Genome-Wide Analysis of the Phospholipase Ds in Perennial Ryegrass Highlights LpABFs-LpPLDδ3 Cascade Modulated Osmotic and Heat Stress Responses.

The phospholipase Ds (PLDs) are crucial for cellular signalling and play roles in plant abiotic stress response. In this study, we identified 12 PLD genes from the genome data of perennial ryegrass (Lolium perenne), which is widely used as forage and turfgrass. Among them, LpPLDδ3 was significantly repressed by ABA treatment, and induced by drought stress and heat stress treatments. The ectopic overexpression (OE) of LpPLDδ3 in Arabidopsis enhanced plant tolerance to osmotic and heat stress as demonstrated by an increased survival rate and reduced malondialdehyde (MDA) accumulation and electrolyte leakage (EL). Arabidopsis endogenous ABA RESPONSIVE ELEMENT BINDING FACTORs (ABFs) and heat stress responsive genes were elevated in LpPLDδ3 OE lines under osmotic and heat stress treatments. Additionally, overexpression of LpPLDδ3 in perennial ryegrass protoplasts could increase heat stress tolerance and elevate expression level of heat stress responsive genes. Moreover, LpABF2 and LpABF4 depressed the LpPLDδ3 expression by directly binding to its ABRE core-binding motif of promoter region. In summary, LpPLDδ3 was repressed by LpABF2 and LpABF4 and positively involved in perennial ryegrass osmotic and heat stress responses.

Tulip transcription factor TgWRKY75 activates salicylic acid and abscisic acid biosynthesis to synergistically promote petal senescence.

WRKY transcription factors play a central role in controlling plant organ senescence; however, it is unclear whether and how they regulate petal senescence in the widely grown ornamental plant tulip (Tulipa gesneriana). In this study, we report that TgWRKY75 promotes petal senescence by enhancing the synthesis of both abscisic acid (ABA) and salicylic acid (SA) in tulip and in transgenic Arabidopsis. The expression level of TgWRKY75 was up-regulated in senescent petals, and exogenous ABA or SA treatment induced its expression. The endogenous contents of ABA and SA significantly increased during petal senescence and in response to TgWRKY75 overexpression. Two SA synthesis-related genes, TgICS1 and TgPAL1, were identified as direct targets of TgWRKY75, which binds to their promoters. In parallel, TgWRKY75 activated the expression of the ABA biosynthesis-related gene TgNCED3 via directly binding to its promoter region. Site mutation of the W-box core motif located in the promoters of TgICS1, TgPAL1, and TgNCED3 eliminated their interactions with TgWRKY75. In summary, our study demonstrates a dual regulation of ABA and SA biosynthesis by TgWRKY75, revealing a synergistic process of tulip petal senescence through feedback regulation between TgWRKY75 and the accumulation of ABA and SA.

Genome-wide identification of DREB1 transcription factors in perennial ryegrass and functional profiling of LpDREB1H2 in response to cold stress.

Perennial ryegrass (Lolium perenne L.) is an outstanding turfgrass and forage cultivated in temperate regions worldwide. However, poor tolerance to extreme cold, heat, or drought limits wide extension and cultivation. DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR1s (DREB1s) play a vital role in enhancing plant tolerance to abiotic stress, specifically for low-temperature stress. In this study, a total of 24 LpDREB1 family members were identified from the released genome of perennial ryegrass. Phylogenetic analysis showed that the LpDREB1 genes are divided into 7 groups that have close relationships with rice homologues. Conserved motif analysis revealed that members within the same group have similar conserved motif compositions. All LpDREB1s lack introns, and the promoter sequences of LpDREB1 genes contain multiple cis-acting elements associated with stress response, phytohormone signal transduction and plant growth and development. The majority of LpDREB1 genes were upregulated by drought, submergence, heat and cold stress treatments, including LpDREB1H2. Further investigation showed that LpDREB1H2 is localized in the nucleus. Overexpression of LpDREB1H2 in Arabidopsis induced the expression of cold-responsive (COR) genes, increased the levels of osmotic adjusting substances, and enhanced antioxidant enzyme activities, thus improving the cold tolerance of Arabidopsis. This study lays a foundation for further understanding the function of LpDREB1 genes in perennial ryegrass and provides insights for plant stress tolerance breeding.

SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts.

Nicotinamide adenine diphosphate (NAD+) is a novel messenger RNA 5' cap in Escherichia coli, yeast, mammals, and Arabidopsis Transcriptome-wide identification of NAD+-capped RNAs (NAD-RNAs) was accomplished through NAD captureSeq, which combines chemoenzymatic RNA enrichment with high-throughput sequencing. NAD-RNAs are enzymatically converted to alkyne-RNAs that are then biotinylated using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Originally applied to E. coli RNA, which lacks the m7G cap, NAD captureSeq was then applied to eukaryotes without extensive verification of its specificity for NAD-RNAs vs. m7G-capped RNAs (m7G-RNAs). In addition, the Cu2+ ion in the CuAAC reaction causes RNA fragmentation, leading to greatly reduced yield and loss of full-length sequence information. We developed an NAD-RNA capture scheme utilizing the copper-free, strain-promoted azide-alkyne cycloaddition reaction (SPAAC). We examined the specificity of CuAAC and SPAAC reactions toward NAD-RNAs and m7G-RNAs and found that both prefer the former, but also act on the latter. We demonstrated that SPAAC-NAD sequencing (SPAAC-NAD-seq), when combined with immunodepletion of m7G-RNAs, enables NAD-RNA identification with accuracy and sensitivity, leading to the discovery of new NAD-RNA profiles in Arabidopsis Furthermore, SPAAC-NAD-seq retained full-length sequence information. Therefore, SPAAC-NAD-seq would enable specific and efficient discovery of NAD-RNAs in prokaryotes and, when combined with m7G-RNA depletion, in eukaryotes.

The R2R3-MYB Transcriptional Repressor TgMYB4 Negatively Regulates Anthocyanin Biosynthesis in Tulips (Tulipa gesneriana L.).

Anthocyanins play a paramount role in color variation and significantly contribute to the economic value of ornamental plants. The conserved activation complex MYB-bHLH-WD40 (MBW; MYB: v-myb avian myeloblastosis viral oncogene homolog; bHLH: basic helix-loop-helix protein; WD40:WD-repeat protein) involved in anthocyanin biosynthesis has been thoroughly researched, but there have been limited investigations into the function of repressor factors. In this study, we characterized TgMYB4, an R2R3-MYB transcriptional repressor which is highly expressed during petal coloration in red petal cultivars. TgMYB4-overexpressing tobaccos exhibited white or light pink petals with less anthocyanin accumulation compared to control plants. TgMYB4 was found to inhibit the transcription of ANTHOCYANIDIN SYNTHASE (TfANS1) and DIHYDRO-FLAVONOL-4-REDUCTASE (AtDFR), although it did not bind to their promoters. Moreover, the TgMYB4 protein was able to compete with the MYB activator to bind to the :bHLHprotein, thereby suppressing the function of the activator MBW complex. These findings demonstrate that TgMYB4 plays a suppressive role in the regulation of anthocyanin synthesis during flower pigmentation.

NAC transcription factor TgNAP promotes tulip petal senescence.

