Plant Physiology

It is known that the transcription factor SPEECHLESS (SPCH) drives entry of epidermal cells into stomatal lineage, and that the activation of subtilisin-like protease SDD1 reduces stomatal density and cluster on the epidermis. However, there is still a big gap in our understanding of the relationship between stomatal development and the establishment of stomatal density and pattern, especially during drought. Interestingly, 1-aminocyclopropane-1-carboxylic acid (ACC) not only promotes stomatal development, but also is involved in the establishment of stomatal density and pattern. ACC generation comes from the activity of ACC synthase (ACS), while ACS activity could be mediated by drought. This work showed that the Arabidopsis SPCH activated ACS2/6 expression and ACC-dependent stomatal generation with an increase of stomatal density and cluster under drought conditions; and the possible mechanisms were that ACC-induced Ca2+ shortage in stomatal lineage reduced the inhibition of the transcription factor GT-2 Like 1 (GTL1) on SDD1 expression. These suggest that ACS2/6-dependent ACC accumulation integrated stomatal development with the establishment of stomatal density and pattern by mediating Ca2+ levels in stomatal lineage cells on the leaf epidermis, and this integration is directly related to the growth or survival of plants under escalated drought stress. HighlightACC synthase ACS2/6 activation integrated stomatal individual development with space setting between stomata by mediating Ca2+ levels in stomatal lineage on the leaf epidermis in response to drought.

Cuscuta, commonly known as dodder, is a parasitic plant that thrives by attaching itself to the stems of other plants. It is found across the globe and is notorious for obstructing crop growth as a weed. Over the past decade, Cuscuta has been used to gain insights into plant-plant interactions and molecular trafficking. Here, we report that two plants connected via a Cuscuta bridge can exchange rapid systemic calcium, electric, and reactive oxygen species signals. These findings suggest that plant interactions with Cuscuta may have beneficial effects to plants, enabling them to rapidly communicate with each other.

Sensing of heat, high light (HL), or mechanical injury by a single leaf of a plant results in the activation of different systemic signals that reach systemic tissues within minutes and trigger systemic acquired acclimation (SAA) or systemic wound responses (SWRs), resulting in a heightened state of stress readiness of the entire plant. Among the different signals associated with rapid systemic responses to stress in plants are electric, calcium and reactive oxygen species (ROS) waves. These signals propagate from the stressed or injured leaf to the rest of the plant through the plant vascular bundles, and trigger SWRs and SAA in systemic tissues. However, whether they can propagate through other cell types, and whether or not they are interlinked, remain open questions. Here we report that in response to wounding or heat stress (HS), but not HL stress, the ROS wave can propagate through mesophyll cells of Arabidopsis thaliana. Moreover, we show that propagation of the ROS wave through mesophyll cells during these stresses is sufficient to restore SWR and SAA transcript accumulation in systemic leaves, as well as SAA to HS (but not HL). We further show that propagation of the ROS wave through mesophyll cells could contribute to systemic signal integration during HL&HS stress combination. Our findings reveal that the ROS wave can propagate through tissues other than the vascular bundles of plants, and that different stresses can trigger different types of systemic signals that propagate through different cell layers and induce stress-specific systemic responses. One-sentence summaryIn addition to vascular bundles, mesophyll cells can mediate the ROS wave during systemic responses to wounding or heat stress in Arabidopsis.

PHYTOCHROME INTERACTING FACTORS (PIFs) are transcription factors that interact with the photoreceptors phytochromes and integrate multiple signaling pathways related to light, temperature, defense and hormone responses. PIFs have been extensively studied in Arabidopsis thaliana, but less is known about their roles in other species. Here, we investigate the role of the two homologs of PIF1 found in tomato (Solanum lycopersicum), namely PIF1a and PIF1b. Analysis of gene expression showed very different patterns, indicating a potential evolutionary divergence in their roles. At the protein level, light regulated the stability of PIF1a, but not PIF1b, further supporting a functional divergence. Phenotypic analyses of CRISPR-Cas9-generated tomato mutants defective in PIF1a or PIF1b or both revealed conserved and newly acquired roles compared to Arabidopsis PIF1. Both PIF1a or PIF1b were found to regulate seed germination, photosynthetic pigment biosynthesis and fruit production. However, only PIF1a-defective mutants showed defects on root hair elongation, flowering time and fruit growth and softening. We did not identify any process altered only in plants lacking PIF1b. Together, these data show that neofunctionalization has taken place in tomato, illustrating the potential of these transcription factors to acquiring new roles in different species.

