Regulatory networks in plant responses to drought and cold stress.

Drought and cold represent distinct types of abiotic stress, each initiating unique primary signaling pathways in response to dehydration and temperature changes, respectively. However, a convergence at the gene regulatory level is observed where a common set of stress-responsive genes is activated to mitigate the impacts of both stresses. In this review, we explore these intricate regulatory networks, illustrating how plants coordinate distinct stress signals into a collective transcriptional strategy. We delve into the molecular mechanisms of stress perception, stress signaling, and the activation of gene regulatory pathways, with a focus on insights gained from model species. By elucidating both the shared and distinct aspects of plant responses to drought and cold, we provide insight into the adaptive strategies of plants, paving the way for the engineering of stress-resilient crop varieties that can withstand a changing climate.

BIL9 Promotes Both Plant Growth via BR Signaling and Drought Stress Resistance by Binding with the Transcription Factor HDG11.

Drought stress is a major threat leading to global plant and crop losses in the context of the climate change crisis. Brassinosteroids (BRs) are plant steroid hormones, and the BR signaling mechanism in plant development has been well elucidated. Nevertheless, the specific mechanisms of BR signaling in drought stress are still unclear. Here, we identify a novel Arabidopsis gene, BRZ INSENSITIVE LONG HYPOCOTYL 9 (BIL9), which promotes plant growth via BR signaling. Overexpression of BIL9 enhances drought and mannitol stress resistance and increases the expression of drought-responsive genes. BIL9 protein is induced by dehydration and interacts with the HD-Zip IV transcription factor HOMEODOMAIN GLABROUS 11 (HDG11), which is known to promote plant resistance to drought stress, in vitro and in vivo. BIL9 enhanced the transcriptional activity of HDG11 for drought-stress-resistant genes. BIL9 is a novel BR signaling factor that enhances both plant growth and plant drought resistance.

Complex plant responses to drought and heat stress under climate change.

Global climate change is predicted to result in increased yield losses of agricultural crops caused by environmental conditions. In particular, heat and drought stress are major factors that negatively affect plant development and reproduction, and previous studies have revealed how these stresses induce plant responses at physiological and molecular levels. Here, we provide a comprehensive overview of current knowledge concerning how drought, heat, and combinations of these stress conditions affect the status of plants, including crops, by affecting factors such as stomatal conductance, photosynthetic activity, cellular oxidative conditions, metabolomic profiles, and molecular signaling mechanisms. We further discuss stress-responsive regulatory factors such as transcription factors and signaling factors, which play critical roles in adaptation to both drought and heat stress conditions and potentially function as 'hubs' in drought and/or heat stress responses. Additionally, we present recent findings based on forward genetic approaches that reveal natural variations in agricultural crops that play critical roles in agricultural traits under drought and/or heat conditions. Finally, we provide an overview of the application of decades of study results to actual agricultural fields as a strategy to increase drought and/or heat stress tolerance. This review summarizes our current understanding of plant responses to drought, heat, and combinations of these stress conditions.

The ability to induce heat shock transcription factor-regulated genes in response to lethal heat stress is associated with thermotolerance in tomato cultivars.

Heat stress is a severe challenge for plant production, and the use of thermotolerant cultivars is critical to ensure stable production in high-temperature-prone environments. However, the selection of thermotolerant cultivars is difficult due to the complex nature of heat stress and the time and space needed for evaluation. In this study, we characterized genome-wide differences in gene expression between thermotolerant and thermosensitive tomato cultivars and examined the possibility of selecting gene expression markers to estimate thermotolerance among different tomato cultivars. We selected one thermotolerant and one thermosensitive cultivar based on physiological evaluations and compared heat-responsive gene expression in these cultivars under stepwise heat stress and acute heat shock conditions. Transcriptomic analyses reveled that two heat-inducible gene expression pathways, controlled by the heat shock element (HSE) and the evening element (EE), respectively, presented different responses depending on heat stress conditions. HSE-regulated gene expression was induced under both conditions, while EE-regulated gene expression was only induced under gradual heat stress conditions in both cultivars. Furthermore, HSE-regulated genes showed higher expression in the thermotolerant cultivar than the sensitive cultivar under acute heat shock conditions. Then, candidate expression biomarker genes were selected based on the transcriptome data, and the usefulness of these candidate genes was validated in five cultivars. This study shows that the thermotolerance of tomato is correlated with its ability to maintain the heat shock response (HSR) under acute severe heat shock conditions. Furthermore, it raises the possibility that the robustness of the HSR under severe heat stress can be used as an indicator to evaluate the thermotolerance of crop cultivars.

