In addition, between these two species, their differences in pCRE site sequences were significantly and positively correlated with differences in wound-responsive gene expression. Furthermore, ∼11-39% of pCREs were specific to only one of the species and likely bound by TFs from different families. These findings indicate substantial regulatory divergence in these two plant species that diverged ∼3-7 million years ago. Our study provides insights into the mechanistic basis of how the transcriptional response to wounding is regulated and, importantly, the contribution of cis-regulatory components to variation in wound-responsive gene expression between a wild and a domesticated plant species.

A G-Box-Like Motif Is Necessary for Transcriptional Regulation by Circadian Pseudo-Response Regulators in Arabidopsis.

PSEUDO-RESPONSE REGULATORs (PRRs) play overlapping and distinct roles in maintaining circadian rhythms and regulating diverse biological processes, including the photoperiodic control of flowering, growth, and abiotic stress responses. PRRs act as transcriptional repressors and associate with chromatin via their conserved C-terminal CCT (CONSTANS, CONSTANS-like, and TIMING OF CAB EXPRESSION 1 [TOC1/PRR1]) domains by a still-poorly understood mechanism. Here, we identified genome-wide targets of PRR9 using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and compared them with PRR7, PRR5, and TOC1/PRR1 ChIP-seq data. We found that PRR binding sites are located within genomic regions of low nucleosome occupancy and high DNase I hypersensitivity. Moreover, conserved noncoding regions among Brassicaceae species are enriched around PRR binding sites, indicating that PRRs associate with functionally relevant cis-regulatory regions. The PRRs shared a significant number of binding regions, and our results indicate that they coordinately restrict the expression of target genes to around dawn. A G-box-like motif was overrepresented at PRR binding regions, and we showed that this motif is necessary for mediating transcriptional regulation of CIRCADIAN CLOCK ASSOCIATED 1 and PRR9 by the PRRs. Our results further our understanding of how PRRs target specific promoters and provide an extensive resource for studying circadian regulatory networks in plants.

Determinants of nucleosome positioning and their influence on plant gene expression.

Nucleosome positioning influences the access of transcription factors (TFs) to their binding sites and gene expression. Studies in plant, animal, and fungal models demonstrate similar nucleosome positioning patterns along genes and correlations between occupancy and expression. However, the relationships among nucleosome positioning, cis-regulatory element accessibility, and gene expression in plants remain undefined. Here we showed that plant nucleosome depletion occurs on specific 6-mer motifs and this sequence-specific nucleosome depletion is predictive of expression levels. Nucleosome-depleted regions in Arabidopsis thaliana tend to have higher G/C content, unlike yeast, and are centered on specific G/C-rich 6-mers, suggesting that intrinsic sequence properties, such as G/C content, cannot fully explain plant nucleosome positioning. These 6-mer motif sites showed higher DNase I hypersensitivity and are flanked by strongly phased nucleosomes, consistent with known TF binding sites. Intriguingly, this 6-mer-specific nucleosome depletion pattern occurs not only in promoter but also in genic regions and is significantly correlated with higher gene expression level, a phenomenon also found in rice but not in yeast. Among the 6-mer motifs enriched in genes responsive to treatment with the defense hormone jasmonate, there are no significant changes in nucleosome occupancy, suggesting that these sites are potentially preconditioned to enable rapid response without changing chromatin state significantly. Our study provides a global assessment of the joint contribution of nucleosome occupancy and motif sequences that are likely cis-elements to the control of gene expression in plants. Our findings pave the way for further understanding the impact of chromatin state on plant transcriptional regulatory circuits.

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.

Widespread translational control contributes to the regulation of Arabidopsis photomorphogenesis.