Petal senescence is a crucial determinant for ornamental quality and economic value of floral crops. Salicylic acid (SA) and reactive oxygen species (ROS) are two prominent factors involved in plant senescence regulation. In this study, tulip TgNAP (NAC-like, activated by APETALA3/PISTILLATA) was characterized as positively regulating tulip petal senescence through dually regulating SA biosynthesis and ROS detoxification pathways. TgNAP was upregulated in senescing petals of tulip while exogenous SA and H2O2 treatments substantially promoted petal senescence in tulip. Silencing of TgNAP by VIGS assay delayed SA and H2O2-induced petal senescence in tulip, whereas overexpression of TgNAP promoted the senescence process in Arabidopsis (Arabidopsis thaliana) plants. Additionally, inhibition of SA biosynthesis prolonged the lifespan of TgNAP-silenced petal discs. Further evidence indicated that TgNAP activates the transcriptions of two key SA biosynthetic genes ISOCHORISMATE SYNTHASE 1 (TgICS1) and PHENYLALANINE AMMONIA-LYASE 1 (TgPAL1) through directly binding to their promoter regions. Meanwhile, TgNAP repressed ROS scavenging by directly inhibiting PEROXIDASE 12 (POD12) and POD17 expression. Taken together, these results indicate that TgNAP enhances SA biosynthesis and ROS accumulation to positively regulate petal senescence in tulip.

Transcription factors TgbHLH42-1 and TgbHLH42-2 positively regulate anthocyanin biosynthesis in Tulip (Tulipa gesneriana L.).

The floral coloration of tulip flowers is one of the most prominent traits contributing to its high ornamental value. The molecular mechanisms of petal coloration remain elusive in tulip species. In this study, we performed comparative metabolome and transcriptome analyses using four tulip cultivars with distinguished petal colors. Four types of anthocyanins were identified, including cyanidin derivatives and pelargonidin derivatives. Comparative transcriptome analysis identified 22,303 differential expressed genes (DEGs) from the four cultivars, and 2589 DEGs were commonly regulated in three comparison groups (colored vs. white cultivar), including anthocyanins biosynthesis-related genes and regulatory transcription factors. Two basic helix-loop-helix (bHLH) transcription factors, TgbHLH42-1 and TgbHLH42-2, with differential expression levels among cultivars and petal developmental stages, have high homology to TRANSPARENT TESTA 8 (AtTT8) of Arabidopsis. The anthocyanins accumulation in TgbHLH42-1 overexpressing (OE) seedlings was markedly greater than that in wild-type seedlings in the presence of methyl jasmonate (MeJA), but not for TgbHLH42-2 OE seedlings. Both TgbHLH42-1 and TgbHLH42-2 restored pigmentation defects in tt8 mutant seeds after complementation assay. TgbHLH42-1 could interact with MYB protein AtPAP1 to synergistically activate the transcription of AtDFR, whereas TgbHLH42-2 failed to. Silencing TgbHLH42-1 or TgbHLH42-2 individually could not, but simultaneously silencing both TgbHLH42 could reduce the anthocyanin in tulip petals. These results indicate that TgbHLH42-1 and TgbHLH42-2 function partially redundantly to positively regulate anthocyanin biosynthesis during tulip petal coloration.

Genome-wide identification of heat shock transcription factor families in perennial ryegrass highlights the role of LpHSFC2b in heat stress response.

Perennial ryegrass (Lolium perenne) is a cool-season turf and forage grass. Heat shock transcription factors (HSFs) play an important role in regulating plant abiotic stress. However, HSFs in perennial ryegrass have rarely been characterized. Here, 25 LpHSFs were identified from the perennial ryegrass genome. Phylogenetic analysis showed that the LpHSFs could be classified into 12 subclasses. Gene structure analysis showed that 22 LpHSFs have only one intron. Cis-acting elements analysis revealed that the promoter of 15 LpHSFs contained hormone-responsive and abiotic stress-responsive elements. Expression profile analysis indicated that 24 LpHSFs were differentially expressed under submerge, drought, heat, and cold stresses. In addition, a subclass C2 gene, LpHSFC2b, was significantly induced by abiotic stresses. The LpHSFC2b protein is localized to the nucleus, and heterologous expression of LpHSFC2b in Arabidopsis improves plant thermotolerance. This study provides insights useful for the breeding of stress tolerance in perennial ryegrass.

Melatonin promotes Arabidopsis primary root growth in an IAA-dependent manner.