Constitutively active B2 Raf-like kinases are required for drought-responsive gene expression upstream of ABA-activated SnRK2 kinases.

Osmotic stresses, such as drought and high salinity, adversely affect plant growth and productivity. The phytohormone abscisic acid (ABA) accumulates in response to osmotic stress and enhances stress tolerance in plants by triggering multiple physiological responses through ABA signaling. Subclass III SNF1-related protein kinases 2 (SnRK2s) are key regulators of ABA signaling. Although SnRK2s have long been considered to be self-activated by autophosphorylation after release from PP2C-mediated inhibition, they were recently revealed to be activated by two independent subfamilies of group B Raf-like kinases, B2-RAFs and B3-RAFs, under osmotic stress conditions. However, the relationship between SnRK2 phosphorylation by these RAFs and SnRK2 autophosphorylation and the individual physiological roles of each RAF subfamily remain unknown. In this study, we indicated that B2-RAFs are constantly active and activate SnRK2s when released from PP2C-mediated inhibition by ABA-binding ABA receptors, whereas B3-RAFs are activated only under stress conditions in an ABA-independent manner and enhance SnRK2 activity. Autophosphorylation of subclass III SnRK2s is not sufficient for ABA responses, and B2-RAFs are needed to activate SnRK2s in an ABA-dependent manner. Using plants grown in soil, we found that B2-RAFs regulate subclass III SnRK2s at the early stage of drought stress, whereas B3-RAFs regulate SnRK2s at the later stage. Thus, B2-RAFs are essential kinases for the activation of subclass III SnRK2s in response to ABA under mild osmotic stress conditions, and B3-RAFs function as enhancers of SnRK2 activity under severe stress conditions.

Clock-regulated coactivators selectively control gene expression in response to different temperature stress conditions in Arabidopsis.

Plants respond to severe temperature changes by inducing the expression of numerous genes whose products enhance stress tolerance and responses. Dehydration-responsive element (DRE)-binding protein 1/C-repeat binding factor (DREB1/CBF) transcription factors act as master switches in cold-inducible gene expression. Since DREB1 genes are rapidly and strongly induced by cold stress, the elucidation of the molecular mechanisms of DREB1 expression is vital for the recognition of the initial responses to cold stress in plants. A previous study indicated that the circadian clock-related MYB-like transcription factors REVEILLE4/LHY-CCA1-Like1 (RVE4/LCL1) and RVE8/LCL5 directly activate DREB1 expression under cold stress conditions. These RVEs function in the regulation of circadian clock-related gene expression under normal temperature conditions. They also activate the expression of HSF-independent heat-inducible genes under high-temperature conditions. Thus, there are thought to be specific regulatory mechanisms whereby the target genes of these transcription factors are switched when temperature changes are sensed. We revealed that NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED (LNK) proteins act as coactivators of RVEs in cold and heat stress responses in addition to regulating circadian-regulated genes at normal temperatures. We found that among the four Arabidopsis LNKs, LNK1 and LNK2 function under normal and high-temperature conditions, and LNK3 and LNK4 function under cold conditions. Thus, these LNK proteins play important roles in inducing specific genes under different temperature conditions. Furthermore, LNK3 and LNK4 are specifically phosphorylated under cold conditions, suggesting that phosphorylation is involved in their activation.

A de novo gene originating from the mitochondria controls floral transition in Arabidopsis thaliana.

