Inference of long-range cell-cell force transmission from ECM remodeling fluctuations.

Cells sense, manipulate and respond to their mechanical microenvironment in a plethora of physiological processes, yet the understanding of how cells transmit, receive and interpret environmental cues to communicate with distant cells is severely limited due to lack of tools to quantitatively infer the complex tangle of dynamic cell-cell interactions in complicated environments. We present a computational method to systematically infer and quantify long-range cell-cell force transmission through the extracellular matrix (cell-ECM-cell communication) by correlating ECM remodeling fluctuations in between communicating cells and demonstrating that these fluctuations contain sufficient information to define unique signatures that robustly distinguish between different pairs of communicating cells. We demonstrate our method with finite element simulations and live 3D imaging of fibroblasts and cancer cells embedded in fibrin gels. While previous studies relied on the formation of a visible fibrous 'band' extending between cells to inform on mechanical communication, our method detected mechanical propagation even in cases where visible bands never formed. We revealed that while contractility is required, band formation is not necessary, for cell-ECM-cell communication, and that mechanical signals propagate from one cell to another even upon massive reduction in their contractility. Our method sets the stage to measure the fundamental aspects of intercellular long-range mechanical communication in physiological contexts and may provide a new functional readout for high content 3D image-based screening. The ability to infer cell-ECM-cell communication using standard confocal microscopy holds the promise for wide use and democratizing the method.

Overexpressing Vitamin C Defective 2 reduces fertility and alters Ca2+ signals in Arabidopsis pollen.

A potential strategy to mitigate oxidative damage in plants is to increase the abundance of antioxidants, such as ascorbate (i.e. vitamin C). In Arabidopsis (A. thaliana), a rate-limiting step in ascorbate biosynthesis is a phosphorylase encoded by Vitamin C Defective 2 (VTC2). To specifically overexpress VTC2 (VTC2 OE) in pollen, the coding region was expressed using a promoter from a gene with ∼150-fold higher expression in pollen, leading to pollen grains with an eight-fold increased VTC2 mRNA. VTC2 OE resulted in a near-sterile phenotype with a 50-fold decrease in pollen transmission efficiency and a five-fold reduction in the number of seeds per silique. In vitro assays revealed pollen grains were more prone to bursting (greater than two-fold) or produced shorter, morphologically abnormal pollen tubes. The inclusion of a genetically encoded Ca2+ reporter, mCherry-GCaMP6fast (CGf), revealed pollen tubes with altered tip-focused Ca2+ dynamics and increased bursting frequency during periods of oscillatory and arrested growth. Despite these phenotypes, VTC2 OE pollen failed to show expected increases in ascorbate or reductions in reactive oxygen species, as measured using a redox-sensitive dye or a roGFP2. However, mRNA expression analyses revealed greater than two-fold reductions in mRNA encoding two enzymes critical to biosynthetic pathways related to cell walls or glyco-modifications of lipids and proteins: GDP-d-mannose pyrophosphorylase (GMP) and GDP-d-mannose 3',5' epimerase (GME). These results support a model in which the near-sterile defects resulting from VTC2 OE in pollen are associated with feedback mechanisms that can alter one or more signaling or metabolic pathways critical to pollen tube growth and fertility.

Reproductive resilience: putting pollen grains in two baskets.

To ensure reproductive success, flowering plants produce an excess of pollen to fertilize a limited number of ovules. Pollen grains mature into two distinct subpopulations - those that display high metabolic activity and elevated reactive oxygen species (ROS) levels immediately after hydration (high-ROS/active), and those that maintain an extended period of dormancy with low metabolic activity (low-ROS/inactive/arrested/dormant). We propose that the dormant pollen serves as a backup to provide a second chance for successful fertilization when the 'first wave' of pollen encounters an unpredictable growth condition such as heat stress. This model provides a framework for considering the role of dormancy in reproductive stress tolerance as well as strategies for mitigating pollen thermovulnerability to daytime and night-time warming that is associated with global climate change.

Enhanced Reproductive Thermotolerance of the Tomato high pigment 2 Mutant Is Associated With Increased Accumulation of Flavonols in Pollen.