Melatonin has been characterized as a growth regulator in plants. Melatonin shares tryptophan as the precursor with the auxin indole-3-acetic acid (IAA), but the interplay between melatonin and IAA remains controversial. In this study, we aimed to dissect the relationship between melatonin and IAA in regulating Arabidopsis primary root growth. We observed that melatonin concentrations ranging from 10-9 to 10-6 M functioned as IAA mimics to promote primary root growth in Arabidopsis wild type, as well as in pin-formed (pin) single and double mutants. Transcriptome analysis showed that changes in gene expression after melatonin and IAA treatment were moderately correlated. Most of the IAA-regulated genes were co-regulated by melatonin, indicating that melatonin and IAA regulated a similar subset of genes. Melatonin partially rescued primary root growth defects in pin single and double mutant plants. However, melatonin treatment had little effect on primary root growth in the presence of high concentrations of auxin biosynthesis inhibitors, or polar transport inhibitor, and could not rescue the root length defect of the IAA biosynthesis quintuple mutant yucQ. Therefore, we propose that melatonin promotes primary root growth in an IAA-dependent manner.

Transcriptional variation analysis of Arabidopsis ecotypes in response to drought and salt stresses dissects commonly regulated networks.

Salinity and drought conditions commonly result in osmotic and oxidative stresses, while salinity additionally causes ionic stress. In this study, we identified specific genes regulated by osmotic and ionic stresses in five Arabidopsis ecotypes. Shahdara (SHA) and C24 ecotypes were more tolerant to salt and drought stresses at the seedling growth stage, as evidenced by lower water loss rate, lower electrolyte leakage, and higher survival rate when compared to the other three ecotypes under drought and salinity conditions. Transcriptomic analysis revealed that 3700 and 2242 genes were differentially regulated by salt and osmotic stresses, respectively. Totally 78.1% of upregulated and 62.0% of downregulated genes by osmotic stress were also commonly regulated by salt stress. Gene ontology term enrichment analysis showed that auxin indole-3-acetic acid (IAA), abscisic acid, cytokinin, and gibberellic acid pathways were regulated by the osmotic stress, while IAA, jasmonic acid, and ethylene pathways were changed by the ionic stress. The nutrient and water uptake pathways were regulated by both the osmotic and ionic stresses, whereas ion transportation and kinase pathways were modulated by the ionic stress. Additionally, we characterized bHLH61 as a negative regulator in response to salt and drought stresses. This study provided new clues of plant responses to salt and drought stresses.

Physiological and metabolomic responses of bermudagrass (Cynodon dactylon) to alkali stress.

Bermudagrass (Cynodon dactylon) is a widely used warm-season turfgrass species with superior stress tolerance except for cold. In this study, a comparative analysis of the responses to alkali stress in bermudagrass at the physiological and metabolomic levels were performed. Mild alkali with relatively low pH slightly inhibited growth of bermudagrass as evidenced by lower electrolyte leakage, more rapid growth and higher survival rate when compared to moderate and severe alkali treatments. Moreover, the amount of 37 metabolites including amino acids, organic acids, sugars and sugar alcohols were modulated by the alkali treatments. Among them, 15 metabolites were involved in carbon and amino acid metabolic pathways. Under mild alkali stress, bermudagrass possibly slowed down metabolisms to maintain basic growth. However, moderate and severe alkali-stressed plants accumulated significantly higher amount of carbohydrates which might result in carbon starvation. Taken together, alkali stress had severely inhibitory effect partially due to combined ionic stress and high pH stress. These results suggested that bermudagrass employed different strategies in response to alkali stresses with different pH and ionic values.

Ectopic expression of Medicago truncatula homeodomain finger protein, MtPHD6, enhances drought tolerance in Arabidopsis.

