Beyond transcription: compelling open questions in plant RNA biology.

The study of RNAs has become one of the most influential research fields in contemporary biology and biomedicine. In the last few years, new sequencing technologies have produced an explosion of new and exciting discoveries in the field but have also given rise to many open questions. Defining these questions, together with old, long-standing gaps in our knowledge, is the spirit of this article. The breadth of topics within RNA biology research is vast, and every aspect of the biology of these molecules contains countless exciting open questions. Here, we asked 12 groups to discuss their most compelling question among some plant RNA biology topics. The following vignettes cover RNA alternative splicing; RNA dynamics; RNA translation; RNA structures; R-loops; epitranscriptomics; long non-coding RNAs; small RNA production and their functions in crops; small RNAs during gametogenesis and in cross-kingdom RNA interference; and RNA-directed DNA methylation. In each section, we will present the current state-of-the-art in plant RNA biology research before asking the questions that will surely motivate future discoveries in the field. We hope this article will spark a debate about the future perspective on RNA biology and provoke novel reflections in the reader.

AtPep3 is a hormone-like peptide that plays a role in the salinity stress tolerance of plants.

Peptides encoded by small coding genes play an important role in plant development, acting in a similar manner as phytohormones. Few hormone-like peptides, however, have been shown to play a role in abiotic stress tolerance. In the current study, 17 Arabidopsis genes coding for small peptides were found to be up-regulated in response to salinity stress. To identify peptides leading salinity stress tolerance, we generated transgenic Arabidopsis plants overexpressing these small coding genes and assessed survivability and root growth under salinity stress conditions. Results indicated that 4 of the 17 overexpressed genes increased salinity stress tolerance. Further studies focused on AtPROPEP3, which was the most highly up-regulated gene under salinity stress. Treatment of plants with synthetic peptides encoded by AtPROPEP3 revealed that a C-terminal peptide fragment (AtPep3) inhibited the salt-induced bleaching of chlorophyll in seedlings. Conversely, knockdown AtPROPEP3 transgenic plants exhibited a hypersensitive phenotype under salinity stress, which was complemented by the AtPep3 peptide. This functional AtPep3 peptide region overlaps with an AtPep3 elicitor peptide that is related to the immune response of plants. Functional analyses with a receptor mutant of AtPep3 revealed that AtPep3 was recognized by the PEPR1 receptor and that it functions to increase salinity stress tolerance in plants. Collectively, these data indicate that AtPep3 plays a significant role in both salinity stress tolerance and immune response in Arabidopsis.

Biological Function of Changes in RNA Metabolism in Plant Adaptation to Abiotic Stress.

Plant growth and productivity are greatly impacted by environmental stresses. Therefore, plants have evolved various sophisticated mechanisms for adaptation to nonoptimal environments. Recent studies using RNA metabolism-related mutants have revealed that RNA processing, RNA decay and RNA stability play an important role in regulating gene expression at a post-transcriptional level in response to abiotic stresses. Studies indicate that RNA metabolism is a unified network, and modification of stress adaptation-related transcripts at multiple steps of RNA metabolism is necessary to control abiotic stress-related gene expression. Recent studies have also demonstrated the important role of noncoding RNAs (ncRNAs) in regulating abiotic stress-related gene expression and revealed their involvement in various biological functions through their regulation of DNA methylation, DNA structural modifications, histone modifications and RNA-RNA interactions. ncRNAs regulate mRNA transcription and their synthesis is affected by mRNA processing and degradation. In the present review, recent findings pertaining to the role of the metabolic regulation of mRNAs and ncRNAs in abiotic stress adaptation are summarized and discussed.

RNA Regulation in Plant Cold Stress Response.