Climate change has created an environment where heat stress conditions are becoming more frequent as temperatures continue to raise in crop production areas around the world. This situation leads to decreased crop production due to plant sensitivity to heat stress. Reproductive success is critically dependent on plants' ability to produce functional pollen grains, which are the most thermo-sensitive tissue. Flavonols are plant secondary metabolites known for their potent antioxidative activity, essential for male fertility in several species including tomato, and implicated in heat stress tolerance. Since flavonols are highly abundant in fruits of the tomato high pigment 2 (hp2) mutant, we tested the level of flavonols in pollen of this mutant, under the hypothesis that increased accumulation of flavonols would render pollen more tolerant to heat stress. Indeed, pollen from two alleles of the hp2 mutant was found to have flavonols levels increased by 18 and 280% compared with wild-type (WT) under moderate chronic heat stress (MCHS) conditions. This mutant produced on average 7.8-fold higher levels of viable pollen and displayed better germination competence under heat stress conditions. The percentage of fully seeded fruits and the number of seeds per fruit were maintained in the mutant under heat stress conditions while decreased in wild-type plants. Our results strongly suggest that increased concentrations of pollen flavonols enhance pollen thermotolerance and reproductive success under heat stress conditions. Thus, the high flavonols trait may help frame the model for improving crop resilience to heat stress.

Exogenous Abscisic Acid Confers Salinity Tolerance in Chlamydomonas reinhardtii During Its Life Cycle.

The plant hormone abscisic acid (ABA) coordinates responses to environmental signals with developmental changes and is important for stress resilience and crop yield. However, fundamental questions remain about how this phytohormone affects microalgal growth and stress regulation throughout the different stages of their life cycle. In this study, the effects of ABA on the physiology of the freshwater microalga Chlamydomonas reinhardtii at its different life cycle stages were investigated. Exogenously added ABA enhanced the growth and photosynthesis of C. reinhardtii during the vegetative stage. The hormone also increased the tolerance of this alga to high-salinity stress during gamete formation under nutrient depletion, as well as it extended their survival. We show that the level of reactive oxygen species (ROS) generated in the ABA-treated cells was significantly less than that in the untreated cells under inhibiting NaCl concentrations. Cell size examination showed that ABA prevents cells from forming palmella when exposed to high salinity. All together, these results suggest that ABA can support the vitality and survival of C. reinhardtii under high salt conditions.

ASCORBATE PEROXIDASE6 delays the onset of age-dependent leaf senescence.

Age-dependent changes in reactive oxygen species (ROS) levels are critical in leaf senescence. While H2O2-reducing enzymes such as catalases and cytosolic ASCORBATE PEROXIDASE1 (APX1) tightly control the oxidative load during senescence, their regulation and function are not specific to senescence. Previously, we identified the role of ASCORBATE PEROXIDASE6 (APX6) during seed maturation in Arabidopsis (Arabidopsis thaliana). Here, we show that APX6 is a bona fide senescence-associated gene. APX6 expression is specifically induced in aging leaves and in response to senescence-promoting stimuli such as abscisic acid (ABA), extended darkness, and osmotic stress. apx6 mutants showed early developmental senescence and increased sensitivity to dark stress. Reduced APX activity, increased H2O2 level, and altered redox state of the ascorbate pool in mature pre-senescing green leaves of the apx6 mutants correlated with the early onset of senescence. Using transient expression assays in Nicotiana benthamiana leaves, we unraveled the age-dependent post-transcriptional regulation of APX6. We then identified the coding sequence of APX6 as a potential target of miR398, which is a key regulator of copper redistribution. Furthermore, we showed that mutants of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7), the master regulator of copper homeostasis and miR398 expression, have a higher APX6 level compared with the wild type, which further increased under copper deficiency. Our study suggests that APX6 is a modulator of ROS/redox homeostasis and signaling in aging leaves that plays an important role in developmental- and stress-induced senescence programs.

Characterization of novel pollen-expressed transcripts reveals their potential roles in pollen heat stress response in Arabidopsis thaliana.