De novo genes created in the plant mitochondrial genome have frequently been transferred into the nuclear genome via intergenomic gene transfer events. Therefore, plant mitochondria might be a source of de novo genes in the nuclear genome. However, the functions of de novo genes originating from mitochondria and the evolutionary fate remain unclear. Here, we revealed that an Arabidopsis thaliana specific small coding gene derived from the mitochondrial genome regulates floral transition. We previously identified 49 candidate de novo genes that induce abnormal morphological changes on overexpression. We focused on a candidate gene derived from the mitochondrial genome (sORF2146) that encodes 66 amino acids. Comparative genomic analyses indicated that the mitochondrial sORF2146 emerged in the Brassica lineage as a de novo gene. The nuclear sORF2146 emerged following an intergenomic gene transfer event in the A. thaliana after the divergence between Arabidopsis and Capsella. Although the nuclear and mitochondrial sORF2146 sequences are the same in A. thaliana, only the nuclear sORF2146 is transcribed. The nuclear sORF2146 product is localized in mitochondria, which may be associated with the pseudogenization of the mitochondrial sORF2146. To functionally characterize the nuclear sORF2146, we performed a transcriptomic analysis of transgenic plants overexpressing the nuclear sORF2146. Flowering transition-related genes were highly regulated in the transgenic plants. Subsequent phenotypic analyses demonstrated that the overexpression and knockdown of sORF2146 in transgenic plants resulted in delayed and early flowering, respectively. These findings suggest that a lineage-specific de novo gene derived from mitochondria has an important regulatory effect on floral transition.

ER Stress and the Unfolded Protein Response: Homeostatic Regulation Coordinate Plant Survival and Growth.

The endoplasmic reticulum (ER), a eukaryotic organelle, is the major site of protein biosynthesis. The disturbance of ER function by biotic or abiotic stress triggers the accumulation of misfolded or unfolded proteins in the ER. The unfolded protein response (UPR) is the best-studied ER stress response. This transcriptional regulatory system senses ER stress, activates downstream genes that function to mitigate stress, and restores homeostasis. In addition to its conventional role in stress responses, recent reports indicate that the UPR is involved in plant growth and development. In this review, we summarize the current knowledge of ER stress sensing and the activation and downstream regulation of the UPR. We also describe how the UPR modulates both plant growth and stress tolerance by maintaining ER homeostasis. Lastly, we propose that the UPR is a major component of the machinery that balances the trade-off between plant growth and survival in a dynamic environment.

Functional genomics in plant abiotic stress responses and tolerance: From gene discovery to complex regulatory networks and their application in breeding.

Land plants have developed sophisticated systems to cope with severe stressful environmental conditions during evolution. Plants have complex molecular systems to respond and adapt to abiotic stress, including drought, cold, and heat stress. Since 1989, we have been working to understand the complex molecular mechanisms of plant responses to severe environmental stress conditions based on functional genomics approaches with Arabidopsis thaliana as a model plant. We focused on the function of drought-inducible genes and the regulation of their stress-inducible transcription, perception and cellular signal transduction of stress signals to describe plant stress responses and adaptation at the molecular and cellular levels. We have identified key genes and factors in the regulation of complex responses and tolerance of plants in response to dehydration and temperature stresses. In this review article, we describe our 30-year experience in research and development based on functional genomics to understand sophisticated systems in plant response and adaptation to environmental stress conditions.

Ethanol-Mediated Novel Survival Strategy against Drought Stress in Plants.

Water scarcity is a serious agricultural problem causing significant losses to crop yield and product quality. The development of technologies to mitigate the damage caused by drought stress is essential for ensuring a sustainable food supply for the increasing global population. We herein report that the exogenous application of ethanol, an inexpensive and environmentally friendly chemical, significantly enhances drought tolerance in Arabidopsis thaliana, rice and wheat. The transcriptomic analyses of ethanol-treated plants revealed the upregulation of genes related to sucrose and starch metabolism, phenylpropanoids and glucosinolate biosynthesis, while metabolomic analysis showed an increased accumulation of sugars, glucosinolates and drought-tolerance-related amino acids. The phenotyping analysis indicated that drought-induced water loss was delayed in the ethanol-treated plants. Furthermore, ethanol treatment induced stomatal closure, resulting in decreased transpiration rate and increased leaf water contents under drought stress conditions. The ethanol treatment did not enhance drought tolerance in the mutant of ABI1, a negative regulator of abscisic acid (ABA) signaling in Arabidopsis, indicating that ABA signaling contributes to ethanol-mediated drought tolerance. The nuclear magnetic resonance analysis using 13C-labeled ethanol indicated that gluconeogenesis is involved in the accumulation of sugars. The ethanol treatment did not enhance the drought tolerance in the aldehyde dehydrogenase (aldh) triple mutant (aldh2b4/aldh2b7/aldh2c4). These results show that ABA signaling and acetic acid biosynthesis are involved in ethanol-mediated drought tolerance and that chemical priming through ethanol application regulates sugar accumulation and gluconeogenesis, leading to enhanced drought tolerance and sustained plant growth. These findings highlight a new survival strategy for increasing crop production under water-limited conditions.

