Dynamics of mRNA fate during light stress and recovery: from transcription to stability and translation.

Transcript stability is an important determinant of its abundance and, consequently, translational output. Transcript destabilisation can be rapid and is well suited for modulating the cellular response. However, it is unclear the extent to which RNA stability is altered under changing environmental conditions in plants. We previously hypothesised that recovery-induced transcript destabilisation facilitated a phenomenon of rapid recovery gene downregulation (RRGD) in Arabidopsis thaliana (Arabidopsis) following light stress, based on mathematical calculations to account for ongoing transcription. Here, we test this hypothesis and investigate processes regulating transcript abundance and fate by quantifying changes in transcription, stability and translation before, during and after light stress. We adapt syringe infiltration to apply a transcriptional inhibitor to soil-grown plants in combination with stress treatments. Compared with measurements in juvenile plants and cell culture, we find reduced stability across a range of transcripts encoding proteins involved in RNA binding and processing. We also observe light-induced destabilisation of transcripts, followed by their stabilisation during recovery. We propose that this destabilisation facilitates RRGD, possibly in combination with transcriptional shut-off that was confirmed for HSP101, ROF1 and GOLS1. We also show that translation remains highly dynamic over the course of light stress and recovery, with a bias towards transcript-specific increases in ribosome association, independent of changes in total transcript abundance, after 30 min of light stress. Taken together, we provide evidence for the combinatorial regulation of transcription and stability that occurs to coordinate translation during light stress and recovery in Arabidopsis.

The role of SWEET4 proteins in the post-phloem sugar transport pathway of Setaria viridis sink tissues.

In the developing seeds of all higher plants, filial cells are symplastically isolated from the maternal tissue supplying photosynthate to the reproductive structure. Photoassimilates must be transported apoplastically, crossing several membrane barriers, a process facilitated by sugar transporters. Sugars Will Eventually be Exported Transporters (SWEETs) have been proposed to play a crucial role in apoplastic sugar transport during phloem unloading and the post-phloem pathway in sink tissues. Evidence for this is presented here for developing seeds of the C4 model grass Setaria viridis. Using immunolocalization, SvSWEET4 was detected in various maternal and filial tissues within the seed along the sugar transport pathway, in the vascular parenchyma of the pedicel, and in the xylem parenchyma of the stem. Expression of SvSWEET4a in Xenopus laevis oocytes indicated that it functions as a high-capacity glucose and sucrose transporter. Carbohydrate and transcriptional profiling of Setaria seed heads showed that there were some developmental shifts in hexose and sucrose content and consistent expression of SvSWEET4 homologues. Collectively, these results provide evidence for the involvement of SWEETs in the apoplastic transport pathway of sink tissues and allow a pathway for post-phloem sugar transport into the seed to be proposed.

The covalent nucleotide modifications within plant mRNAs: What we know, how we find them, and what should be done in the future.

Although covalent nucleotide modifications were first identified on the bases of transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), a number of these epitranscriptome marks have also been found to occur on the bases of messenger RNAs (mRNAs). These covalent mRNA features have been demonstrated to have various and significant effects on the processing (e.g. splicing, polyadenylation, etc.) and functionality (e.g. translation, transport, etc.) of these protein-encoding molecules. Here, we focus our attention on the current understanding of the collection of covalent nucleotide modifications known to occur on mRNAs in plants, how they are detected and studied, and the most outstanding future questions of each of these important epitranscriptomic regulatory signals.

Enzymes degraded under high light maintain proteostasis by transcriptional regulation in Arabidopsis.

Photoinhibitory high light stress in Arabidopsis leads to increases in markers of protein degradation and transcriptional up-regulation of proteases and proteolytic machinery, but proteostasis is largely maintained. We find significant increases in the in vivo degradation rate for specific molecular chaperones, nitrate reductase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase and other plastid, mitochondrial, peroxisomal, and cytosolic enzymes involved in redox shuttles. Coupled analysis of protein degradation rates, mRNA levels, and protein abundance reveal that 57% of the nuclear-encoded enzymes with higher degradation rates also had high light­induced transcriptional responses to maintain proteostasis. In contrast, plastid-encoded proteins with enhanced degradation rates showed decreased transcript abundances and must maintain protein abundance by other processes. This analysis reveals a light-induced transcriptional program for nuclear-encoded genes, beyond the regulation of the photosystem II (PSII) D1 subunit and the function of PSII, to replace key protein degradation targets in plants and ensure proteostasis under high light stress.

Elucidating the role of SWEET13 in phloem loading of the C4 grass Setaria viridis.

