Arabidopsis Response Research Articles

RNA-binding protein MAC5A interacts with the 26S proteasome to regulate DNA damage response in Arabidopsis.

DNA damage response (DDR) in eukaryotes is essential for the maintenance of genome integrity in challenging environments. The regulatory mechanisms of DDR have been well-established in yeast and humans. However, increasing evidence supports the idea that plants seem to employ different signaling pathways that remain largely unknown. Here, we report the role of MODIFIER OF SNC1, 4-ASSOCIATED COMPLEX SUBUNIT 5A (MAC5A) in DDR in Arabidopsis (Arabidopsis thaliana). Lack of MAC5A in mac5a mutants causes hypersensitive phenotypes to methyl methanesulfonate (MMS), a DNA damage inducer. Consistent with this observation, MAC5A can regulate alternative splicing of DDR genes to maintain the proper response to genotoxic stress. Interestingly, MAC5A interacts with the 26S proteasome (26SP) and is required for its proteasome activity. MAC core subunits are also involved in MMS-induced DDR. Moreover, we find that MAC5A, the MAC core subunits, and 26SP may act collaboratively to mediate high-boron-induced growth repression through DDR. Collectively, our findings uncover the crucial role of MAC in MMS-induced DDR in orchestrating growth and stress adaptation in plants.

Chromosome-scale assemblies of the male and female Populus euphratica genomes reveal the molecular basis of sex determination and sexual dimorphism

Reference-quality genomes of both sexes are essential for studying sex determination and sex-chromosome evolution, as their gene contents and expression profiles differ. Here, we present independent chromosome-level genome assemblies for the female (XX) and male (XY) genomes of desert poplar (Populus euphratica), resolving a 22.7-Mb X and 24.8-Mb Y chromosome. We also identified a relatively complete 761-kb sex-linked region (SLR) in the peritelomeric region on chromosome 14 (Y). Within the SLR, recombination around the partial repeats for the feminizing factor ARR17 (ARABIDOPSIS RESPONSE REGULATOR 17) was potentially suppressed by flanking palindromic arms and the dense accumulation of retrotransposons. The inverted small segments S1 and S2 of ARR17 exhibited relaxed selective pressure and triggered sex determination by generating 24-nt small interfering RNAs that induce male-specific hyper-methylation at the promoter of the autosomal targeted ARR17. We also detected two male-specific fusion genes encoding proteins with NB-ARC domains at the breakpoint region of an inversion in the SLR that may be responsible for the observed sexual dimorphism in immune responses. Our results show that the SLR appears to follow proposed evolutionary dynamics for sex chromosomes and advance our understanding of sex determination and the evolution of sex chromosomes in Populus.

HISTONE DEACETYLASE 9 transduces heat signal in plant cells

Heat stress limits plant growth, development, and crop yield, but how plant cells precisely sense and transduce heat stress signals remains elusive. Here, we identified a conserved heat stress response mechanism to elucidate how heat stress signal is transmitted from the cytoplasm into the nucleus for epigenetic modifiers. We demonstrate that HISTONE DEACETYLASE 9 (HDA9) transduces heat signals from the cytoplasm to the nucleus to play a positive regulatory role in heat responses in Arabidopsis. Heat specifically induces HDA9 accumulation in the nucleus. Under heat stress, the phosphatase PP2AB'β directly interacts with and dephosphorylates HDA9 to protect HDA9 from 26S proteasome-mediated degradation, leading to the translocation of nonphosphorylated HDA9 to the nucleus. This heat-induced enrichment of HDA9 in the nucleus depends on the nucleoporin HOS1. In the nucleus, HDA9 binds and deacetylates the target genes related to signaling transduction and plant development to repress gene expression in a transcription factor YIN YANG 1-dependent and -independent manner, resulting in rebalance of plant development and heat response. Therefore, we uncover an HDA9-mediated positive regulatory module in the heat shock signal transduction pathway. More important, this cytoplasm-to-nucleus translocation of HDA9 in response to heat stress is conserved in wheat and rice, which confers the mechanism significant implication potential for crop breeding to cope with global climate warming.

Titanium nanoparticles activate a transcriptional response in Arabidopsis that enhances tolerance to low phosphate, osmotic stress and pathogen infection.