In addition to plants, all organisms react to environmental stimuli via the perception of signals and subsequently respond through alterations of gene expression. However, genes/mRNAs are usually not the functional unit themselves, and instead, resultant protein products with individual functions result in various acquired phenotypes. In order to fully characterize the adaptive responses of plants to environmental stimuli, it is essential to determine the level of proteins, in addition to the regulation of mRNA expression. This regulatory step, which is referred to as "mRNA posttranscriptional regulation," occurs subsequent to mRNA transcription and prior to translation. Although these RNA regulatory mechanisms have been well-studied in many organisms, including plants, it is not fully understood how plants respond to environmental stimuli, such as cold stress, via these RNA regulations.A recent study described several RNA regulatory factors in relation to environmental stress responses, including plant cold stress tolerance. In this chapter, the functions of RNA regulatory factors and comprehensive analyses related to the RNA regulations involved in cold stress response are summarized, such as mRNA maturation, including capping, splicing, polyadenylation of mRNA, and the quality control system of mRNA; mRNA degradation, including the decapping step; and mRNA stabilization. In addition, the putative roles of messenger ribonucleoprotein (mRNP) granules, such as processing bodies (PBs) and stress granules (SGs), which are cytoplasmic particles, are described in relation to RNA regulations under stress conditions. These RNA regulatory systems are important for adjusting or fine-tuning and determining the final levels of mRNAs and proteins in order to adapt or respond to environmental stresses. Collectively, these new areas of study revealed that plants possess precise novel regulatory mechanisms which specifically function in the response to cold stress.

Analysis of differential expression patterns of mRNA and protein during cold-acclimation and de-acclimation in Arabidopsis.

Overwintering plants are capable of exhibiting high levels of cold tolerance, which is acquired through the process of cold acclimation (CA). In contrast to CA, the acquired freezing tolerance is rapidly reduced during cold de-acclimation (DA) and plants resume growth after sensing warm temperatures. In order to better understand plant growth and development, and to aid in the breeding of cold-tolerant plants, it is important to decipher the functional mechanisms of the DA process. In this study, we performed comparative transcriptomic and proteomic analyses during CA and DA. As revealed by shotgun proteomics, we identified 3987 peptides originating from 1569 unique proteins and the corresponding mRNAs were analyzed. Among the 1569 genes, 658 genes were specifically induced at the transcriptional level during the process of cold acclimation. In order to investigate the relationship between mRNA and the corresponding protein expression pattern, a Pearson correlation was analyzed. Interestingly, 199 genes showed a positive correlation of mRNA and protein expression pattern, indicating that both their transcription and translation occurred during CA. However, 226 genes showed a negative correlation of mRNA and protein expression pattern, indicating that their mRNAs were transcribed during CA and were stored for the subsequent DA step. Under this scenario, those proteins were specifically increased during DA without additional transcription of mRNA. In order to confirm the negative correlation of mRNA and protein expression patterns, qRT-PCR and western blot analyses were performed. Mitochondrial malate dehydrogenase 1 (mMDH1) exhibited a negative correlation of mRNA and protein levels, which was characterized by CA-specific mRNA induction and protein accumulation specifically during DA. These data indicate that the expression of specific mRNAs and subsequent accumulation of corresponding proteins are not always in accordance under low temperature stress conditions in plants.

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.

Loss of Arabidopsis 5'-3' Exoribonuclease AtXRN4 Function Enhances Heat Stress Tolerance of Plants Subjected to Severe Heat Stress.

mRNA degradation plays an important role in the rapid and dynamic alteration of gene expression in response to environmental stimuli. Arabidopsis 5'-3' exoribonuclease (AtXRN4), a homolog of yeast Xrn1p, functions after a de-capping step in the degradation of uncapped RNAs. While Xrn1p-dependent degradation of mRNA is the main process of mRNA decay in yeast, information pertaining to the targets of XRN4-based degradation in plants is limited. In order to better understand the biological function of AtXRN4, the current study examined the survivability of atxrn4 mutants subjected to heat stress. The results indicated that atxrn4 mutants, compared with wild-type plants, exhibited an increased survival rate when subjected to a short-term severe heat stress. A microarray and mRNA decay assay showed that loss of AtXRN4 function caused a reduction in the degradation of heat shock factor A2 (HSFA2) and ethylene response factor 1 (ERF1) mRNA. The heat stress tolerance phenotype of atxrn4 mutants was significantly reduced or lost by mutation of HSFA2, a known key regulator of heat acclimation, thus indicating that HSFA2 is a target gene of AtXRN4-mediated mRNA degradation both under non-stress conditions and during heat acclimation. These results demonstrate that AtXRN4-mediated mRNA degradation is linked to the suppression of heat acclimation.

RNA regulation in plant abiotic stress responses.

RNA regulatory processes such as transcription, degradation and stabilization control are the major mechanisms that determine the levels of mRNAs in plants. Transcriptional and post-transcriptional regulations of RNAs are drastically altered during plant stress responses. As a result of these molecular processes, plants are capable of adjusting to changing environmental conditions. Understanding the role of these mechanisms in plant stress responses is important and necessary for the engineering of stress-tolerant plants. Recent studies in the area of RNA regulation have increased our understanding of how plants respond to environmental stresses. This review highlights recent progress in RNA regulatory processes that are involved in plant stress responses, such as small RNAs, alternative splicing, RNA granules and RNA-binding proteins. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress.

