INTRODUCTION:Plants have evolved complex defense systems to cope with herbivore attack, yet how these defense responses are coordinated across distinct cell types and time scales remains unclear. Understanding the cellular and spatial organization of such defenses is critical for elucidating the mechanisms underlying plant-insect interactions. OBJECTIVES:This study aimed to dissect the spatiotemporal regulation of tobacco leaf defense responses to herbivory by integrating time-series single-cell transcriptomics and spatial metabolomics. METHODS:We generated a high-resolution single-cell atlas of 28,318 tobacco leaf cells under simulated herbivory. Transcriptional dynamics, cell-cell communication networks, and spatial metabolite distributions were analyzed using co-expression, pseudotime, and ligand-receptor inference approaches. Functional validation of key regulators was conducted through qRT-PCR and VIGS assays. RESULTS:Our analyses revealed rapid transcriptional and metabolic remodeling following herbivore stress, with epidermal subpopulations serving as early signaling hubs. Spatial metabolomics confirmed epidermis-enriched accumulation of defense metabolites. Pseudotime and co-expression analyses identified epidermal subcluster 6 as an early-responsive population characterized by elevated WRKY81 expression. Silencing WRKY81 impaired defense activation, increasing herbivore feeding efficiency and metabolic assimilation. CONCLUSION:This study establishes the first single-cell-resolved dynamic defense network of tobacco leaves against herbivory. The findings uncover the central role of epidermal transcriptional reprogramming and identify WRKY81 as a critical regulator of early defense commitment, offering a mechanistic basis for the development of pest-resistant crops.

INTRODUCTION:Pseudomonas syringae pv. tabaci, a Gram-negative bacterial pathogen, causes devastating tobacco wildfire disease with global economic impacts. While its pathogenicity is well documented, the dynamic defense mechanisms of tobacco against infection remain poorly understood. OBJECTIVE:This study aimed to decipher phased defense mechanisms of tobacco against P. syringae infection through multi-omics integration, with emphasis on elucidating spatiotemporal coordination between transcriptional reprogramming and metabolic remodeling, and functionally validating critical regulatory modules. METHODS:Time-series transcriptomic and metabolomic profiling was integrated to reconstruct dynamic response patterns. Stage-specific regulatory modules were explored via TO-GCN and WGCNA, and the roles of WRKY6 and WRKY23 in disease resistance were validated by generating transgenic lines. RESULTS:Early infection (12-24 hpi) prioritized stress signaling and hormone pathway activation (salicylic acid/jasmonate), transitioning to cellular homeostasis regulation at late stages (48-60 hpi). WRKY, ERF, and NAC families orchestrated stage-specific gene expression. Notably, WRKY6 and WRKY23 functioned as negative regulators, with their silencing leading to a reduction in lesion area by 42-58% and pathogen load by 3.2-4.5 fold. Metabolomic analysis revealed sustained activation of phenylpropanoid metabolism, specifically regulating L-phenylalanine homeostasis and biosynthesis of its defense derivative xanthosine. Additionally, core modules involved in sphingolipid metabolism, light responses, and hormone cross-talk were also identified. CONCLUSION:We demonstrate that WRKY-mediated transcriptional reprogramming coordinates phytohormone signaling, sphingolipid dynamics, and light responses to spatiotemporally regulate secondary metabolite production. The identified WRKY6 and WRKY23 regulatory module establishes a molecular framework for engineering disease resistance, and the proposed two-phase defense model (early signaling → late metabolic remodeling) advances understanding of plant-pathogen interactions and offers targets for precision breeding.

Dehydration-Responsive Element Binding Protein 2C (DREB2C) is a vital transcription factor that responds to drought, cold and heat stresses in Arabidopsis. Previous studies have suggested that DREB2C might be involved in abscisic acid (ABA) signaling, but the detailed molecular mechanism remains unclear. In this study, we find that DREB2C positively regulates ABA-mediated seed germination and root elongation. Degradation assays show that the degradation of DREB2C relies on the 26S proteasome pathway and ABA promotes the accumulation of DREB2C. Further analyses confirm that two novel E3 ligases DREB2C-Degrading E3 Ligase 1/2 (DEL1/2) interact with DREB2C and mediate its ubiquitination. Functional analyses demonstrate that DEL1 or DEL2 negatively regulates ABA-mediated seed germination and root elongation, while overexpression of DEL1 or DEL2 in DREB2C-overexpressing lines results in reduced ABA hypersensitivity. Genetic analysis indicates that DEL1 and DEL2 function redundantly in the degradation of DREB2C and act upstream of DREB2C in the ABA signaling pathway. In addition, DREB2C directly binds to the promoters of DEL1 and DEL2 and activates their expression, thereby shutting down the transmission of ABA signals. Collectively, we uncover a novel feedback regulatory loop between DREB2C and DEL1/DEL2 that balances the initial growth stage and ABA responses in Arabidopsis.