Titanium is a ubiquitous element with a wide variety of beneficial effects in plants, including enhanced nutrient uptake and resistance to pathogens and abiotic stresses. While there is numerous evidence supporting the beneficial effects that Ti fertilization give to plants, there is little information on which genetic signaling pathways the Ti application activate in plant tissues. In this study, we utilize RNA-seq and ionomics technologies to unravel the molecular signals that Arabidopsis plants unleash when treated with Ti. RNA-seq analysis showed that Ti activates abscisic acid and salicylic acid signaling pathways and the expression of NUCLEOTIDE BINDING SITE-LEUCINE RICH REPEAT receptors likely by acting as a chemical priming molecule. This activation results in enhanced resistance to drought, high salinity, and infection with Botrytis cinerea in Arabidopsis. Ti also grants an enhanced nutritional state, even at suboptimal phosphate concentrations by upregulating the expression of multiple nutrient and membrane transporters and by modifying or increasing the production root exudates. Our results suggest that Ti might act similarly to the beneficial element Silicon in other plant species.

Spermine inhibits PAMP-induced ROS and Ca2+ burst and reshapes the transcriptional landscape of PAMP-triggered immunity in Arabidopsis.

Polyamines are small polycationic amines whose levels increase during defense. Previous studies support the contribution of the polyamine spermine to defense responses. However, the potential contribution of spermine to pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) has not been completely established. Here, we compared the contribution of spermine and putrescine to early and late PTI responses in Arabidopsis. We found that putrescine and spermine have opposite effects on PAMP-elicited reactive oxygen species (ROS) production, with putrescine increasing and spermine lowering the flg22-stimulated ROS burst. Through genetic and pharmacological approaches, we found that the inhibitory effect of spermine on flg22-elicited ROS production is independent of polyamine oxidation, nitric oxide, and salicylic acid signaling but resembles chemical inhibition of RBOHD (RESPIRATORY BURST OXIDASE HOMOLOG D). Spermine can also suppress ROS elicited by FLS2-independent but RBOHD-dependent pathways, thus pointing to compromised RBOHD activity. Consistent with this, we found that spermine but not putrescine dampens flg22-stimulated cytosolic Ca2+ influx. Finally, we found that both polyamines differentially reshape transcriptional responses during PTI and disease resistance to Pseudomonas syringae. Overall, we provide evidence for the differential contributions of putrescine and spermine to PTI, with an impact on plant defense.

Ozone and nitrogen dioxide regulate similar gene expression responses in Arabidopsis but natural variation in the extent of cell death is likely controlled by different genetic loci.

High doses of ozone (O3) and nitrogen dioxide (NO2) cause damage and cell death in plants. These two gases are among the most harmful air pollutants for ecosystems and therefore it is important to understand how plant resistance or sensitivity to these gases work at the molecular level and its genetic control. We compared transcriptome data from O3 and NO2 fumigations to other cell death related treatments, as well as individual marker gene transcript level in different Arabidopsis thaliana accessions. Our analysis revealed that O3 and NO2 trigger very similar gene expression responses that include genes involved in pathogen resistance, cell death and ethylene signaling. However, we also identified exceptions, for example RBOHF encoding a reactive oxygen species producing RESPIRATORY BURST OXIDASE PROTEIN F. This gene had increased transcript levels by O3 but decreased transcript levels by NO2, showing that plants can identify each of the gases separately and activate distinct signaling pathways. To understand the genetics, we conducted a genome wide association study (GWAS) on O3 and NO2 tolerance of natural Arabidopsis accessions. Sensitivity to both gases seem to be controlled by several independent small effect loci and we did not find an overlap in the significantly associated regions. Further characterization of the GWAS candidate loci identified new regulators of O3 and NO2 induced cell death including ABH1, a protein that functions in abscisic acid signaling, mRNA splicing and miRNA processing. The GWAS results will facilitate further characterization of the control of programmed cell death and differences between oxidative and nitrosative stress in plants.

Distribution, expression of hexaploid wheat Fes1s and functional characterization of two TaFes1As in Arabidopsis.