Transcriptional regulatory network of plant cold-stress responses.

Recent studies have revealed the complex and flexible transcriptional regulatory network involved in cold-stress responses. Focusing on two major signaling pathways that respond to cold stress, we outline current knowledge of the transcriptional regulatory network and the post-translational regulation of transcription factors in the network. Cold-stress signaling pathways are closely associated with other signaling pathways such as those related to the circadian clock, and large amounts of data on their crosstalk and tradeoffs are available. However, it remains unknown how plants sense and transmit cold-stress signals to regulate gene expression. We discuss recent reports on cold-stress sensing and associated signaling pathways that regulate the network. We also emphasize future directions for developing abiotic stress-tolerant crop plants.

Characterization of photosystem II assembly complexes containing ONE-HELIX PROTEIN1 in Arabidopsis thaliana.

The assembly process of photosystem II (PSII) requires several auxiliary proteins to form assembly intermediates. In plants, early assembly intermediates comprise D1 and D2 subunits of PSII together with a few auxiliary proteins including at least ONE-HELIX PROTEIN1 (OHP1), OHP2, and HIGH-CHLOROPHYLL FLUORESCENCE 244 (HCF244) proteins. Herein, we report the basic characterization of the assembling intermediates, which we purified from Arabidopsis transgenic plants overexpressing a tagged OHP1 protein and named the OHP1 complexes. We analyzed two major forms of OHP1 complexes by mass spectrometry, which revealed that the complexes consist of OHP1, OHP2, and HCF244 in addition to the PSII subunits D1, D2, and cytochrome b559. Analysis of chlorophyll fluorescence showed that a major form of the complex binds chlorophyll a and carotenoids and performs quenching with a time constant of 420 ps. To identify the localization of the auxiliary proteins, we solubilized thylakoid membranes using a digitonin derivative, glycodiosgenin, and separated them into three fractions by ultracentrifugation, and detected these proteins in the loose pellet containing the stroma lamellae and the grana margins together with two chlorophyll biosynthesis enzymes. The results indicated that chlorophyll biosynthesis and assembly may take place in the same compartments of thylakoid membranes. Inducible suppression of the OHP2 mRNA substantially decreased the OHP2 protein in mature Arabidopsis leaves without a significant reduction in the maximum quantum yield of PSII under low-light conditions, but it compromised the yields under high-light conditions. This implies that the auxiliary protein is required for acclimation to high-light conditions.

Affinity Purification Followed by Liquid Chromatography-Tandem Mass Spectrometry to Identify Proteins Interacting with ABA Signaling Components.

Abscisic acid (ABA) is a key phytohormone involved in plant development, seed germination and responses to osmotic stresses, such as drought and high salinity. SNF1-related protein kinases (SnRK2s) play important roles in ABA-dependent and ABA-independent osmotic stress signaling. SnRK2s phosphorylate transcription factors and ion channels in response to ABA or osmotic stress to induce the expression of stress-responsive genes and stomatal closure, respectively, to confer osmotic stress tolerance. The activity of SnRK2s is directly or indirectly regulated by several protein factors. Identification of downstream substrates or upstream regulators of SnRK2s is very useful for elucidating protein components that regulate ABA and osmotic stress signaling. Here, we describe the use of affinity purification by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry to identify protein complexes involved in ABA and osmotic stress signaling in plants. We previously identified several protein factors that regulate ABA and osmotic stress signaling by using this method.

Arabidopsis TBP-ASSOCIATED FACTOR 12 ortholog NOBIRO6 controls root elongation with unfolded protein response cofactor activity.