KEY MESSAGE: Arabidopsis pollen transcriptome analysis revealed new intergenic transcripts of unknown function, many of which are long non-coding RNAs, that may function in pollen-specific processes, including the heat stress response. The male gametophyte is the most heat sensitive of all plant tissues. In recent years, long noncoding RNAs (lncRNAs) have emerged as important components of cellular regulatory networks involved in most biological processes, including response to stress. While examining RNAseq datasets of developing and germinating Arabidopsis thaliana pollen exposed to heat stress (HS), we identified 66 novel and 246 recently annotated intergenic expressed loci (XLOCs) of unknown function, with the majority encoding lncRNAs. Comparison with HS in cauline leaves and other RNAseq experiments indicated that 74% of the 312 XLOCs are pollen-specific, and at least 42% are HS-responsive. Phylogenetic analysis revealed that 96% of the genes evolved recently in Brassicaceae. We found that 50 genes are putative targets of microRNAs and that 30% of the XLOCs contain small open reading frames (ORFs) with homology to protein sequences. Finally, RNAseq of ribosome-protected RNA fragments together with predictions of periodic footprint of the ribosome P-sites indicated that 23 of these ORFs are likely to be translated. Our findings indicate that many of the 312 unknown genes might be functional and play a significant role in pollen biology, including the HS response.

A Ratiometric Calcium Reporter CGf Reveals Calcium Dynamics Both in the Single Cell and Whole Plant Levels Under Heat Stress.

Land plants evolved to quickly sense and adapt to temperature changes, such as hot days and cold nights. Given that calcium (Ca2+) signaling networks are implicated in most abiotic stress responses, heat-triggered changes in cytosolic Ca2+ were investigated in Arabidopsis leaves and pollen. Plants were engineered with a reporter called CGf, a ratiometric, genetically encoded Ca2+ reporter with an mCherry reference domain fused to an intensiometric Ca2+ reporter GCaMP6f. Relative changes in [Ca2+]cyt were estimated based on CGf's apparent K D around 220 nM. The ratiometric output provided an opportunity to compare Ca2+ dynamics between different tissues, cell types, or subcellular locations. In leaves, CGf detected heat-triggered cytosolic Ca2+ signals, comprised of three different signatures showing similarly rapid rates of Ca2+ influx followed by differing rates of efflux (50% durations ranging from 5 to 19 min). These heat-triggered Ca2+ signals were approximately 1.5-fold greater in magnitude than blue light-triggered signals in the same leaves. In contrast, growing pollen tubes showed two different heat-triggered responses. Exposure to heat caused tip-focused steady growth [Ca2+]cyt oscillations to shift to a pattern characteristic of a growth arrest (22%), or an almost undetectable [Ca2+]cyt (78%). Together, these contrasting examples of heat-triggered Ca2+ responses in leaves and pollen highlight the diversity of Ca2+ signals in plants, inviting speculations about their differing kinetic features and biological functions.

Large-Scale Analysis of Pollen Viability and Oxidative Level Using H2DCFDA-Staining Coupled with Flow Cytometry.

Determining pollen viability and other physiological parameters is of critical importance for evaluating the reproductive capacity of plants, both for fundamental and applied sciences. Flow cytometry is a powerful high-performance high-throughput tool for analyzing large populations of cells that has been in restricted use in plant cell research and in pollen-related studies, it has been minimized mostly for determination of DNA content. Recently, we developed a flow cytometry-based approach for robust and rapid evaluation of pollen viability that utilizes the reactive oxygen species (ROS) fluorescent reporter dye H2DCFDA (Luria et al., Plant J 98(5):942-952, 2019). This new approach revealed that pollen from Arabidopsis thaliana and Solanum lycopersicum naturally distribute into two subpopulations with different ROS levels. This method can be employed for a myriad of pollen-related studies, primarily in response to stimuli such as biotic or abiotic stress. In this chapter, we describe the protocol for H2DCFDA staining coupled with flow cytometry analysis providing specific guidelines. These guidelines are broadly applicable to many other types of cellular reporters to further develop this novel approach in the field of pollen biology.

Whole-Plant Live Imaging of Reactive Oxygen Species.

Reactive oxygen species (ROS) are key regulators of numerous subcellular, cellular, and systemic signals. They function in plants as an integral part of many different hormonal, physiological, and developmental pathways, as well as play a critical role in defense and acclimation responses to different biotic and abiotic conditions. Although many ROS imaging techniques have been developed and utilized in plants, a whole-plant imaging platform for the dynamic detection of ROS in mature plants is lacking. Here we report a robust and straightforward method for the whole-plant live imaging of ROS in mature plants grown in soil. This new method could be used to study local and systemic ROS signals in different genetic variants, conduct phenotyping studies to identify new pathways for ROS signaling, monitor the stress level of different plants and mutants, and unravel novel routes of ROS integration into stress, growth regulation, and development in plants. We demonstrate the utility of this new method for studying systemic ROS signals in different Arabidopsis mutants and wound responses in cereals such as wheat and corn.