Photosynthetic efficiency and sink demand are tightly correlated with rates of phloem loading, where maintaining low cytosolic sugar concentrations is paramount to prevent the downregulation of photosynthesis. Sugars Will Eventually be Exported Transporters (SWEETs) are thought to have a pivotal role in the apoplastic phloem loading of C4 grasses. SWEETs have not been well studied in C4 species, and their investigation is complicated by photosynthesis taking place across two cell types and, therefore, photoassimilate export can occur from either one. SWEET13 homologues in C4 grasses have been proposed to facilitate apoplastic phloem loading. Here, we provide evidence for this hypothesis using the C4 grass Setaria viridis. Expression analyses on the leaf gradient of C4 species Setaria and Sorghum bicolor show abundant transcript levels for SWEET13 homologues. Carbohydrate profiling along the Setaria leaf shows total sugar content to be significantly higher in the mature leaf tip compared with the younger tissue at the base. We present the first known immunolocalization results for SvSWEET13a and SvSWEET13b using novel isoform-specific antisera. These results show localization to the bundle sheath and phloem parenchyma cells of both minor and major veins. We further present the first transport kinetics study of C4 monocot SWEETs by using a Xenopus laevis oocyte heterologous expression system. We demonstrate that SvSWEET13a and SvSWEET13b are high-capacity transporters of glucose and sucrose, with a higher apparent Vmax for sucrose, compared with glucose, typical of clade III SWEETs. Collectively, these results provide evidence for an apoplastic phloem loading pathway in Setaria and possibly other C4 species.

Autophagy mutants show delayed chloroplast development during de-etiolation in carbon limiting conditions.

Autophagy is a conserved catabolic process that plays an essential role under nutrient starvation conditions and influences different developmental processes. We observed that seedlings of autophagy mutants (atg2, atg5, atg7, and atg9) germinated in the dark showed delayed chloroplast development following illumination. The delayed chloroplast development was characterized by a decrease in photosynthetic and chlorophyll biosynthetic proteins, lower chlorophyll content, reduced chloroplast size, and increased levels of proteins involved in lipid biosynthesis. Confirming the biological impact of these differences, photosynthetic performance was impaired in autophagy mutants 12 h post-illumination. We observed that while gene expression for photosynthetic machinery during de-etiolation was largely unaffected in atg mutants, several genes involved in photosystem assembly were transcriptionally downregulated. We also investigated if the delayed chloroplast development could be explained by lower lipid import to the chloroplast or lower triglyceride (TAG) turnover. We observed that the limitations in the chloroplast lipid import imposed by trigalactosyldiacylglycerol1 are unlikely to explain the delay in chloroplast development. However, we found that lower TAG mobility in the triacylglycerol lipase mutant sugardependent1 significantly affected de-etiolation. Moreover, we showed that lower levels of carbon resources exacerbated the slow greening phenotype whereas higher levels of carbon resources had an opposite effect. This work suggests a lack of autophagy machinery limits chloroplast development during de-etiolation, and this is exacerbated by limited lipid turnover (lipophagy) that physically or energetically restrains chloroplast development.

Extending the Genotype in Brachypodium by Including DNA Methylation Reveals a Joint Contribution with Genetics on Adaptive Traits.

Epigenomic changes have been considered a potential missing link underlying phenotypic variation in quantitative traits but is potentially confounded with the underlying DNA sequence variation. Although the concept of epigenetic inheritance has been discussed in depth, there have been few studies attempting to directly dissect the amount of epigenomic variation within inbred natural populations while also accounting for genetic diversity. By using known genetic relationships between Brachypodium lines, multiple sets of nearly identical accession families were selected for phenotypic studies and DNA methylome profiling to investigate the dual role of (epi)genetics under simulated natural seasonal climate conditions. Despite reduced genetic diversity, appreciable phenotypic variation was still observable in the measured traits (height, leaf width and length, tiller count, flowering time, ear count) between as well as within the inbred accessions. However, with reduced genetic diversity there was diminished variation in DNA methylation within families. Mixed-effects linear modeling revealed large genetic differences between families and a minor contribution of DNA methylation variation on phenotypic variation in select traits. Taken together, this analysis suggests a limited but significant contribution of DNA methylation toward heritable phenotypic variation relative to genetic differences.

Excess Light Priming in Arabidopsis thaliana Genotypes with Altered DNA Methylomes.