Hexaploid wheat is a major food crop and is sensitive to heat stress. It is necessary to discover genes related to thermotolerance in wheat. Fes1s is a class of nucleotide exchange factor of heat shock protein 70s, proven to be participated in heat response in human, yeast, and Arabidopsis. However, little is known about Fes1s in hexaploid wheat. In this study, we identified nine Fes1s in hexaploid wheat (TaFes1s) and found that they present as three triads. A phylogenetic relationship analysis revealed that these Fes1s grouped into Fes1A, Fes1B and Fes1C subclades, and Fes1As and Fes1Bs were divergent in monocots, but possibly not in dicots. The sequences, gene structures and protein motifs of TaFes1s homoeologues within a triad were highly conserved. Through cis-elements analysis including heat shock elements, and miRNA targets prediction, we found that regulation of three TaFes1s homoeologues may be different, while the expression patterns of three homoeologues were similar. The expression levels of TaFes1As were higher than those of TaFes1Bs and TaFes1Cs, and based on these expressions, TaFes1As were chosen for functional characterization. Intriguingly, neither TaFes1A-5A nor TaFes1A-5D could not rescue the thermotolerance defect of Arabidopsis fes1a mutants at seedling stage, but in the transgenic plants seed germination was accelerated under normal and heat stress condition. The functional characterization indicated that roles of Fes1As would be different in Arabidopsis and hexaploid wheat, and function retention of TaFes1As may occur during wheat evolution. In conclusion, our study comprehensively characterized the distribution and expression of Fes1s in hexaploid wheat and found that two TaFes1As could accelerate seed germination under normal and heat stress condition.

Ectopic expression of WOX5 promotes cytokinin signaling and de novo shoot regeneration.

WOX5 has a potential in activating cytokinin signaling and shoot regeneration, in addition to its role in pluripotency acquisition. Thus, overexpression of WOX5 maximizes plant regeneration capacity during tissue culture. In vitro plant regeneration involves two steps: callus formation and de novo shoot organogenesis. The WUSCHEL-RELATED HOMEOBOX 5 (WOX5) homeodomain transcription factor is known to be mainly expressed during incubation on callus-inducing medium (CIM) and involved in pluripotency acquisition in callus, but whether WOX5 also affects de novo shoot regeneration on cytokinin-rich shoot-inducing medium (SIM) remains unknown. Based on the recent finding that WOX5 promotes cytokinin signaling, we hypothesized that ectopic expression of WOX5 beyond CIM would further enhance overall plant regeneration capacity, because intense cytokinin signaling is particularly required for shoot regeneration on SIM. Here, we found that overexpression of the WOX5 gene on SIM drastically promoted de novo shoot regeneration from callus with the repression of type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, negative regulators of cytokinin signaling. The enhanced shoot regeneration phenotypes were indeed dependent on cytokinin signaling, which were partially suppressed in the progeny derived from crossing WOX5-overexpressing plants with cytokinin-insensitive 35S:ARR7 plants. The function of WOX5 in enhancing cytokinin-dependent shoot regeneration is evolutionarily conserved, as conditional overexpression of OsWOX5 on SIM profoundly enhanced shoot regeneration in rice callus. Overall, our results provide the technical advance that maximizes in vitro plant regeneration by constitutively expressing WOX5, which unequivocally promotes both callus pluripotency and de novo shoot regeneration.

Touch-Induced Transcriptional Changes in Flower Buds of a Non-Model Horticultural Plant Dianthus hybrida

Touch stimulus responses are common in plants. Some flowering plants sense the arrival of their pollinators and secrete nectar or release pollen sacs, facilitating successful pollination. Molecular mechanisms for mechanical stimulus responses in plants are well characterized in Arabidopsis leaves, but not in non-model plants or other organs such as flowers. Here, we performed RNA-seq analysis of touched flower buds of Dianthus hybrida, a major ornamental plant. Upon touch treatment, 931 and 132 genes were upregulated and downregulated, respectively. GO enrichment analysis revealed that genes encoding serine/threonine protein kinases were significantly abundant among the upregulated genes, which is consistent with previous studies that demonstrated the pivotal role of protein phosphorylation in the touch stimulus response of Arabidopsis leaves. In comparison with the gene expression profile of touched Arabidopsis leaves, the same families but different homologs of the representative touch-induced genes encoding protein kinases were upregulated, showing that phosphorelay signaling was the common mechanism for touch stimulus response in flowers and leaves, but the players of the phosphorelay signaling were different. These results will contribute to further studies on the mechanical stimulus responses of ornamental flowers and the utilization of this mechanism for breeding programs.