Tobacco (Nicotiana tabacum, 2n = 48) is a key non-food economic crop, yet its stress response and gene regulatory mechanisms remain poorly understood. By analyzing 603 transcriptome datasets, this study identified 1405 tissue-specific genes, revealing tissue-specific synthesis of terpenoids and other ecologically important secondary metabolites in sepals and other tissues. Comparative stress-response analysis highlighted distinct gene expression patterns in leaves and roots under biotic and abiotic stresses. Additionally, 28,396 expression quantitative trait loci (eQTLs) were mapped in leaves, offering valuable genetic regulatory markers. These findings provide crucial insights into tobacco's gene expression characteristics and their functional implications, serving as a foundation for future research.

Saline-alkali stress is one of the major abiotic factors limiting crop production and affecting the ecological environment. The plasma membrane (PM) H+-ATPases are involved in modulating the membrane potential in response to alkaline stress. The central loop (cytoplasmic domain) of the PM H+-ATPase AHA2, in contrast to its well-studied C-terminal regulatory domain, remains poorly understood in terms of its regulatory function. In this study, we found that CARK1 and CARK3 (cytosolic ABA receptor kinase 1 and 3) positively modulate saline-alkali stress tolerance in Arabidopsis. Using molecular biology and biochemistry approaches, we reveal that CARK1 and CARK3 interact with and phosphorylate AHA2 at Thr469 in the central loop domain. Molecular mechanism indicates that CARK1/3-mediated phosphorylation elevates AHA2 activity through two key actions: First, by increasing Thr947 phosphorylation and promoting binding to 14-3-3 protein, and second, by releasing autoinhibitory interaction between the C-terminus and the central loop of AHA2. Functional and genetic analyses reveal that the phosphorylation-mimicking mutation AHA2T469D dramatically rescues hypersensitivity to alkali tolerance, H+ efflux, and cytosolic ROS accumulation in aha2 and cark1/3aha2 triple mutants. Collectively, our work reveals the central regulatory loop of AHA2 in response to alkali stress and reports that its activity is enhanced through Thr469 phosphorylation by CARK1/3.

Tobacco is a vital economic crop, and its cultivation is severely threatened by black shank disease, caused by Phytophthora nicotianae (P. nicotianae). Actin depolymerizing factor (ADF) proteins are key regulators of actin dynamics and play crucial roles in plant growth, intracellular transport, and stress responses. Although the ADF gene family has been extensively studied in various plant species, characterization in tobacco remains limited, particularly regarding its role in disease resistance. In this study, 24 NtADF genes were identified in the tobacco genome. Phylogenetic analysis classified them into six groups, with structural and motif compositions supporting functional conservation within groups. Evolutionary analysis revealed that whole-genome duplication (WGD) or segmental duplication (SDR) was the primary drivers of NtADFs duplication, with strong purification selection. Promoter analysis identified numerous stress- and hormone-related cis-elements, and the regulatory network predictions indicated potential interactions with transcription factors (e.g., ERF, MYB, WRKY) and microRNAs (miRNAs). Expression profiling demonstrated tissue-specific patterns and differential responses to abiotic and biotic stresses. Notably, functional characterization demonstrated that NtADF22 played a critical role in conferring resistance to black shank disease. This study provides comprehensive insights into the NtADF gene family, highlighting its important roles in plant development and stress responses. These findings offer both theoretical and practical significance for molecular breeding and genetic improvement of tobacco.

O-acetylation of cell wall polysaccharides is determined by plant species and tissue type and plays an instrumental role in the growth of plant cells and their interaction with the environment. The trichome birefringence-like (TBL) gene family encodes the acetyltransferases that facilitate O-acetylation. However, no report on the TBL gene family in tobacco (Nicotiana tabacum) can be found. In this study, 130 TBL genes were identified and characterized; they are distributed across all 24 tobacco chromosomes. Analysis of gene structure and conserved domains and motifs identified five groups of N. tabacum TBL genes (NtTBL). The NtTBL genes underwent segmental duplication and purifying selection. Functional analysis of cis-elements, interaction networks, and expression patterns of NtTBL genes indicated their significant roles in growth and development and stress responses. Furthermore, we validated the pivotal role of NtTBL31 in tobacco growth and drought resistance. This study lays a foundation for further functional characterization of the TBL gene family in tobacco.

Actin depolymerizing factors (ADFs), like other actin-binding proteins (ABPs), are modified by phosphorylation to regulate the dynamics of the actin filaments, thereby functioning in various processes throughout the plant lifecycle. In this study, we found that the Arabidopsis thaliana cytoplasmic kinase AGC1.7 interacts with ADF7 in vitro and in vivo. AGC1.7 phosphorylates ADF7 at its Ser-6, Ser-103 and Ser-104 residues in vitro, while replacing these residues with alanine promotes ADF7-mediated actin depolymerization in vitro. Expression of the phosphorylation-mimetic mutant protein ADF7S6/103/104D driven by the pollen-specific LAT52 promoter fully rescues the defects in germination rate, silique length and seeds per silique in both adf7-2 and agc1.5 agc1.7 (agcdm) mutants. Our data establish a model whereby AGC1.7-mediated ADF7 phosphorylation plays an important role in pollen germination and pollen tube growth.