Plants must continuously react to the ever-fluctuating nature of their environment. Repeated exposure to stressful conditions can lead to priming, whereby prior encounters heighten a plant's ability to respond to future events. A clear example of priming is provided by the model plant Arabidopsis thaliana (Arabidopsis), in which photosynthetic and photoprotective responses are enhanced following recurring light stress. While there are various post-translational mechanisms underpinning photoprotection, an unresolved question is the relative importance of transcriptional changes toward stress priming and, consequently, the potential contribution from DNA methylation - a heritable chemical modification of DNA capable of influencing gene expression. Here, we systematically investigate the potential molecular underpinnings of physiological priming against recurring excess-light (EL), specifically DNA methylation and transcriptional regulation: the latter having not been examined with respect to EL priming. The capacity for physiological priming of photosynthetic and photoprotective parameters following a recurring EL treatment was not impaired in Arabidopsis mutants with perturbed establishment, maintenance, or removal of DNA methylation. Importantly, no differences in development or basal photoprotective capacity were identified in the mutants that may confound the above result. Little evidence for a causal transcriptional component of physiological priming was identified; in fact, most alterations in primed plants presented as a transcriptional 'dampening' in response to an additional EL exposure, likely a consequence of physiological priming. However, a set of transcripts uniquely regulated in primed plants provide preliminary evidence for a novel transcriptional component of recurring EL priming, independent of physiological changes. Thus, we propose that physiological priming of recurring EL in Arabidopsis occurs independently of DNA methylation; and that the majority of the associated transcriptional alterations are a consequence, not cause, of this physiological priming.

RNA Polymerase II Read-Through Promotes Expression of Neighboring Genes in SAL1-PAP-XRN Retrograde Signaling.

In plants, the molecular function(s) of the nucleus-localized 5'-3' EXORIBONUCLEASES (XRNs) are unclear; however, their activity is reported to have a significant effect on gene expression and SAL1-mediated retrograde signaling. Using parallel analysis of RNA ends, we documented a dramatic increase in uncapped RNA substrates of the XRNs in both sal1 and xrn2xrn3 mutants. We found that a major consequence of reducing SAL1 or XRN activity was RNA Polymerase II 3' read-through. This occurred at 72% of expressed genes, demonstrating a major genome-wide role for the XRN-torpedo model of transcription termination in Arabidopsis (Arabidopsis thaliana). Read-through is speculated to have a negative effect on transcript abundance; however, we did not observe this. Rather, we identified a strong association between read-through and increased transcript abundance of tandemly orientated downstream genes, strongly correlated with the proximity (less than 1,000 bp) and expression of the upstream gene. We observed read-through in the proximity of 903 genes up-regulated in the sal1-8 retrograde signaling mutant; thus, this phenomenon may account directly for up to 23% of genes up-regulated in sal1-8 Using APX2 and AT5G43770 as exemplars, we genetically uncoupled read-through loci from downstream genes to validate the principle of read-through-mediated mRNA regulation, providing one mechanism by which an ostensibly posttranscriptional exoribonuclease that targets uncapped RNAs could modulate gene expression.

Maintenance of pre-existing DNA methylation states through recurring excess-light stress.

The capacity for plant stress priming and memory and the notion of this being underpinned by DNA methylation-mediated memory is an appealing hypothesis for which there is mixed evidence. We previously established a lack of drought-induced methylome variation in Arabidopsis thaliana (Arabidopsis); however, this was tied to only minor observations of physiological memory. There are numerous independent observations demonstrating that photoprotective mechanisms, induced by excess-light stress, can lead to robust programmable changes in newly developing leaf tissues. Although key signalling molecules and transcription factors are known to promote this priming signal, an untested question is the potential involvement of chromatin marks towards the maintenance of light stress acclimation, or memory. Thus, we systematically tested our previous hypothesis of a stress-resistant methylome using a recurring excess-light stress, then analysing new, emerging, and existing tissues. The DNA methylome showed negligible stress-associated variation, with the vast majority attributable to stochastic differences. Yet, photoacclimation was evident through enhanced photosystem II performance in exposed tissues, and nonphotochemical quenching and fluorescence decline ratio showed evidence of mitotic transmission. Thus, we have observed physiological acclimation in new and emerging tissues in the absence of substantive DNA methylome changes.

The Arabidopsis DNA Methylome Is Stable under Transgenerational Drought Stress.