Plant root growth is indeterminate but continuously responds to environmental changes. We previously reported on the severe root growth defect of a double mutant in bZIP17 and bZIP28 (bz1728) modulating the unfolded protein response (UPR). To elucidate the mechanism by which bz1728 seedlings develop a short root, we obtained a series of bz1728 suppressor mutants, called nobiro, for rescued root growth. We focused here on nobiro6, which is defective in the general transcription factor component TBP-ASSOCIATED FACTOR 12b (TAF12b). The expression of hundreds of genes, including the bZIP60-UPR regulon, was induced in the bz1728 mutant, but these inductions were markedly attenuated in the bz1728nobiro6 mutant. In view of this, we assigned transcriptional cofactor activity via physical interaction with bZIP60 to NOBIRO6/TAF12b. The single nobiro6/taf12b mutant also showed an altered sensitivity to endoplasmic reticulum stress for both UPR and root growth responses, demonstrating that NOBIRO6/TAF12b contributes to environment-responsive root growth control through UPR.

CIN-like TCP13 is essential for plant growth regulation under dehydration stress.

KEY MESSAGE: A dehydration-inducible Arabidopsis CIN-like TCP gene, TCP13, acts as a key regulator of plant growth in leaves and roots under dehydration stress conditions. Plants modulate their shape and growth in response to environmental stress. However, regulatory mechanisms underlying the changes in shape and growth under environmental stress remain elusive. The CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family of transcription factors (TFs) are key regulators for limiting the growth of leaves through negative effect of auxin response. Here, we report that stress-inducible CIN-like TCP13 plays a key role in inducing morphological changes in leaves and growth regulation in leaves and roots that confer dehydration stress tolerance in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing TCP13 (35Spro::TCP13OX) exhibited leaf rolling, and reduced leaf growth under osmotic stress. The 35Spro::TCP13OX transgenic leaves showed decreased water loss from leaves, and enhanced dehydration tolerance compared with their control counterparts. Plants overexpressing a chimeric repressor domain SRDX-fused TCP13 (TCP13pro::TCP13SRDX) showed severely serrated leaves and enhanced root growth. Transcriptome analysis of TCP13pro::TCP13SRDX transgenic plants revealed that TCP13 affects the expression of dehydration- and abscisic acid (ABA)-regulated genes. TCP13 is also required for the expression of dehydration-inducible auxin-regulated genes, INDOLE-3-ACETIC ACID5 (IAA5) and LATERAL ORGAN BOUNDARIES (LOB) DOMAIN 1 (LBD1). Furthermore, tcp13 knockout mutant plants showed ABA-insensitive root growth and reduced dehydration-inducible gene expression. Our findings provide new insight into the molecular mechanism of CIN-like TCP that is involved in both auxin and ABA response under dehydration stress.

Inter-tissue and inter-organ signaling in drought stress response and phenotyping of drought tolerance.

Plant response to drought stress includes systems for intracellular regulation of gene expression and signaling, as well as inter-tissue and inter-organ signaling, which helps entire plants acquire stress resistance. Plants sense water-deficit conditions both via the stomata of leaves and roots, and transfer water-deficit signals from roots to shoots via inter-organ signaling. Abscisic acid is an important phytohormone involved in the drought stress response and adaptation, and is synthesized mainly in vascular tissues and guard cells of leaves. In leaves, stress-induced abscisic acid is distributed to various tissues by transporters, which activates stomatal closure and expression of stress-related genes to acquire drought stress resistance. Moreover, the stepwise stress response at the whole-plant level is important for proper understanding of the physiological response to drought conditions. Drought stress is sensed by multiple types of sensors as molecular patterns of abiotic stress signals, which are transmitted via separate parallel signaling networks to induce downstream responses, including stomatal closure and synthesis of stress-related proteins and metabolites. Peptide molecules play important roles in the inter-organ signaling of dehydration from roots to shoots, as well as signaling of osmotic changes and reactive oxygen species/Ca2+ . In this review, we have summarized recent advances in research on complex plant drought stress responses, focusing on inter-tissue signaling in leaves and inter-organ signaling from roots to shoots. We have discussed the mechanisms via which drought stress adaptations and resistance are acquired at the whole-plant level, and have proposed the importance of quantitative phenotyping for measuring plant growth under drought conditions.

Corrigendum: Drought Stress Responses and Resistance in Plants: From Cellular Responses to Long-Distance Intercellular Communication.

[This corrects the article DOI: 10.3389/fpls.2020.556972.].