Protein targeting and insertion into membranes are essential for cellular organization and organelle function. The Guided Entry of Tail-anchored (GET) pathway facilitates the post-translational targeting and insertion of tail-anchored (TA) membrane proteins. Arabidopsis thaliana has four GET3 homologues, including AtGET3B and AtGET3D localized to chloroplasts. These photosynthetic organelles possess complex membrane systems, and the mechanisms underlying their protein targeting and membrane biogenesis are not fully understood. This study conducted a comprehensive proteomic analysis of get3b mutant plastids, which displayed significant alterations. Fluorometric based complex assembly as well as CO2 assimilation analyses confirmed that disruption of GET3B function displayed a significant impact on photosystem II assembly as well as carbon fixation, respectively, indicating a functional role in chloroplast biogenesis. Additionally, genetic interactions were found between GET3B and the two component STIC system, which cooperates with the cpSRP pathway and is involved in the co-translational sorting of thylakoid proteins. Further, physical interactions were observed between GET3B and the C-terminus of ALB3 and ALB4 in vitro and the full length proteins in vivo, indicating a role of GET3B in protein targeting and membrane integration within chloroplasts. These findings enhance our understanding of GET3Bs involvement in stromal protein targeting and thylakoidal biogenesis.

Pollen tubes exhibit one of the fastest apical growth rates among plant cells. Maintaining the proper balance between turgor pressure and cell wall synthesis at the pollen tube tip is crucial for this rapid growth, and any disruption can result in pollen tube rupture. In our study, we reveal that exposure to short-wavelength visible light, specifically blue light, induces pollen tube rupture. The frequency of pollen tube rupture increases in an intensity-dependent manner. Additionally, we observed Ca2+ influx after blue light irradiation, accompanying with either pollen tube rupture or a temporary halt in elongation. These findings offer insights into the interplay between pollen tube integrity maintenance and Ca2+ influx at the pollen tube tip, presenting a novel and efficient method to control pollen tube burst. Subject Areas(1) growth and development (11) new methodology

Ripening is a complex developmental change of a mature organ, the fruit. In plants like a tomato, it involves softening, pigmentation, and biosynthesis of metabolites beneficial for the human diet. Examination of the transcriptional changes towards ripening suggests that redundant uncharacterized factors may be involved in the coordination of the ripening switch. Previous studies have demonstrated that Arabidopsis CLASS-II KNOX genes play a significant role in controlling the maturation of siliques and their transition to senescence. Here we examined the combined role of all four tomato CLASS-II KNOX genes in the maturation and ripening of fleshy fruits using an artificial microRNA targeting them simultaneously. As expected, the knockdown plants (35S::amiR-TKN-CL-II) exhibited leaves with increased complexity, reminiscent of the leaf phenotype of plants overexpressing CLASS-I KNOX, which antagonize CLASS-II KNOX gene functions. The fruits of 35S::amiR-TKN-CL-II plants were notably smaller than the control. While their internal gel/placenta tissue softened and accumulated the typical pigmentation, the pericarp color break took place ten days later than control, and eventually, it turned yellow instead of red. Additionally, the pericarp of 35S::amiR-TKN-CL-II fruits remained significantly firmer than control even after three weeks of shelf storage. Strikingly, the 35S::amiR-TKN-CL-II fruits showed early ethylene release and respiration peak, but these were correlated only with liquefaction and pigmentation of the internal tissues. Our findings suggest that CLASS-II KNOX genes are required to coordinate the spatial and temporal patterns of tomato fruit ripening. One sentence summaryTomato CLASS-II KNOX genes play antagonistic roles in the regulation of ripening at the internal fruit domains and pericarp.