BACKGROUND: The plant homeodomain (PHD) finger is a Cys4HisCys3-type zinc finger which promotes protein-protein interactions and binds to the cis-acting elements in the promoter regions of target genes. In Medicago truncatula, five PHD homologues with full-length sequence were identified. However, the detailed function of PHD genes was not fully addressed. RESULTS: In this study, we characterized the function of MtPHD6 during plant responses to drought stress. MtPHD6 was highly induced by drought stress. Ectopic expression of MtPHD6 in Arabidopsis enhanced tolerance to osmotic and drought stresses. MtPHD6 transgenic plants exhibited decreased water loss rate, MDA and ROS contents, and increased leaf water content and antioxidant enzyme activities under drought condition. Global transcriptomic analysis revealed that MtPHD6 reprogramed transcriptional networks in transgenic plants. Expression levels of ABA receptor PYR/PYLs, ZINC FINGER, AP2/EREBP and WRKY transcription factors were mainly up-regulated after transformation of MtPHD6. Interaction network analysis showed that ZINC FINGER, AP2/EREBP and WRKY interacted with each other and downstream stress induced proteins. CONCLUSIONS: We proposed that ZINC FINGER, AP2/EREBP and WRKY transcription factors were activated through ABA dependent and independent pathways to increase drought tolerance of MtPHD6 transgenic plants.

Comparative physiological and metabolomic analyses reveal natural variations of tulip in response to storage temperatures.

MAIN CONCLUSION: Three tulip cultivars were screened out with successful bloom after a short-term cold treatment, and the differential responses to postharvest cold treatment were analyzed between two contrasting tulip cultivars. Tulip is one of the most important ornamental bulbous plants in the world. A precious precooling treatment during bulb postharvest is required for optimal floral stalk elongation and flower development in tulip. In this study, the naturally growing and flowering variations of tulip to storage temperatures were analyzed after long-term cold (LTC) and short-term cold (STC) treatments. Three cultivars were screened out with successful blooming after STC, which included 'Dow Jones' (DJ), 'Van Eijk' (VE) and 'World's Favourite' (WF) (5 °C for 2 weeks). Comparative analysis revealed that DJ cultivar maintained normal and intact reproductive organs under STC condition, while the 'Orange Emperor' (OE) cultivar, which failed blooming after STC treatment, showed gradually destroyed reproductive organs under STC condition. In addition, the DJ cultivar accumulated lower ROS levels and higher antioxidant enzyme activities, as well as significantly higher contents of total primary metabolites than OE to maintain normal shoot growth and floral organ development under STC condition. The relative expression levels of genes involved in vernalization and/or flower time regulation in DJ were significantly higher than those in OE after STC treatment. This study provides new insights into understanding the underlying mechanism of natural variation of tulip cultivars during postharvest storage treatment.

ABA receptor PYL9 promotes drought resistance and leaf senescence.

Drought stress is an important environmental factor limiting plant productivity. In this study, we screened drought-resistant transgenic plants from 65 promoter-pyrabactin resistance 1-like (PYL) abscisic acid (ABA) receptor gene combinations and discovered that pRD29A::PYL9 transgenic lines showed dramatically increased drought resistance and drought-induced leaf senescence in both Arabidopsis and rice. Previous studies suggested that ABA promotes senescence by causing ethylene production. However, we found that ABA promotes leaf senescence in an ethylene-independent manner by activating sucrose nonfermenting 1-related protein kinase 2s (SnRK2s), which subsequently phosphorylate ABA-responsive element-binding factors (ABFs) and Related to ABA-Insensitive 3/VP1 (RAV1) transcription factors. The phosphorylated ABFs and RAV1 up-regulate the expression of senescence-associated genes, partly by up-regulating the expression of Oresara 1. The pyl9 and ABA-insensitive 1-1 single mutants, pyl8-1pyl9 double mutant, and snrk2.2/3/6 triple mutant showed reduced ABA-induced leaf senescence relative to the WT, whereas pRD29A::PYL9 transgenic plants showed enhanced ABA-induced leaf senescence. We found that leaf senescence may benefit drought resistance by helping to generate an osmotic potential gradient, which is increased in pRD29A::PYL9 transgenic plants and causes water to preferentially flow to developing tissues. Our results uncover the molecular mechanism of ABA-induced leaf senescence and suggest an important role of PYL9 and leaf senescence in promoting resistance to extreme drought stress.