Arabidopsis non-coding RNA regulation in abiotic stress responses.

Plant growth and productivity are largely affected by environmental stresses. Therefore, plants have evolved unique adaptation mechanisms to abiotic stresses through fine-tuned adjustment of gene expression and metabolism. Recent advanced technologies, such as genome-wide transcriptome analysis, have revealed that a vast amount of non-coding RNAs (ncRNAs) apart from the well-known housekeeping ncRNAs such as rRNAs, tRNAs, small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs) are expressed under abiotic stress conditions. These various types of ncRNAs are involved in chromatin regulation, modulation of RNA stability and translational repression during abiotic stress response. In this review, we summarize recent progress that has been made on ncRNA research in plant abiotic stress response.

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.

Arabidopsis cold shock domain proteins: relationships to floral and silique development.

Cold shock domain proteins (CSPs) are highly conserved from bacteria to higher plants and animals. Bacterial cold shock proteins function as RNA chaperones by destabilizing RNA secondary structures and promoting translation as an adaptative mechanism to low temperature stress. In animals, cold shock domain proteins exhibit broad functions related to growth and development. In order to understand better the function of CSPs in planta, detailed analyses were performed for Arabidopsis thaliana CSPs (AtCSPs) on the transcript and protein levels using an extensive series of tissue harvested throughout developmental stages within the entire life cycle of Arabidopsis. On both the transcript and protein levels, AtCSPs were enriched in shoot apical meristems and siliques. Although all AtCSPs exhibited similar expression patterns, AtCSP2 was the most abundantly expressed gene. In situ hybridization analyses were also used to confirm that AtCSP2 and AtCSP4 transcripts accumulate in developing embryos and shoot apices. AtCSPs transcripts were also induced during a controlled floral induction study. In vivo ChIP analysis confirmed that an embryo expressed MADS box transcription factor, AGL15, interacts within two AtCSP promoter regions and alters the respective patterns of AtCSP transcription. Comparative analysis of AtCSP gene expression between Landsberg and Columbia ecotypes confirmed a 1000-fold reduction of AtCSP4 gene expression in the Landsberg background. Analysis of the AtCSP4 genomic locus identified multiple polymorphisms in putative regulatory cis-elements between the two ecotypes. Collectively, these data support the hypothesis that AtCSPs are involved in the transition to flowering and silique development in Arabidopsis.

Germination of photoblastic lettuce seeds is regulated via the control of endogenous physiologically active gibberellin content, rather than of gibberellin responsiveness.

Phytochrome regulates lettuce (Lactuca sativa L. cv. Grand Rapids) seed germination via the control of the endogenous level of bioactive gibberellin (GA). In addition to the previously identified LsGA20ox1, LsGA20ox2, LsGA3ox1, LsGA3ox2, LsGA2ox1, and LsGA2ox2, five cDNAs were isolated from lettuce seeds: LsCPS, LsKS, LsKO1, LsKO2, and LsKAO. Using an Escherichia coli expression system and functional assays, it is shown that LsCPS and LsKS encode ent-copalyl diphosphate synthase and ent-kaurene synthase, respectively. Using a Pichia pastoris system, it was found that LsKO1 and LsKO2 encode ent-kaurene oxidases and LsKAO encodes ent-kaurenoic acid oxidase. A comprehensive expression analysis of GA metabolism genes using the quantitative reverse transcription polymerase chain reaction suggested that transcripts of LsGA3ox1 and LsGA3ox2, both of which encode GA 3-oxidase for GA activation, were primarily expressed in the hypocotyl end of lettuce seeds, were expressed at much lower levels than the other genes tested, and were potently up-regulated by phytochrome. Furthermore, LsDELLA1 and LsDELLA2 cDNAs that encode DELLA proteins, which act as negative regulators in the GA signalling pathway, were isolated from lettuce seeds. The transcript levels of these two genes were little affected by light. Lettuce seeds in which de novo GA biosynthesis was suppressed responded almost identically to exogenously applied GA, irrespective of the light conditions, suggesting that GA responsiveness is not significantly affected by light in lettuce seeds. It is proposed that lettuce seed germination is regulated mainly via the control of the endogenous content of bioactive GA, rather than the control of GA responsiveness.