Responding to the environment is a core aspect of plant growth and development. Mounting an effective response is important for plants to balance growth and survival. The HEAT SHOCK FACTOR (HSF) transcription factor family is a central and required component of plant heat stress responses and acquired thermotolerance. The HSF family has dramatically expanded in plant lineages, often including a repertoire of 20 or more genes. Here we assess the composition and heat responsiveness of the HSF family in Setaria viridis (Setaria), a model C4 panicoid grass, and make targeted comparisons between the HSF families of Setaria and maize. Examples of both conserved and variable expression responses to a heat stress event were observed when comparing the two species. Novel and existing data on chromatin accessibility, histone modifications, and genome-wide DNA binding profiles were utilized to assess the chromatin of HSF family members with distinct responses to heat stress. We observed significant variability for both expression and chromatin state within syntenic and orthologous sets of HSFs between Setaria and maize, as well as between syntenic pairs of maize HSFs retained following its most recent genome duplication event. These observations collectively support a complex scenario of expansion and sub-functionalization within this transcription factor family that has significant untapped potential for better understanding the evolution of large gene families. Significance StatementA comparison of the Heat Shock Factor transcription factors in maize and Setaria reveals examples of consistent and variable expression responses to heat stress and provides insights into the role of chromatin in predicting expression responses.

Deciphering signal transduction processes is crucial for understanding how plants sense and respond to environmental changes. Various chemical compounds function as central messengers within deeply intertwined signaling networks. How such compounds act in concert remains to be elucidated. We have developed dual-sensing genetically encoded fluorescent indicators (2-In-1-GEFIs) for multiparametric in vivo analyses of the phytohormone abscisic acid (ABA), Ca2+, protons (H+), chloride (anions), the glutathione redox potential (EGSH) and hydrogen peroxide (H2O2). Simultaneous analyses of two signaling compounds in Arabidopsis (Arabidopsis thaliana) roots revealed that ABA treatment and uptake did not trigger rapid cytosolic Ca2+ or H+ fluxes. Glutamate, ATP, Arabidopsis PLANT ELICITOR PEPTIDE (AtPEP1) and glutathione disulfide (GSSG) treatments induced rapid spatiotemporally overlapping cytosolic Ca2+, H+ and anion fluxes, but except for GSSG only weakly affected the cytosolic redox state. Overall, 2-In-1-GEFIs enable complementary high-resolution in vivo analyses of signaling compound dynamics and facilitate an advanced understanding of the spatiotemporal coordination of signal transduction processes in Arabidopsis.

As multicellular organisms, plants constantly balance and coordinate many metabolic, physiological, and molecular responses between different cell types and tissues. This process is essential for plant development, growth, and response to different environmental cues. Because plants lack a nervous system, they transmit different signals over long distances via cell-to-cell signaling. Recent studies revealed that reactive oxygen species (ROS), produced by respiratory burst oxidase homologs (RBOHs) at the apoplast play a key role in cell-to-cell signaling. A state of enhanced ROS production by one cell is thereby sensed by a neighboring cell, causing it to produce ROS, creating a continuous chain of cell-to-cell ROS accumulation termed the ROS wave. This process was found to mediate systemic signals throughout the plant and is required for plant acclimation to different stresses. Although RBOHs were found to produce ROS essential for this process, the identity of the receptor(s) perceiving the apoplastic ROS signal is currently unknow. Here we reveal that the leucine-rich-repeat receptor-like kinase HPCA1 (H2O2-induced Ca2+ increases 1) acts as a central ROS receptor required for the propagation of cell-to-cell ROS signals, systemic signaling in response to different biotic and abiotic stresses, and plant acclimation to stress. We further report that HPCA1 is required for systemic calcium signals, but not systemic membrane depolarization responses, and identify key calcium-dependent signal transduction proteins involved in this process. Our findings reveal that HPCA1 plays a key role in mediating and coordinating systemic cell-to-cell ROS and calcium signals that are required for plant acclimation to stress.

O_LIContradictory results indicate that aquaporins might facilitate the diffusion of both water and H2O2 during abscisic acid (ABA) triggered stomatal closure. Here, we tested whether maize plasma membrane PIP2;5 aquaporin regulates stomatal closure under water deficit or ABA treatment in intact plants, detached leaves, and peeled epidermis. C_LIO_LITranspiration, stomatal conductance and aperture, as well as reactive oxygen species (ROS) in stomatal complexes were studied in maize lines deregulated in PIP2;5 gene expression, under water deficit and/or ABA treatments. C_LIO_LIIn well-watered conditions, the PIP2;5 overexpressing (OE) plants transpired more than the wild-type plants (WT), while no significant difference in transpiration was observed between pip2;5 KO and WT plants. Upon mild-water deficit or low ABA concentration treatment, the transpiration and stomatal conductance decreased more in PIP2;5 OE, and less in pip2;5 KO lines, in comparison with WT plants. Using isolated epidermis, ABA treatment induced faster stomatal closing in PIP2;5 OE lines compared to the WT, while pip2;5 KO stomata were ABA insensitive. These phenotypes were associated with guard cell ROS accumulation. C_LIO_LITogether, these data indicate that maize PIP2;5 regulates early stomatal closure for water conservation upon a water deficit environment. C_LI