Environmental 'light' has a vital role in regulating plant growth and development. Transcriptomic profiling has been widely used to examine how light regulates mRNA levels on a genome-wide scale, but the global role of translational regulation in the response to light is unknown. Through a transcriptomic comparison of steady-state and polysome-bound mRNAs, we reveal a clear impact of translational control on thousands of genes, in addition to transcriptomic changes, during photomorphogenesis. Genes encoding ribosomal protein are preferentially regulated at the translational level, which possibly contributes to the enhanced translation efficiency. We also reveal that mRNAs regulated at the translational level share characteristics of longer half-lives and shorter cDNA length, and that transcripts with a cis-element, TAGGGTTT, in their 5' untranslated region have higher translatability. We report a previously neglected aspect of gene expression regulation during Arabidopsis photomorphogenesis. The identities and molecular signatures associated with mRNAs regulated at the translational level also offer new directions for mechanistic studies of light-triggered translational enhancement in Arabidopsis.

A glutathione S-transferase regulated by light and hormones participates in the modulation of Arabidopsis seedling development.

Glutathione S-transferases (GSTs) have been well documented to be involved in diverse aspects of biotic and abiotic stresses, especially detoxification processes. Whether they regulate plant development remains unclear. Here, we report on our isolation by reverse transcription-polymerase chain reaction of a plant GST, AtGSTU17, from Arabidopsis (Arabidopsis thaliana) and demonstrate that its expression is regulated by multiple photoreceptors, especially phytochrome A (phyA) under all light conditions. Further physiological studies indicated that AtGSTU17 participates in various aspects of seedling development, including hypocotyl elongation, anthocyanin accumulation, and far-red light-mediated inhibition of greening with a requirement of functional phyA. The loss-of-function mutant of AtGSTU17 (atgstu17) resulted in reduced biomass of seedlings and number of lateral roots in the presence of auxin, as well as insensitivity to abscisic acid (ABA)-mediated inhibition of root elongation, with similarity to different phyA mutant alleles. Moreover, the root phenotype conferred by atgstu17 was reflected by histochemical ß-glucuronidase staining of AtGSTU17 promoter activity with the addition of auxin or ABA. Further microarray analysis of wild-type Columbia and atgstu17 seedlings treated with far-red irradiation or ABA revealed that AtGSTU17 might modulate hypocotyl elongation by positively regulating some light-signaling components and negatively regulating a group of auxin-responsive genes and modulate root development by negatively controlling an auxin transport protein in the presence of ABA. Therefore, our data reveal that AtGSTU17 participates in light signaling and might modulate various aspects of Arabidopsis development by affecting glutathione pools via a coordinated regulation with phyA and phytohormones.

Glutathione S-transferase interacting with far-red insensitive 219 is involved in phytochrome A-mediated signaling in Arabidopsis.

Far-red (FR) insensitive 219 (FIN219) was previously shown to be involved in phytochrome A-mediated FR light signaling. To further understand its function and regulatory relation with other light-signaling components, a yeast two-hybrid approach was used to isolate FIN219-interacting partners. Here, we demonstrate that FIN219-interacting protein 1 (FIP1) interacts with FIN219 in vitro and in vivo and is composed of 217 amino acids that belong to the tau class of the large glutathione S-transferase gene family. FIP1 was further shown to have glutathione S-transferase activity. The gain of function and partial loss of function of FIP1 resulted in a hyposensitive hypocotyl phenotype under continuous FR (cFR) light and a delayed flowering phenotype under long-day conditions, which suggests that FIP1 may exist in a complex to function in the regulation of Arabidopsis (Arabidopsis thaliana) development. In addition, FIP1 mRNA was down-regulated in the suppressor of phytochrome A-105 1 mutant and differentially expressed in constitutive photomorphogenic 1-4 (cop1-4) and cop1-5 mutants under cFR. Intriguingly, FIP1 expression was up-regulated in the fin219 mutant under all light conditions, except cFR. Furthermore, promoter activity assays revealed that FIP1 expression was light dependent, mainly associated with vascular tissues, and developmentally regulated. Subcellular localization studies revealed that the beta-glucuronidase-FIP1 fusion protein was localized in the nucleus and cytoplasm. Taken together, these data indicate that FIP1 may interact with FIN219 to regulate cell elongation and flowering in response to light.