Overexpression of oligouridylate binding protein 1b results in ABA hypersensitivity.

Oligouridylate binding protein 1b (UBP1b), a marker protein of plant stress granules (SGs), plays a role in heat stress tolerance in plants. A previous microarray analysis revealed that the expression of several ABA signaling-related genes is higher in UBP1b-overexpressing Arabidopsis plants (UBP1b-ox) subjected to both non-stressed and heat stress conditions. Root elongation and seed germination assays demonstrated that UBP1b-ox exhibited hypersensitivity to ABA. RT-qPCR analysis confirmed that mitogen-activated protein kinase (MAPK) cascade genes, such as MPK3, MKK4, and MKK9 were upregulated in UBP1b-ox plants. ABA receptor genes, including PYL5 and PYL6, were also upregulated in UBP1b-ox plants. mRNA of WRKY33 - a downstream gene of MPK3 and an upstream gene of ethylene biosynthesis, exhibited high levels of accumulation, although the level of endogenous ABA was not significantly different between UBP1b-ox and control plants. In addition, RNA decay analysis revealed that WRKY33 was more stable in UBP1b-ox plants, indicating that the mRNA of WRKY33 was protected within UBP1b SGs. Collectively, these data demonstrate that UBP1b plays an important role in plant response to ABA.

Drought stress differentially regulates the expression of small open reading frames (sORFs) in Arabidopsis roots and shoots.

Characterizing the molecular mechanisms governing the response of plant roots and shoots to drought stress could aid the development of strategies aiming to ameliorate drought stress. Small open reading frames (sORFs), putatively encoding small peptides, may play a significant role in the response to different abiotic stresses. Microarray analyses revealed that after 5, 7 and 9 d of a drought treatment, 2, 77, and 104 sORFs were up-regulated in roots, respectively; while the number of upregulated sORFs in shoots was 12, 45, and 158, respectively. RT-qPCR analysis confirmed the up-regulated expression of ATRIKEN29196 and ATRIKEN32280 specifically in roots. The identified upregulated sORFs, particularly those in roots, may contribute to drought stress tolerance.

Oligouridylate Binding Protein 1b Plays an Integral Role in Plant Heat Stress Tolerance.

Stress granules (SGs), which are formed in the plant cytoplasm under stress conditions, are transient dynamic sites (particles) for mRNA storage. SGs are actively involved in protecting mRNAs from degradation. Oligouridylate binding protein 1b (UBP1b) is a component of SGs. The formation of microscopically visible cytoplasmic foci, referred to as UBP1b SG, was induced by heat treatment in UBP1b-overexpressing Arabidopsis plants (UBP1b-ox). A detailed understanding of the function of UBP1b, however, is still not clear. UBP1b-ox plants displayed increased heat tolerance, relative to control plants, while ubp1b mutants were more sensitive to heat stress than control plants. Microarray analysis identified 117 genes whose expression was heat-inducible and higher in the UBP1b-ox plants. RNA decay analysis was performed using cordycepin, a transcriptional inhibitor. In order to determine if those genes serve as targets of UBP1b, the rate of RNA degradation of a DnaJ heat shock protein and a stress-associated protein (AtSAP3) in UBP1b-ox plants was slower than in control plants; indicating that the mRNAs of these genes were protected within the UBP1b SG granule. Collectively, these data demonstrate that UBP1b plays an integral role in heat stress tolerance in plants.

Heat stable ssDNA/RNA-binding activity of a wheat cold shock domain protein.

The cold-induced wheat WCSP1 protein belongs to the cold shock domain (CSD) protein family. In prokaryotes and eukaryotes, the CSD functions as a nucleic acid-binding domain. Here, we demonstrated that purified recombinant WCSP1 is boiling soluble and binds ss/dsDNA and mRNA. Furthermore, boiled-WCSP1 retained its characteristic nucleic acid-binding activity. A WCSP1 deletion mutant, containing only a CSD, lost ssDNA/RNA-binding activity; while a mutant containing the CSD and the first glycine-rich region (GR) displayed the activity. These data indicated that the first GR of WCSP1 is necessary for the binding activity but is not for the heat stability of the protein.

A distinctive class of spermidine synthase is involved in chilling response in rice.