In C4 photosynthesis, incoming CO2 is incorporated in mesophyll cells (MC) into 4-carbon acids that diffuse to bundle sheath cells (BSC) and decarboxylated to generate a high CO2 concentration that suppresses the oxygenation reaction of Rubisco. Decarboxylation can occur by NADP-malic enzyme, (NADP-ME), NAD-malic enzyme (NAD-ME) or phosphoenolpyruvate carboxykinase (PEPCK). NADP-ME generates NADPH in the BSC chloroplast and species that use it as the major route for decarboxylation typically have dimorphic BSC chloroplasts with little or no photosystem II. They operate an energy shuttle: much of the 3-phosphoglycerate formed in the Calvin-Benson cycle diffuses to the MC, enters the chloroplasts and is reduced to triose phosphates that return to the BSC. In species where carboxylation occurs mainly via NAD-ME or PEPCK, BSC chloroplasts possess photosystem II. Indirect evidence indicates they nevertheless have the capacity to operate an energy shuttle. We show here that NAD-ME and PEPCK species possess large pools of 3PGA and triose phosphates and, for two examples of each subtype, opposed concentration gradients of 3-phosphoglycerate and triose phosphates to drive rapid exchange between the BSC and MC. Reasons for and consequences of the widespread operation of the intercellular energy shuttle in C4 plants are discussed. Highlight StatementAn intercellular energy shuttle in which 3-phosphoglycerate moves from the bundle sheath to the mesophyll and triose phosphates return to the bundle sheath is a general feature of C4 photosynthesis.

Aldoximes are amino acid-derived metabolites that serve as precursors of auxins and modulate phenylpropanoid production in Arabidopsis. However, the enzymes responsible for aldoxime production in Solanaceae remain unknown. Here, we report the identification of aldoxime-producing enzymes in tomato (Solanum lycopersicum) and examine how altered aldoxime production affects auxin production and phenylpropanoid metabolism. Through homology-based analysis, we identified five putative CYP79 homologs in tomato, among which SlCYP79DB32 and SlCYP79DB52 exhibited aldoxime-producing activity toward multiple amino acids, including phenylalanine and tryptophan. SlCYP79DB32 and SlCYP79DB52 converted phenylalanine into phenylacetaldoxime (PAOx), whereas only SlCYP79DB52 converted tryptophan into indole-3-acetaldoxime (IAOx). Stable isotope-labeled feeding experiments revealed that IAOx and PAOx can be converted to the auxins indole-3-acetic acid (IAA) and phenylacetic acid (PAA), respectively. Consistently, tomato plants engineered to overproduce IAOx and PAOx accumulated elevated levels of IAA and PAA. These plants also accumulated lower levels of phenylpropanoids. In Brassicaceae plants such as Arabidopsis and Camelina, aldoxime accumulation represses phenylpropanoid production by promoting degradation of phenylalanine ammonia-lyase (PAL). However, aldoxime accumulation did not reduce PAL activity in tomato, suggesting an alternative mechanism in this species. Transcriptome analysis revealed extensive transcriptional reprogramming in aldoxime-overaccumulating tomato plants, including upregulation of stress- and defense-related genes. Despite the observed reduction in phenylpropanoid levels, transcript levels of most phenylpropanoid biosynthetic genes were not decreased, suggesting possible post-transcriptional regulation of this repression. Together, our findings demonstrate that aldoximes can serve as intermediates in auxin biosynthesis in tomato and reveal that aldoxime-mediated repression of phenylpropanoid metabolism extends beyond Brassicaceae.