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress.

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H2 DCFDA-staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into 'low-ROS' and 'high-ROS' subpopulations. Pollen germination assays following fluorescence-activated cell sorting revealed that the high-ROS pollen germinated with a frequency that was 35-fold higher than the low-ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose-dependent alterations in DCF-fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry-based approaches to investigate metabolic changes during stress responses in pollen.

A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16).

BACKGROUND: In flowering plants, the male gametophyte (pollen) is one of the most vulnerable cells to temperature stress. In Arabidopsis thaliana, a pollen-specific Cyclic Nucleotide-Gated cation Channel 16 (cngc16), is required for plant reproduction under temperature-stress conditions. Plants harboring a cncg16 knockout are nearly sterile under conditions of hot days and cold nights. To understand the underlying cause, RNA-Seq was used to compare the pollen transcriptomes of wild type (WT) and cngc16 under normal and heat stress (HS) conditions. RESULTS: Here we show that a heat-stress response (HSR) in WT pollen resulted in 2102 statistically significant transcriptome changes (≥ 2-fold changes with adjusted p-value ≤0.01), representing approximately 15% of 14,226 quantified transcripts. Of these changes, 89 corresponded to transcription factors, with 27 showing a preferential expression in pollen over seedling tissues. In contrast to WT, cngc16 pollen showed 1.9-fold more HS-dependent changes (3936 total, with 2776 differences between WT and cngc16). In a quantitative direct comparison between WT and cngc16 transcriptomes, the number of statistically significant differences increased from 21 pre-existing differences under normal conditions to 192 differences under HS. Of the 20 HS-dependent changes in WT that were most different in cngc16, half corresponded to genes encoding proteins predicted to impact cell wall features or membrane dynamics. CONCLUSIONS: Results here define an extensive HS-dependent reprogramming of approximately 15% of the WT pollen transcriptome, and identify at least 27 transcription factor changes that could provide unique contributions to a pollen HSR. The number of statistically significant transcriptome differences between WT and cngc16 increased by more than 9-fold under HS, with most of the largest magnitude changes having the potential to specifically impact cell walls or membrane dynamics, and thereby potentiate cngc16 pollen to be hypersensitive to HS. However, HS-hypersensitivity could also be caused by the extensive number of differences throughout the transcriptome having a cumulative effect on multiple cellular pathways required for tip growth and fertilization. Regardless, results here support a model in which a functional HS-dependent reprogramming of the pollen transcriptome requires a specific calcium-permeable Cyclic Nucleotide-Gated cation Channel, CNGC16.

SELENOPROTEIN O is a chloroplast protein involved in ROS scavenging and its absence increases dehydration tolerance in Arabidopsis thaliana.

The evolutionary conserved family of Selenoproteins performs redox-regulatory functions in bacteria, archaea and eukaryotes. Among them, members of the SELENOPROTEIN O (SELO) subfamily are located in mammalian and yeast mitochondria, but their functions are thus far enigmatic. Screening of T-DNA knockout mutants for resistance to the proline analogue thioproline (T4C), identified mutant alleles of the plant SELO homologue in Arabidopsis thaliana. Absence of SELO resulted in a stress-induced transcriptional activation instead of silencing of mitochondrial proline dehydrogenase, and also high elevation of Δ(1)-pyrroline-5-carboxylate dehydrogenase involved in degradation of proline, thereby alleviating T4C inhibition and lessening drought-induced proline accumulation. Unlike its animal homologues, SELO was localized to chloroplasts of plants ectopically expressing SELO-GFP. The protein was co-fractionated with thylakoid membrane complexes, and co-immunoprecipitated with FNR, PGRL1 and STN7, all involved in regulating PSI and downstream electron flow. The selo mutants displayed extended survival under dehydration, accompanied by longer photosynthetic activity, compared with wild-type plants. Enhanced expression of genes encoding ROS scavenging enzymes in the unstressed selo mutant correlated with higher oxidant scavenging capacity and reduced methyl viologen damage. The study elucidates SELO as a PSI-related component involved in regulating ROS levels and stress responses.