A cDNA for a putative 42 kD spermidine synthase (OsSPDS2) was cloned from rice. The deduced OsSPDS2 sequence showed highest similarity with Arabidopsis AtSPDS3. Phylogenetic analysis revealed that OsSPDS2 and AtSPDS3 form a distinctive subclass in the spermidine synthase family in plants. OsSPDS2 mRNA accumulated in roots during long term exposure to chilling temperature (12 degrees C). In contrast, no such induction of the paralogous OsSPDS1 was observed during the chilling treatment. ABA treatment up-regulated OsSPDS2, whereas salt stress did not change OsSPDS2 levels significantly. Data suggested a distinct function of OsSPDS2 in chilling response in rice.

Arabidopsis tiling array analysis to identify the stress-responsive genes.

Plants respond and adapt to drought, cold, and high-salinity stresses. Stress-inducible gene products function in the stress response and tolerance in plants. Using cDNA microarrays and oligonucleotide microarrays, stress-inducible genes have been identified in various plant species so far. Recently, tiling array technology has become a powerful tool for the whole-genome transcriptome analysis. We applied the Arabidopsis Affymetrix tiling arrays to study the whole-genome transcriptome under drought, cold, and high-salinity stresses and identified a large number of drought, cold, and high-salinity stress-inducible genes and transcriptional units (TUs).

Phytochrome- and gibberellin-mediated regulation of abscisic acid metabolism during germination of photoblastic lettuce seeds.

Germination of lettuce (Lactuca sativa) 'Grand Rapids' seeds is regulated by phytochrome. The action of phytochrome includes alterations in the levels of gibberellin (GA) and abscisic acid (ABA). To determine the molecular mechanism of phytochrome regulation of ABA metabolism, we isolated four lettuce cDNAs encoding 9-cis-epoxycarotenoid dioxygenase (biosynthesis; LsNCED1-LsNCED4) and four cDNAs for ABA 8'-hydroxylase (catabolism; LsABA8ox1-LsABA8ox4). Measurements of ABA and its catabolites showed that a decrease in ABA level coincided with a slight increase in the level of the ABA catabolite phaseic acid after red light treatment. Quantitative reverse transcription-polymerase chain reaction analysis indicated that ABA levels are controlled by phytochrome through down-regulation of LsNCED2 and LsNCED4 expression and up-regulation of LsABA8ox4 expression in lettuce seeds. Furthermore, the expression levels of LsNCED4 decreased after GA(1) treatment, whereas the levels of expression of the other two genes were unaffected. The LsNCED4 expression was also down-regulated by red light in lettuce seeds in which GA biosynthesis was suppressed by AMO-1618, a specific GA biosynthesis inhibitor. These results indicate that phytochrome regulation of ABA metabolism is mediated by both GA-dependent and -independent mechanisms. Spatial analysis showed that after red light treatment, the ABA decrease on the hypocotyl side was greater than that on the cotyledon side of lettuce seeds. Moreover, phytochrome-regulated expression of ABA and GA biosynthesis genes was observed on the hypocotyl side, rather than the cotyledon side, suggesting that this regulation occurs near the photoperceptive site.

Functional conservation of cold shock domains in bacteria and higher plants.

In Escherichia coli, a family of cold shock proteins (CSPs) function as transcription antiterminators or translational enhancers at low temperature by destabilizing RNA secondary structure. A wheat nucleic acid-binding protein (WCSP1) was found to contain a cold shock domain (CSD) bearing high similarity to E. coli cold shock proteins. In the present study, a series of mutations were introduced into WCSP1, and its functionality was investigated by using in vivo and in vitro assays in the context of functional conservation with E. coli CSPs. Constitutive expression of WT WCSP1 in an E. coli cspA, cspB, cspE, cspG quadruple deletion mutant complemented its cold-sensitive phenotype, suggesting that WCSP1 shares a function with E. coli CSPs for cold adaptation. In addition, transcription antitermination activity was demonstrated for WCSP1 by using an E. coli strain that has a hairpin loop upstream of a chloramphenicol resistance gene. In vitro dsDNA melting assays clearly demonstrated that WCSP1 melts dsDNA, an activity that was positively correlated to the ability to bind ssDNA. When mutations were introduced at critical residues within the consensus RNA binding motifs (RNP1 and RNP2) of WCSP1, it failed to melt dsDNA. Studies with WCSP1-GFP fusion proteins documented patterns that are consistent with ER and nuclear localization. In vivo and in vitro functional analyses, coupled with subcellular localization data, suggest that WCSP1 may function as a RNA chaperone to destabilize secondary structure and is involved in the regulation of translation under low temperature.