C4 grasses often outperform C3 species under hot, arid conditions due to superior water and nitrogen use efficiencies and lower rates of photorespiration. A method of concentrating CO2 around the site of carbon fixation in the bundle sheath (BS) is required to realize these gains. In NADP-malic enzyme (NADP-ME)-type C4 grasses such as maize, suberin deposition in the BS cell wall is hypothesized to act as a diffusion barrier to CO2 escape and O2 entry from surrounding mesophyll cells. Suberin is a heteropolyester comprised of acyl-lipid-derived aliphatic and phenylpropanoid-derived aromatic components. To disrupt BS suberization, we mutated two paralogously duplicated, unlinked maize orthologues of Arabidopsis thaliana ALIPHATIC SUBERIN FERULOYL TRANSFERASE, ZmAsft1 and ZmAsft2, using closely linked Dissociation transposons. Loss-of-function double mutants revealed a 97% reduction in suberin-specific omega-hydroxy fatty acids without a stoichiometric decrease in ferulic acid. However, BS suberin lamellae were deficient in electron opaque material, and cohesion between the suberin lamellae and polysaccharide cell walls was attenuated in double mutants. There were no other morphological phenotypes under ambient conditions. Furthermore, there was no significant effect on net CO2 assimilation at any intercellular CO2 concentration, and no effect on 13C isotope discrimination relative to wild type. Thus, ZmAsft expression is not required to establish a functional CO2 concentrating mechanism in in maize. Double mutant leaves exhibit elevated cell wall elasticity, transpirational, and stomatal conductance relative to WT. Thus, the ZmAsft genes are dispensable for gas exchange barrier function but may be involved in regulation of leaf water movement. One-sentence SummaryDouble mutants of two paralogously duplicated maize Aliphatic Suberin Feruloyl Transferase (ZmAsft) genes exhibit reduced aliphatic suberin content, cell wall cohesion defects, and elevated leaf transpiration, but no changes in CO2 assimilation relative to wild type.

Auxin response is regulated by a group of AUX/IAA transcriptional inhibitors that suppress auxin signaling in the absence of the hormone. While the degradation of these proteins upon auxin signaling has been well studied, the molecular control of their rapid turn-over is not clearly understood. Here, we report that the TARANI/ UBIQUITIN PROTEASE 14 protein in Arabidopsis thaliana (Arabidopsis) is required for AUX/IAA degradation. The tni mutation was originally identified in a forward genetic screen to isolate mutants with altered leaf shape. Detailed phenotypic analysis revealed that tni displays pleiotropic phenotypic alterations that resemble auxin-related defects. The activity of auxin responsive reporters DR5::GUS, DR5::nYFP and IAA2::GUS was reduced in tni organs, implying that TNI is required for normal auxin response. Genetic interaction studies suggested that TNI acts along with TIR1, ARF7, AUX1 and PIN1 - molecules involved in auxin signaling or transport. A map-based cloning approach combined with next-generation sequencing identified TNI as UBIQUITIN SPECIFIC PROTEASE14 which is involved in ubiquitin recycling. In tni, the mutant primary transcript is spliced inefficiently, which is predicted to produce an aberrant protein product in addition to the normal protein, where a polypeptide corresponding to the 3rd intron in inserted in-frame within the Zn-finger domain of UBP14. The tni plants accumulated poly-ubiquitin chains and excess poly-ubiquitinated proteins due to reduced TNI activity. Improper ubiquitin recycling affected the degradation of DII:VENUS, IAA18:GUS and HS::AXR3-NT:GUS, resulting in their stabilization in the tni mutant. Thus, our study identified a function for TNI/UBP14 in regulating auxin response through ubiquitin recycling.

Ethylene is a gaseous plant hormone crucial for regulating plant growth, development, and stress adaptation, yet the molecular basis underlying ethylene receptor function remains elusive. Here, we show that subfamily I receptors constitute core ethylene-sensing module and function epistatically to subfamily II receptors. Notably, we discover that only subfamily I receptors possess Ca2+-permeable channel activity, which are indispensable for ethylene-induced cytosolic calcium influx. Overall, this work supports a mechanistic framework in which subfamily I receptors integrate ethylene sensing with Ca2+ influx, providing new insight into how plants translate hormonal cues into downstream signaling events.

Rapid Alkalinization Factor (RALF) peptides regulate plant growth and cell wall signaling, but their roles in fruit development remain unclear. Here, we characterized tomato (Solanum lycopersicum) fruit-associated RALF peptides and their interactions with leucine-rich repeat extensins (LRXs). Expression analyses identified SlRALF5, SlRALF7, and SlRALF10 as the main fruit-expressed RALFs. SlRALF10 was associated with early fruit development, whereas SlRALF5 and SlRALF7 remained expressed during ripening. Sequence analyses showed that SlRALF5/7 retain conserved motifs of canonical RALFs, while SlRALF10 displays divergent structural features and altered charge distribution. Synthetic SlRALF5 and SlRALF7 inhibited root growth and induced extracellular alkalinization, whereas SlRALF10 lacked both activities. Co-immunoprecipitation assays showed that all three peptides interact with the fruit-expressed proteins SlLRX2 and SlLRX5. Structural modeling predicted distinct electrostatic properties for the SlLRX5/SlRALF10 complex compared with SlRALF5. These results reveal structural and functional specialization among tomato fruit RALF peptides and suggest that distinct SlRALFs may differentially respond to cell wall remodeling during fruit development and ripening.