Genome-wide characterization of C2H2 zinc-finger gene family provides insight into the mechanisms and evolution of the dehydration-rehydration responses in Physcomitrium and Arabidopsis.

Dehydration tolerance is a vital factor for land plant evolution and world agricultural production. Numerous studies enlightened that the plant-specific C2H2-type zinc-finger proteins (C2H2-ZFPs) as master regulators played pivotal roles in the abiotic stress responses of plants. However, a comprehensive understanding of the evolution of C2H2-ZFPs in terrestrial plants and its regulatory mechanism in dehydration and rehydration response remains a mystery. In this study, the genome-wide identification of C2H2-ZFP genes revealed 549 homologs in the representatives of terrestrial plant lineages from liverwort to angiosperms. Based on the characteristics of the conserved C2H2-ZF domains, four major C2H2-ZF types (M-, Z-, Q-, and D-type) were identified in the C2H2-ZFPs, with the dominants of M-type in all selected species and followed by Z-type in non-seed plants and Q-type in seed plants, respectively. Phylogenetic analyses of the identified C2H2-ZFPs supported four major groups in the land plant representatives, among which the members from the desiccation-tolerant Physcomitrium patens and the dehydration-sensitive Arabidopsis thaliana displayed different topological relationships in the phylogenies reconstructed for a single species. C2H2-ZFPs clustered in the same subclades shared similar features in their conserved domains and gene structures. Approximately, 81% of the C2H2-ZFP promoters of all 549 identified C2H2-ZFPs harbored the conserved ABA-responsive elements (ABREs) and/or dehydration-responsive elements (DREs). Comparative transcriptomic analyses showed that 50 PpZFPs and 56 AtZFPs significantly changed their transcripts abundance. Interestingly, most of the dehydration- and rehydration-responsive PpZPFs and AtZFPs had been predicted to contain the ABRE and DRE elements in their promoter regions and with over half of which phylogenetically belonging to group III. The differences in the expression patterns of C2H2-ZFPs in responses to dehydration and rehydration between P. patens and A. thaliana reflected their different strategies to adapt to dehydration. The identified candidate PpZFPs were specifically induced by moderate dehydration and reached the peak transcript abundance in severe dehydration. Our study lays the foundations for further functional investigation of C2H2-ZFPs in dehydration responses from an evolutionary perspective in land plants. The findings will provide us with genetic resources and potential targets for drought tolerance breeding in crops and beyond.

ALKBH9C, a potential RNA m6 A demethylase, regulates the response of Arabidopsis to abiotic stresses and abscisic acid.

Although several studies have shown that AlkB homolog (ALKBH) proteins are potential RNA demethylases (referred to as 'erasers'), biological functions of only a few ALKBH proteins have been characterized to date. In this study, we determined the function of ALKBH9C (At4g36090) in seed germination and seedling growth of Arabidopsis thaliana in response to abiotic stress and abscisic acid (ABA). Seed germination of the alkbh9c mutant was delayed in response to salt, drought, coldand ABA. Moreover, seedling growth of the mutant was repressed under salt stress or ABA but enhanced under drought conditions. Notably, the stress-responsive phenotypes were associated with the altered expression of several m6 A-modified transcripts related to salt, droughtor ABA response. Global m6 A levels were increased in the alkbh9c mutant, and ALKBH9C bound to m6 A-modified RNAs and had in vitro m6 A demethylase activity, suggesting its potential role as an m6 A eraser. The m6 A levels in several stress-responsive genes were increased in the alkbh9c mutant, and the stability of m6 A-modified transcripts was altered in the mutant. Collectively, our results suggest that m6 A eraser ALKBH9C is crucial for seed germination and seedling growth of Arabidopsis in response to abiotic stresses or ABA via affecting the stability of stress-responsive transcripts.

Evolutionary analysis of the LORELEI gene family in plants reveals regulatory subfunctionalization.