Transcriptomic profiling of tall fescue in response to heat stress and improved thermotolerance by melatonin and 24-epibrassinolide.

BACKGROUND: Tall fescue is a widely used cool season turfgrass and relatively sensitive to high temperature. Chemical compounds like melatonin (MT) and 24-epibrassinolide (EBL) have been reported to improve plant heat stress tolerance effectively. RESULTS: In this study, we reported that MT and EBL pretreated tall fescue seedlings showed decreased reactive oxygen species (ROS), electrolyte leakage (EL) and malondialdehide (MDA), but increased chlorophyll (Chl), total protein and antioxidant enzyme activities under heat stress condition, resulting in improved plant growth. Transcriptomic profiling analysis showed that 4311 and 8395 unigenes were significantly changed after 2 h and 12 h of heat treatments, respectively. Among them, genes involved in heat stress responses, DNA, RNA and protein degradation, redox, energy metabolisms, and hormone metabolism pathways were highly induced after heat stress. Genes including FaHSFA3, FaAWPM and FaCYTC2 were significantly upregulated by both MT and EBL treatments, indicating that these genes might function as the putative target genes of MT and EBL. CONCLUSIONS: These findings indicated that heat stress caused extensively transcriptomic reprogramming of tall fescue and exogenous application of MT and EBL effectively improved thermotolerance in tall fescue.

The zinc-finger transcription factor ZAT6 is essential for hydrogen peroxide induction of anthocyanin synthesis in Arabidopsis.

The accumulation of flavonoids is activated by various abiotic stresses, and the induction of reactive oxygen species (ROS) especially hydrogen peroxide (H2O2) is a general response to abiotic stress in plants. However, the direct link between flavonoids and H2O2 and underlying mechanism remain elusive. In this study, we found that the concentrations of anthocyanin and flavonoids were significantly induced by H2O2 treatment. Furthermore, we found that the transcript level of ZINC FINGER of ARABIDOPSIS THALIANA 6 (ZAT6) was significantly activated after exogenous H2O2 treatment, and modulation of AtZAT6 expression positively affected the concentrations of both anthocyanin and total flavonoids. Notably, exogenous H2O2-induced anthocyanin synthesis was largely alleviated in AtZAT6 knockdown plants, but showed higher level in AtZAT6 overexpressing plants. AtZAT6 directly activated the expressions of TT5, TT7, TT3, TT18, MYB12, and MYB111 through binding to their promoters with TACAAT elements of these genes, and the activation of MYB12 and MYB111 up-regulated the expressions of TT4 and TT6. Taken together, this study indicates that AtZAT6 plays important role in H2O2-activated anthocyanin synthesis, via directly binding to the promoters of several genes that involved in anthocyanin synthesis.

Phytomelatonin: a universal abiotic stress regulator.

Melatonin, a derivative of tryptophan, was first detected in plant species in 1995 and it has been shown to be a diverse regulator during plant growth and development, and in stress responses. Recently, great progress has been made towards determining the detailed functions of melatonin in plant responses to abiotic stress. Melatonin priming improves plant tolerance to cold, heat, salt, and drought stresses through regulation of genes involved in the DREB/CBF, HSF, SOS, and ABA pathways, respectively. As a scavenger of free radicals, melatonin also directly detoxifies reactive oxygen species, thus alleviating membrane oxidation. Abiotic stress-inhibited photosynthesis is partially recovered and metabolites accumulate in the presence of melatonin, leading to improved plant growth, delayed leaf senescence, and increased stress tolerance. In this review, we summarize the interactions of melatonin with phytohormones to regulate downstream gene expression, protein stabilization, and epigenetic modification in plants. Finally, we consider the need for, and approaches to, the identification of melatonin receptors and components during signaling transduction pathways.

Zinc finger of Arabidopsis thaliana 6 is involved in melatonin-mediated auxin signaling through interacting INDETERMINATE DOMAIN15 and INDOLE-3-ACETIC ACID 17.