Cotton fibers are single-celled trichomes initiated from ovule epidermis prior to anthesis. Thereafter, the fibers undergo rapid elongation for 20 d before switching to intensive cell wall cellulose synthesis. The final length attained determines fiber yield and quality. As such, cotton fiber represents an excellent single cell model to study regulation of cell growth and differentiation, with significant agronomical implications. One major unresolved question is whether fiber elongation follows a diffusive or a tip growth pattern. We addressed this issue by using cell biology and electrophysiological approaches. Confocal imaging of Ca2+ binding dye, fluo-3 acetoxymethyl (Fluo-3), and in situ microelectrode ion flux measurement revealed that cytosolic Ca2+ was evenly distributed along the elongating fiber cells with Ca2+ and H+ fluxes oscillating from apical to basal regions of the elongating fibers. These findings demonstrate that, contrary to growing pollen tubes or root hairs, cotton fiber growth follows a diffusive, but not the tip growth, pattern. Further analyses showed that the elongating fibers exhibited substantial net H+ efflux, indicating a strong activity of the plasma membrane H+-ATPase required for energy dependent solute uptake. Interestingly, the growing cotton fibers were responding to H2O2 treatment, know to promote fiber elongation, by a massive increase in the net Ca2+ and H+ efflux in both tip and basal zones, while non-growing cells lacked this ability. These observations suggest that desensitization of the cell and a loss of its ability to respond to H2O2 may be causally related to the termination of the cotton fiber elongation. One sentence summaryConfocal imaging of Ca2+ patterning and in situ microelectrode ion flux measurements demonstrate that, contrary to growing pollen tubes or root hairs, cotton fiber growth follows a diffusive, but not the tip growth, pattern.

Many plants accumulate transitory starch reserves in their leaves during the day to buffer their carbohydrate supply against fluctuating light conditions, and to provide carbon and energy for survival at night. It is universally accepted that transitory starch is synthesized from ADP-glucose (ADPG) in the chloroplasts. However, the consensus that ADPG is made in the chloroplasts by ADPG pyrophosphorylase has been challenged by a controversial proposal that ADPG is made primarily in the cytosol, probably by sucrose synthase (SUS), and then imported into the chloroplasts. To resolve this long-standing controversy, we critically re-examined the experimental evidence that appears to conflict with the consensus pathway. We show that when precautions are taken to avoid artefactual changes during leaf sampling, Arabidopsis thaliana mutants that lack SUS activity in mesophyll cells (quadruple sus1234) or have no SUS activity (sextuple sus123456) have wild-type levels of ADPG and starch, while ADPG is 20 times lower in the pgm and adg1 mutants that are blocked in the classical pathway of starch synthesis. We conclude that the ADPG needed for starch synthesis in leaves is synthesized primarily by ADPG pyrophosphorylase in the chloroplasts. Significance statementMutant analysis shows that sucrose synthase makes no significant contribution to transitory starch synthesis in Arabidopsis leaves, resolving a 20-year old controversy about one of the most important pathways of photosynthetic metabolism.

In nature, plants frequently encounter concurrent stresses, particularly the simultaneous occurrence of cold and drought stress poses a challenge to plants in middle and high latitudes. However, the molecular mechanisms underlying the plants response to this double-stress scenario remain unclear. Although some responses suggest that drought stress can improve cold resistance in plants, through ABA signaling pathways. In our study, we discovered that moderate low temperature treatment significantly enhanced Arabidopsis drought tolerance. Low temperature rapidly triggers the transcription factor CCA1, a prototypical response to cold stress, which inturn directly regulates the expression of OST1 and P5CS1 by binding to their promoters. This leads to the premature closure of stomata and accumulation of proline through a non-ABA-dependent pathway even before plants experience drought stress, ultimately improving plant resistance against drought and cold. Moreover, this mechanism is conserved across plant species, and the synergistic resistance mechanism enables perennial plants to survive winter conditions and annual plants to withstand multi-stresses.