A signaling complex comprising members of the LORELEI (LRE)-LIKE GPI-anchored protein (LLG) and Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) families perceive RAPID ALKALINIZATION FACTOR (RALF) peptides and regulate growth, reproduction, immunity, and stress responses in Arabidopsis (Arabidopsis thaliana). Genes encoding these proteins are members of multigene families in most angiosperms and could generate thousands of signaling complex variants. However, the links between expansion of these gene families and the functional diversification of this critical signaling complex as well as the evolutionary factors underlying the maintenance of gene duplicates remain unknown. Here, we investigated LLG gene family evolution by sampling land plant genomes and explored the function and expression of angiosperm LLGs. We found that LLG diversity within major land plant lineages is primarily due to lineage-specific duplication events, and that these duplications occurred both early in the history of these lineages and more recently. Our complementation and expression analyses showed that expression divergence (i.e. regulatory subfunctionalization), rather than functional divergence, explains the retention of LLG paralogs. Interestingly, all but one monocot and all eudicot species examined had an LLG copy with preferential expression in male reproductive tissues, while the other duplicate copies showed highest levels of expression in female or vegetative tissues. The single LLG copy in Amborella trichopoda is expressed vastly higher in male compared to in female reproductive or vegetative tissues. We propose that expression divergence plays an important role in retention of LLG duplicates in angiosperms.

Type-A response regulators negatively mediate heat stress response by altering redox homeostasis in Arabidopsis.

Besides the long-standing role of cytokinins (CKs) as growth regulators, their current positioning at the interface of development and stress responses is coming into recognition. The current evidence suggests the notion that CKs are involved in heat stress response (HSR), however, the role of CK signaling components is still elusive. In this study, we have identified a role of the CK signaling components type-A Arabidopsis response regulators (ARRs) in HSR in Arabidopsis. The mutants of multiple type-A ARR genes exhibit improved basal and acquired thermotolerance and, altered response to oxidative stress in our physiological analyses. Through proteomics profiling, we show that the type-A arr mutants experience a ‘stress-primed’ state enabling them to respond more efficiently upon exposure to real stress stimuli. A substantial number of proteins that are involved in the heat-acclimatization process such as the proteins related to cellular redox status and heat shock, are already altered in the type-A arr mutants without a prior exposure to stress conditions. The metabolomics analyses further reveal that the mutants accumulate higher amounts of α-and γ-tocopherols, which are important antioxidants for protection against oxidative damage. Collectively, our results suggest that the type-A ARRs play an important role in heat stress response by affecting the redox homeostasis in Arabidopsis.

The type-B response regulators ARR10, ARR12, and ARR18 specify the central cell in Arabidopsis.

In Arabidopsis thaliana, the female gametophyte consists of two synergid cells, an egg cell, a diploid central cell, and three antipodal cells. CYTOKININ INDEPENDENT 1 (CKI1), a histidine kinase constitutively activating the cytokinin signaling pathway, specifies the central cell and restricts the egg cell. However, the mechanism regulating CKI1-dependent central cell specification is largely unknown. Here, we showed that the type-B ARABIDOPSIS RESPONSE REGULATORS10, 12, and 18 (ARR10/12/18) localize at the chalazal pole of the female gametophyte. Phenotypic analysis showed that the arr10 12 18 triple mutant is female sterile. We examined the expression patterns of embryo sac marker genes and found that the embryo sac of arr10 12 18 plants had lost central cell identity, a phenotype similar to that of the Arabidopsis cki1 mutant. Genetic analyses demonstrated that ARR10/12/18, CKI1, and ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN2, 3, and 5 (AHP2/3/5) function in a common pathway to regulate female gametophyte development. In addition, constitutively activated ARR10/12/18 in the cki1 embryo sac partially restored the fertility of cki1. Results of transcriptomic analysis supported the conclusion that ARR10/12/18 and CKI1 function together to regulate the identity of the central cell. Our results demonstrated that ARR10/12/18 function downstream of CKI1-AHP2/3/5 as core factors to determine cell fate of the female gametophyte.

AtHD2D, a plant-specific histone deacetylase involved in abscisic acid response and lateral root development.