The IDA-LIKE peptides IDL6 and IDL7 are negative modulators of stress responses in Arabidopsis thaliana.

Small signalling peptides have emerged as important cell to cell messengers in plant development and stress responses. However, only a few of the predicted peptides have been functionally characterized. Here, we present functional characterization of two members of the IDA-LIKE (IDL) peptide family in Arabidopsis thaliana, IDL6 and IDL7. Localization studies suggest that the peptides require a signal peptide and C-terminal processing to be correctly transported out of the cell. Both IDL6 and IDL7 appear to be unstable transcripts under post-transcriptional regulation. Treatment of plants with synthetic IDL6 and IDL7 peptides resulted in down-regulation of a broad range of stress-responsive genes, including early stress-responsive transcripts, dominated by a large group of ZINC FINGER PROTEIN (ZFP) genes, WRKY genes, and genes encoding calcium-dependent proteins. IDL7 expression was rapidly induced by hydrogen peroxide, and idl7 and idl6 idl7 double mutants displayed reduced cell death upon exposure to extracellular reactive oxygen species (ROS). Co-treatment of the bacterial elicitor flg22 with IDL7 peptide attenuated the rapid ROS burst induced by treatment with flg22 alone. Taken together, our results suggest that IDL7, and possibly IDL6, act as negative modulators of stress-induced ROS signalling in Arabidopsis.

Orchestrating rapid long-distance signaling in plants with Ca2+ , ROS and electrical signals.

Plants show a rapid systemic response to a wide range of environmental stresses, where the signals from the site of stimulus perception are transmitted to distal organs to elicit plant-wide responses. A wide range of signaling molecules are trafficked through the plant, but a trio of potentially interacting messengers, reactive oxygen species (ROS), Ca2+ and electrical signaling ('trio signaling') appear to form a network supporting rapid signal transmission. The molecular components underlying this rapid communication are beginning to be identified, such as the ROS producing NAPDH oxidase RBOHD, the ion channel two pore channel 1 (TPC1), and glutamate receptor-like channels GLR3.3 and GLR3.6. The plant cell wall presents a plant-specific route for possible propagation of signals from cell to cell. However, the degree to which the cell wall limits information exchange between cells via transfer of small molecules through an extracellular route, or whether it provides an environment to facilitate transmission of regulators such as ROS or H+ remains to be determined. Similarly, the role of plasmodesmata as both conduits and gatekeepers for the propagation of rapid cell-to-cell signaling remains a key open question. Regardless of how signals move from cell to cell, they help prepare distant parts of the plant for impending challenges from specific biotic or abiotic stresses.

Reactive Oxygen Species Tune Root Tropic Responses.

The default growth pattern of primary roots of land plants is directed by gravity. However, roots possess the ability to sense and respond directionally to other chemical and physical stimuli, separately and in combination. Therefore, these root tropic responses must be antagonistic to gravitropism. The role of reactive oxygen species (ROS) in gravitropism of maize and Arabidopsis (Arabidopsis thaliana) roots has been previously described. However, which cellular signals underlie the integration of the different environmental stimuli, which lead to an appropriate root tropic response, is currently unknown. In gravity-responding roots, we observed, by applying the ROS-sensitive fluorescent dye dihydrorhodamine-123 and confocal microscopy, a transient asymmetric ROS distribution, higher at the concave side of the root. The asymmetry, detected at the distal elongation zone, was built in the first 2 h of the gravitropic response and dissipated after another 2 h. In contrast, hydrotropically responding roots show no transient asymmetric distribution of ROS Decreasing ROS levels by applying the antioxidant ascorbate, or the ROS-generation inhibitor diphenylene iodonium attenuated gravitropism while enhancing hydrotropism. Arabidopsis mutants deficient in Ascorbate Peroxidase 1 showed attenuated hydrotropic root bending. Mutants of the root-expressed NADPH oxidase RBOH C, but not rbohD, showed enhanced hydrotropism and less ROS in their roots apices (tested in tissue extracts with Amplex Red). Finally, hydrostimulation prior to gravistimulation attenuated the gravistimulated asymmetric ROS and auxin signals that are required for gravity-directed curvature. We suggest that ROS, presumably H2O2, function in tuning root tropic responses by promoting gravitropism and negatively regulating hydrotropism.