Although accumulating evidence demonstrates the cross talk between melatonin and auxin as derivatives of tryptophan, the underlying signaling events remain unclear. In this study, we found that melatonin and auxin mediated the transcriptional levels of zinc finger of Arabidopsis thaliana (ZAT6) in a mutually antagonistic manner. ZAT6 negatively modulated the endogenous auxin level, and ZAT6 knockdown plants were less sensitive to melatonin-regulated auxin biosynthesis, indicating its involvement in melatonin-mediated auxin accumulation. Additionally, the identification of INDETERMINATE DOMAIN15 (IDD15) and INDOLE-3-ACETIC ACID 17 (IAA17) in Arabidopsis that interacted with ZAT6 in vivo provided new insight of ZAT6-mediated auxin signaling. Further investigation showed that ZAT6 repressed the transcription activation of IDD15 on the YUC2 promoter, while ZAT6 inhibited the interaction of TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and IAA17 through competitively binding to IAA17. Thus, both auxin synthesis and the auxin response were negatively modulated by ZAT6. Taken together, ZAT6 is involved in melatonin-mediated auxin signaling through forming an interacting complex of auxin signaling pathway in Arabidopsis.

The Arabidopsis Cys2/His2 zinc finger transcription factor ZAT18 is a positive regulator of plant tolerance to drought stress.

Environmental stress poses a global threat to plant growth and reproduction, especially drought stress. Zinc finger proteins comprise a family of transcription factors that play essential roles in response to various abiotic stresses. Here, we found that ZAT18 (At3g53600), a nuclear C2H2 zinc finger protein, was transcriptionally induced by dehydration stress. Overexpression (OE) of ZAT18 in Arabidopsis improved drought tolerance while mutation of ZAT18 resulted in decreased plant tolerance to drought stress. ZAT18 was preferentially expressed in stems, siliques, and vegetative rosette leaves. Subcellular location results revealed that ZAT18 protein was predominantly localized in the nucleus. ZAT18 OE plants exhibited less leaf water loss, lower content of reactive oxygen species (ROS), higher leaf water content, and higher antioxidant enzyme activities after drought treatment when compared with the wild type (WT). RNA sequencing analysis showed that 423 and 561 genes were transcriptionally modulated by the ZAT18 transgene before and after drought treatment, respectively. Pathway enrichment analysis indicated that hormone metabolism, stress, and signaling were over-represented in ZAT18 OE lines. Several stress-responsive genes including COR47, ERD7, LEA6, and RAS1, and hormone signaling transduction-related genes including JAZ7 and PYL5 were identified as putative target genes of ZAT18. Taken together, ZAT18 functions as a positive regulator and plays a crucial role in the plant response to drought stress.

RDM4 modulates cold stress resistance in Arabidopsis partially through the CBF-mediated pathway.

The C-REPEAT-BINDING FACTOR (CBF) pathway has important roles in plant responses to cold stress. How the CBF genes themselves are activated after cold acclimation remains poorly understood. In this study, we characterized cold tolerance of null mutant of RNA-DIRECTED DNA METHYLATION 4 (RDM4), which encodes a protein that associates with RNA polymerases Pol V and Pol II, and is required for RNA-directed DNA methylation (RdDM) in Arabidopsis. The results showed that dysfunction of RDM4 reduced cold tolerance, as evidenced by decreased survival and increased electrolyte leakage. Mutation of RDM4 resulted in extensive transcriptomic reprogramming. CBFs and CBF regulon genes were down-regulated in rdm4 but not nrpe1 (the largest subunit of PolV) mutants, suggesting that the role of RDM4 in cold stress responses is independent of the RdDM pathway. Overexpression of RDM4 constitutively increased the expression of CBFs and regulon genes and decreased cold-induced membrane injury. A great proportion of genes affected by rdm4 overlapped with those affected by CBFs. Chromatin immunoprecipitation results suggested that RDM4 is important for Pol II occupancy at the promoters of CBF2 and CBF3. We present evidence of a considerable role for RDM4 in regulating gene expression at low temperature, including the CBF pathway in Arabidopsis.