In eukaryotes, histone acetylation levels directly regulate downstream gene expression. As a plant-specific histone deacetylase (HDAC), HD2D is involved in plant development and abiotic stress. However, the response of HD2D to drought stress and its interacting proteins, is still unclear. In this study, we analysed HD2D gene expression patterns in Arabidopsis, revealing that HD2D gene was highly expressed in roots and rosette leaves, but poorly expressed in other tissues such as stems, flowers, and young siliques. The HD2D gene expression was induced by d-mannitol. We investigated the responses to drought stress in the wild-type plant, HD2D overexpression lines, and hd2d mutants. HD2D-overexpressing lines showed abscisic acid (ABA) hypersensitivity and drought tolerance, and these phenotypes were not present in hd2d mutants. RNA-seq analysis revealed the transcriptome changes caused by HD2D under drought stress, and showed that HD2D responded to drought stress via the ABA signalling pathway. In addition, we demonstrated that CASEIN KINASE II (CKA4) directly interacted with HD2D. The phosphorylation of Ser residues on HD2D by CKA4 enhanced HD2D enzymatic activity. Furthermore, the phosphorylation of HD2D was shown to contribute to lateral root development and ABA sensing in Arabidopsis, but, these phenotypes could not be reproduced by the overexpression of Ser-phospho-null HD2D lines. Collectively, this study suggests that HD2D responded to drought stress by regulating the ABA signalling pathway, and the expression of drought stress-related genes. The regulatory mechanism of HD2D mediated by CKII phosphorylation provides new insights into the ABA response and lateral root development in Arabidopsis.

A 1-aminocyclopropane-1-carboxylic-acid (ACC) dipeptide elicits ethylene responses through ACC-oxidase mediated substrate promiscuity.

Plants produce the volatile hormone ethylene to regulate many developmental processes and to deal with (a)biotic stressors. In seed plants, ethylene is synthesized from 1-aminocyclopropane-1-carboxylic acid (ACC) by the dedicated enzyme ACC oxidase (ACO). Ethylene biosynthesis is tightly regulated at the level of ACC through ACC synthesis, conjugation and transport. ACC is a non-proteinogenic amino acid, which also has signaling roles independent from ethylene. In this work, we investigated the biological function of an uncharacterized ACC dipeptide. The custom-synthesized di-ACC molecule can be taken up by Arabidopsis in a similar way as ACC, in part via Lysine Histidine Transporters (e.g., LHT1). Using Nano-Particle Assisted Laser Desoprtion/Ionization (Nano-PALDI) mass-spectrometry imaging, we revealed that externally fed di-ACC predominantly localizes to the vasculature tissue, despite it not being detectable in control hypocotyl segments. Once taken up, the ACC dimer can evoke a triple response phenotype in dark-grown seedlings, reminiscent of ethylene responses induced by ACC itself, albeit less efficiently compared to ACC. Di-ACC does not act via ACC-signaling, but operates via the known ethylene signaling pathway. In vitro ACO activity and molecular docking showed that di-ACC can be used as an alternative substrate by ACO to form ethylene. The promiscuous nature of ACO for the ACC dimer also explains the higher ethylene production rates observed in planta, although this reaction occurred less efficiently compared to ACC. Overall, the ACC dipeptide seems to be transported and converted into ethylene in a similar way as ACC, and is able to augment ethylene production levels and induce subsequent ethylene responses in Arabidopsis.

The cytokinin type-B response regulator PeRR12 is a negative regulator of adventitious rooting and salt tolerance in poplar

Adventitious root (AR) development is an ecologically and economically important biological process that maintains ecological balance, improves plant survivability, and allows for massive vegetative propagation, but its genetic mechanisms are not well understood. Here, eight Arabidopsis response regulator (ARR) genes were cloned and identified in poplar, most of which were detected in the AR, phloem, and xylem and showed remarkable induction at different time points during AR development. Subcellular localization indicated that most of these PeRR genes are in the nucleus. Based on qRT-PCR expression analysis of some genes related to AR development, we inferred that overexpression of PeRR12 (OE_PeRR12) may inhibited AR formation by suppressing the transcription of PeWOX11, PeWOX5, PePIN1 and PePIN3 in poplar while promoting type-A RR transcripts. Correspondingly, exogenous auxin partially restored the rooting of OE_PeRR12 poplar by inhibiting PeRR12 expression. Moreover, the activities of the antioxidant systems of OE_PeRR12 poplars were lower than those of wild-type poplars under salt stress conditions, indicating that PeRR12 may acts as a repressor that mediates salt tolerance by suppressing the expression of PeHKT1;1. Altogether, these results suggest that PeRR12 plays essential roles in mediating AR formation and salinity tolerance in poplar.

The MYB33, MYB65, and MYB101 transcription factors affect Arabidopsis and potato responses to drought by regulating the ABA signaling pathway.