Desert Perennial Shrubs Shape the Microbial-Community Miscellany in Laimosphere and Phyllosphere Space.

Microbial function, composition, and distribution play a fundamental role in ecosystem ecology. The interaction between desert plants and their associated microbes is expected to greatly affect their response to changes in this harsh environment. Using comparative analyses, we studied the impact of three desert shrubs, Atriplex halimus (A), Artemisia herba-alba (AHA), and Hammada scoparia (HS), on soil- and leaf-associated microbial communities. DNA extracted from the leaf surface and soil samples collected beneath the shrubs were used to study associated microbial diversity using a sequencing survey of variable regions of bacterial 16S rRNA and fungal ribosomal internal transcribed spacer (ITS1). We found that the composition of bacterial and fungal orders is plant-type-specific, indicating that each plant type provides a suitable and unique microenvironment. The different adaptive ecophysiological properties of the three plant species and the differential effect on their associated microbial composition point to the role of adaptation in the shaping of microbial diversity. Overall, our findings suggest a link between plant ecophysiological adaptation as a "temporary host" and the biotic-community parameters in extreme xeric environments.

Identification of novel transcriptional regulators of Zat12 using comprehensive yeast one-hybrid screens.

To appropriately acclimate to environmental stresses, plants have to rapidly activate a specific transcriptional program. Yet, the identity and function of many of the transcriptional regulators that mediate early responses to abiotic stress stimuli is still unknown. In this work we employed the promoter of the multi-stress-responsive zinc-finger protein Zat12 in yeast one-hybrid (Y1H) screens to identify early abiotic stress-responsive transcriptional regulators. Analysis of Zat12 promoter fragments fused to luciferase underlined an approximately 200 bp fragment responsive to NaCl and to reactive oxygen species (ROS). Using these segments and others as baits against Y1H control or stress Arabidopsis prey libraries, we identified 15 potential Zat12 transcriptional regulators. Among the prominent proteins identified were known transcription factors including bZIP29 and ANAC91 as well as unknown function proteins such as a homolog of the human USB1, a U6 small nuclear RNA (snRNA) processing protein, and dormancy/auxin-associated family protein 2 (DRM2). Altered expression of Zat12 during high light stress in the knockout mutants further indicated the involvement of these proteins in the regulation of Zat12. Using a state of the art microfluidic approach we showed that AtUSB1 and DRM2 can specifically bind dsDNA and were able to identify the preferred DNA-binding motif of all four proteins. Overall, the proteins identified in this work provide an important start point for charting the earliest signaling network of Zat12 and of other genes required for acclimation to abiotic stresses.

Ultra-fast alterations in mRNA levels uncover multiple players in light stress acclimation in plants.

The acclimation of plants to changes in light intensity requires rapid responses at several different levels. These include biochemical and biophysical responses as well as alterations in the steady-state level of different transcripts and proteins. Recent studies utilizing promoter::reporter constructs suggested that transcriptional responses to changes in light intensity could occur within seconds, rates for which changes in mRNA expression are not routinely measured or functionally studied. To identify and characterize rapid changes in the steady-state level of different transcripts in response to light stress we performed RNA sequencing analysis of Arabidopsis thaliana plants subjected to light stress. Here we report that mRNA accumulation of 731 transcripts occurs as early as 20-60 sec following light stress application, and that at least five of these early response transcripts play an important biological role in the acclimation of plants to light stress. More than 20% of transcripts accumulating in plants within 20-60 sec of initiation of light stress are H2 O2 - and ABA-response transcripts, and the accumulation of several of these transcripts is inhibited by transcriptional inhibitors. In accordance with the association of rapid response transcripts with H2 O2 and ABA signaling, a mutant impaired in ABA sensing (abi-1) was found to be more tolerant to light stress, and the response of several of the rapid response transcripts was altered in mutants impaired in reactive oxygen metabolism. Our findings reveal that transcriptome reprogramming in plants could occur within seconds of initiation of abiotic stress and that this response could invoke known as well as unknown proteins and pathways.