Improving the responsiveness, acclimation, and memory of plants to abiotic stress holds substantive potential for improving agriculture. An unresolved question is the involvement of chromatin marks in the memory of agriculturally relevant stresses. Such potential has spurred numerous investigations yielding both promising and conflicting results. Consequently, it remains unclear to what extent robust stress-induced DNA methylation variation can underpin stress memory. Using a slow-onset water deprivation treatment in Arabidopsis (Arabidopsis thaliana), we investigated the malleability of the DNA methylome to drought stress within a generation and under repeated drought stress over five successive generations. While drought-associated epi-alleles in the methylome were detected within a generation, they did not correlate with drought-responsive gene expression. Six traits were analyzed for transgenerational stress memory, and the descendants of drought-stressed lineages showed one case of memory in the form of increased seed dormancy, and that persisted one generation removed from stress. With respect to transgenerational drought stress, there were negligible conserved differentially methylated regions in drought-exposed lineages compared with unstressed lineages. Instead, the majority of observed variation was tied to stochastic or preexisting differences in the epigenome occurring at repetitive regions of the Arabidopsis genome. Furthermore, the experience of repeated drought stress was not observed to influence transgenerational epi-allele accumulation. Our findings demonstrate that, while transgenerational memory is observed in one of six traits examined, they are not associated with causative changes in the DNA methylome, which appears relatively impervious to drought stress.

Rapid Recovery Gene Downregulation during Excess-Light Stress and Recovery in Arabidopsis.

Stress recovery may prove to be a promising approach to increase plant performance and, theoretically, mRNA instability may facilitate faster recovery. Transcriptome (RNA-seq, qPCR, sRNA-seq, and PARE) and methylome profiling during repeated excess-light stress and recovery was performed at intervals as short as 3 min. We demonstrate that 87% of the stress-upregulated mRNAs analyzed exhibit very rapid recovery. For instance, HSP101 abundance declined 2-fold every 5.1 min. We term this phenomenon rapid recovery gene downregulation (RRGD), whereby mRNA abundance rapidly decreases promoting transcriptome resetting. Decay constants (k) were modeled using two strategies, linear and nonlinear least squares regressions, with the latter accounting for both transcription and degradation. This revealed extremely short half-lives ranging from 2.7 to 60.0 min for 222 genes. Ribosome footprinting using degradome data demonstrated RRGD loci undergo cotranslational decay and identified changes in the ribosome stalling index during stress and recovery. However, small RNAs and 5'-3' RNA decay were not essential for recovery of the transcripts examined, nor were any of the six excess light-associated methylome changes. We observed recovery-specific gene expression networks upon return to favorable conditions and six transcriptional memory types. In summary, rapid transcriptome resetting is reported in the context of active recovery and cellular memory.

A chloroplast retrograde signal, 3'-phosphoadenosine 5'-phosphate, acts as a secondary messenger in abscisic acid signaling in stomatal closure and germination.

Organelle-nuclear retrograde signaling regulates gene expression, but its roles in specialized cells and integration with hormonal signaling remain enigmatic. Here we show that the SAL1-PAP (3'-phosphoadenosine 5'- phosphate) retrograde pathway interacts with abscisic acid (ABA) signaling to regulate stomatal closure and seed germination in Arabidopsis. Genetically or exogenously manipulating PAP bypasses the canonical signaling components ABA Insensitive 1 (ABI1) and Open Stomata 1 (OST1); priming an alternative pathway that restores ABA-responsive gene expression, ROS bursts, ion channel function, stomatal closure and drought tolerance in ost1-2. PAP also inhibits wild type and abi1-1 seed germination by enhancing ABA sensitivity. PAP-XRN signaling interacts with ABA, ROS and Ca2+; up-regulating multiple ABA signaling components, including lowly-expressed Calcium Dependent Protein Kinases (CDPKs) capable of activating the anion channel SLAC1. Thus, PAP exhibits many secondary messenger attributes and exemplifies how retrograde signals can have broader roles in hormone signaling, allowing chloroplasts to fine-tune physiological responses.

Uncoupling High Light Responses from Singlet Oxygen Retrograde Signaling and Spatial-Temporal Systemic Acquired Acclimation.

Distinct ROS signaling pathways initiated by singlet oxygen ((1)O2) or superoxide and hydrogen peroxide have been attributed to either cell death or acclimation, respectively. Recent studies have revealed that more complex antagonistic and synergistic relationships exist within and between these pathways. As specific chloroplastic ROS signals are difficult to study, rapid systemic signaling experiments using localized high light (HL) stress or ROS treatments were used in this study to uncouple signals required for direct HL and ROS perception and distal systemic acquired acclimation (SAA). A qPCR approach was chosen to determine local perception and distal signal reception. Analysis of a thylakoidal ascorbate peroxidase mutant (tapx), the (1)O2-retrograde signaling double mutant (ex1/ex2), and an apoplastic signaling double mutant (rbohD/F) revealed that tAPX and EXECUTER 1 are required for both HL and systemic acclimation stress perception. Apoplastic membrane-localized RBOHs were required for systemic spread of the signal but not for local signal induction in directly stressed tissues. Endogenous ROS treatments revealed a very strong systemic response induced by a localized 1 h induction of (1)O2 using the conditional flu mutant. A qPCR time course of (1)O2 induced systemic marker genes in directly and indirectly connected leaves revealed a direct vascular connection component of both immediate and longer term SAA signaling responses. These results reveal the importance of an EXECUTER-dependent (1)O2 retrograde signal for both local and long distance RBOH-dependent acclimation signaling that is distinct from other HL signaling pathways, and that direct vascular connections have a role in spatial-temporal SAA induction.