Drought is one of the main climate threats limiting crop production. Potato is one of the four most important food crop species worldwide and is sensitive to water shortage. The CBP80 gene was shown to affect Arabidopsis and potato responses to drought by regulating the level of microRNA159 and, consequently, the levels of the MYB33 and MYB101 transcription factors (TFs). Here, we show that three MYB TFs, MYB33, MYB65, and MYB101, are involved in plant responses to water shortage. Their downregulation in Arabidopsis causes stomatal hyposensitivity to abscisic acid (ABA), leading to reduced tolerance to drought. Transgenic Arabidopsis and potato plants overexpressing these genes, with a mutated recognition site in miR159, show hypersensitivity to ABA and relatively high tolerance to drought conditions. Thus, the MYB33, MYB65, and MYB101 genes may be potential targets for innovative breeding to obtain crops with relatively high tolerance to drought.

AtS40-1, a group I DUF584 protein positively regulates ABA response and salt tolerance in Arabidopsis

Abiotic stresses such as salt and drought affect plants growth and development, whereas the plant hormone ABA is able to regulate plant responses to abiotic stresses by regulating downstream gene expression. Therefore characterization of unknown function ABA responsive genes is able to identify novel regulators of plant abiotic stress responses. We report here the characterization of AtS40-1, a Group I DUF584 protein in the regulation of ABA and salt responses in Arabidopsis. RT-PCR results show that the expression of AtS40-1 was dramatically induced by ABA, but only slightly increase, if any, was observed for other three Group I DUF584 genes including AtS40-1L, AtS40-2 and AtS40-3. Transfection assays in Arabidopsis protoplasts show that all the four Group I DUF584 proteins were predominately localized in nucleus and were able to repress the expression of the co-transfected reporter gene. The roles of AtS40-1 in regulating plant response to ABA and abiotic stress responses were analyzed, by using transgenic plants and inactivation mutants. The results show that the ABA responses were increased in the 35S:AtS40-1 transgenic plants, but decreased in the ats40-1 mutants. Similar to AtS40-1, the results indicate that AtS40-1L, the most closely related DUF584 protein to AtS40-1, positively regulates ABA responses in Arabidopsis. However, further decreased ABA responses were not observed in the ats40-1 ats40-1L double mutants. On the other hand, salt tolerance was increased in the transgenic plants overexpressing AtS40-1 or AtS40-1L, but decreased in the ats40-1 and ats40-1L mutants. Quantitative RT-PCR results show that the ABA induced expression of the ABA signaling regulator genes ABI3, ABI4 and ABA responsive gene RAB18 was decreased, where as ABA signaling gene ABI1 was increased in the ats40-1 mutants. These results suggest that AtS40-1 regulates ABA and salt responses in Arabidopsis, possibly by affecting ABA induced expression of some ABA signaling regulator genes.

Improved abiotic stress tolerance in Arabidopsis by constitutive active form of a banana DREB2 type transcription factor, MaDREB20.CA, than its native form, MaDREB20.

Banana is grown as one of the important fruit crops in tropical and subtropical regions of the world. In this study, we report induced expression of a dehydration responsive element binding 2 (DREB2) gene (MaDREB20) under individual heat, drought, and combined drought and heat stress in root of two banana genotypes Grand Nain (GN) and Hill Banana (HB). Motif analysis of MaDREB20 protein demonstrated the presence of a negative regulatory domain (NRD) or PEST motif between 150 and 184 amino acids. Transgenic Arabidopsis overexpressing MaDREB20 gene showed more survival rate, above-ground biomass, seed yield, leaf relative water content, and proline content but less ion leakage and malonaldehyde content, revealing improved tolerance against heat and drought as well as their combination than the wild-type. Overexpression of MaDREB20.CA (constitutive active form of MaDREB20 after removal of PEST region) showed better abiotic stress tolerance in Arabidopsis than its native form (MaDREB20). Transgenic Arabidopsis overexpressing MaDREB20 and MaDREB20.CA genes appeared to be associated with reduced stomatal densities under normal condition, better regulation of stomatal aperture under drought than in wild-type plants, and differential regulation of downstream target (AtTCH4 and AtIAA1) genes under heat, drought, and combined stress. Taken together, our findings revealed important functions of MaDREB20 in abiotic stress responses in transgenic Arabidopsis and could form a basis for CRISPR/Cas9-mediated removal of its NRD to enhance stress tolerance in banana.