Reconsidering plant memory: Intersections between stress recovery, RNA turnover, and epigenetics.

Plants grow in dynamic environments where they can be exposed to a multitude of stressful factors, all of which affect their development, yield, and, ultimately, reproductive success. Plants are adept at rapidly acclimating to stressful conditions and are able to further fortify their defenses by retaining memories of stress to enable stronger or more rapid responses should an environmental perturbation recur. Indeed, one mechanism that is often evoked regarding environmental memories is epigenetics. Yet, there are relatively few examples of such memories; neither is there a clear understanding of their duration, considering the plethora of stresses in nature. We propose that this field would benefit from investigations into the processes and mechanisms enabling recovery from stress. An understanding of stress recovery could provide fresh insights into when, how, and why environmental memories are created and regulated. Stress memories may be maladaptive, hindering recovery and affecting development and potential yield. In some circumstances, it may be advantageous for plants to learn to forget. Accordingly, the recovery process entails a balancing act between resetting and memory formation. During recovery, RNA metabolism, posttranscriptional gene silencing, and RNA-directed DNA methylation have the potential to play key roles in resetting the epigenome and transcriptome and in altering memory. Exploration of this emerging area of research is becoming ever more tractable with advances in genomics, phenomics, and high-throughput sequencing methodology that will enable unprecedented profiling of high-resolution stress recovery time series experiments and sampling of large natural populations.

Insights into chloroplast biogenesis and development.

In recent years many advances have been made to obtain insight into chloroplast biogenesis and development. In plants several plastids types exist such as the proplastid (which is the progenitor of all plastids), leucoplasts (group of colourless plastids important for storage including elaioplasts (lipids), amyloplasts (starch) or proteinoplasts (proteins)), chromoplasts (yellow to orange-coloured due to carotenoids, in flowers or in old leaves as gerontoplasts), and the green chloroplasts. Chloroplasts are indispensable for plant development; not only by performing photosynthesis and thus rendering the plant photoautotrophic, but also for biochemical processes (which in some instances can also take place in other plastids types), such as the synthesis of pigments, lipids, and plant hormones and sensing environmental stimuli. Although we understand many aspects of these processes there are gaps in our understanding of the establishment of functional chloroplasts and their regulation. Why is that so? Even though chloroplast function is comparable in all plants and most of the algae, ferns and moss, detailed analyses have revealed many differences, specifically with respect to its biogenesis. As an update to our prior review on the genetic analysis of chloroplast biogenesis and development [1] herein we will focus on recent advances in Angiosperms (monocotyledonous and dicotyledonous plants) that provide novel insights and highlight the challenges and prospects for unravelling the regulation of chloroplast biogenesis specifically during the establishment of the young plants. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

Genetic suppression of plant development and chloroplast biogenesis via the Snowy Cotyledon 3 and Phytochrome B pathways.

Plant development is regulated by external and internal factors such as light and chloroplast development. A revertant of the Arabidopsis thaliana (L.) Heyhn. chloroplast biogenesis mutant snowy cotyledon 3 (sco3-1) was isolated partially recovering the impaired chloroplast phenotype. The mutation was identified in the Phytochrome B (PhyB) gene and is a result of an amino acid change within the PAS repeat domain required for light-induced nuclear localisation. An independent phyB-9 mutation was crossed into sco3-1 mutants, resulting in the same partial reversion of sco3-1. Further analysis demonstrated that SCO3 and PhyB influence the greening process of seedlings and rosette leaves, embryogenesis, rosette formation and flowering. Interestingly, the functions of these proteins are interwoven in various ways, suggesting a complex genetic interaction. Whole-transcriptome profiling of sco3-1phyB-9 indicated that a completely distinct set of genes was differentially regulated in the double mutant compared with the single sco3-1 or phyB-9 mutants. Thus, we hypothesise that PhyB and SCO3 genetically suppress each other in plant and chloroplast development.