The polar auxin transport is required for proper plant growth and development. D6 PROTEIN KINASE (D6PK) is required for the phosphorylation of PIN-FORMED (PIN) auxin efflux carriers to regulate auxin transport, while the regulation of D6PK stabilization is still poorly understood. Here, we found that Cytosolic ABA Receptor Kinases (CARKs) redundantly interact with D6PK, and the interactions are dependent on CARKs' kinase activities. Similarly, CARK3 also could interact with paralogs of D6PK, including D6PKL1, D6PKL2, and D6PKL3. The genetic analysis shows that D6PK acts the downstream of CARKs to regulate Arabidopsis growth, including hypocotyl, leaf area, vein formation, and the length of silique. Loss-of-function of CARK3 in overexpressing GFP-D6PK plants leads to reduce the level of D6PK protein, thereby rescues plant growth. In addition, the cell-free degradation assays indicate that D6PK is degraded through 26 S proteasome pathway, while the phosphorylation by CARK3 represses this process in cells. In summary, D6PK stabilization by the CARK family is required for auxin-mediated plant growth and development.

R2R3-MYB transcription factors play important roles in response to abiotic stresses in planta, such as salt, drought, and osmotic stress. However, the role of FtMYB11 in Tartary buckwheat (Fagopyrum tataricum) in drought and osmotic tolerance has not yet been elucidated. In this study, we found that FtMYB11 was markedly induced by exogenous abscisic acid (ABA), salinity, and mannitol. Further, FtMYB11-overexpressing Arabidopsis showed hypersensitivity to ABA-mediated seed germination and seedling establishment through regulating transcripts of AtCBF1, AtDREB2A, and AtRD20, compared with wild type, indicating that FtMYB11 plays a positive role in ABA signaling. In contrast, transgenic lines overexpressing FtMYB11 were sensitive to mannitol and NaCl treatments, suggesting that FtMYB11 plays a negative role in osmotic tolerance. Intriguingly, the transcripts of ABA biosynthetic enzyme genes were significantly elevated in plants overexpressing FtMYB11 after exposure to osmotic stresses, such as AtABA3 and AtNCED3. In addition, flavonoid biosynthesis genes were also upregulated in transgenic Arabidopsis under ABA, salt, and drought treatments, including AtC4H, AtF3H, AtANS, AtFLS, and At4CL. The drought tolerance assay showed that plants overexpressing FtMYB11 displayed greater tolerance to water deficit through regulating MDA and proline content. Taken together, FtMYB11 has opposite roles in response to abiotic stresses, but it may mediate flavonoid biosynthesis through regulation of related enzyme genes.

As a regulator of actin filament turnover, Arabidopsis thaliana CAP1 plays an important role in plant growth and development. Here, we analyzed the phenotypes of two Arabidopsis cap1 mutants: cap1-1 (a T-DNA insertion mutant) and Cas9-CAP1 (generated by CRISPR-Cas9 gene editing). Phenotypic analysis demonstrated that loss of CAP1 results in defects in seed germination and seedling morphology, with some seedlings exhibiting one or three cotyledons. The cap1-1 mutant took longer than the wild type to complete its life cycle, but its flowering time was normal, indicating that loss of CAP1 prolongs reproductive but not vegetative growth. Moreover, loss of CAP1 severely reduces seed production in self-pollinated plants, due to disruption of pollen tube elongation. RNA-seq and qRT-PCR analyses demonstrated that CAP1 may be involved in osmotic stress responses. Indeed, the cap1-1 mutant showed increased tolerance of salt and mannitol treatment, indicating that CAP1 plays a negative role in osmotic stress tolerance in Arabidopsis. Taken together, our results demonstrate that CAP1 functions not only in plant growth and development, but also in Arabidopsis responses to osmotic stress.

Abstract Background Fagopyrum (Polygonaceae) is a small plant lineage comprised of more than fifteen economically and medicinally important species. However, the phylogenetic relationships of the genus are not well explored, and the characteristics of Fagopyrum chloroplast genomes (plastomes) remain poorly understood so far. It restricts the comprehension of species diversity in Fagopyrum. Therefore, a comparative plastome analysis and comprehensive phylogenomic analyses are required to reveal the taxonomic relationship among species of Fagopyrum. Results In the current study, 12 plastomes were sequenced and assembled from eight species and two varieties of Fagopyrum. In the comparative analysis and phylogenetic analysis, eight previously published plastomes of Fagopyrum were also included. A total of 49 plastomes of other genera in Polygonaceae were retrieved from GenBank and used for comparative analysis with Fagopyrum. The variation of the Fagopyrum plastomes is mainly reflected in the size and boundaries of inverted repeat/single copy (IR/SC) regions. Fagopyrum is a relatively basal taxon in the phylogenomic framework of Polygonaceae comprising a relatively smaller plastome size (158,768–159,985 bp) than another genus of Polygonaceae (158,851–170,232 bp). A few genera of Polygonaceae have nested distribution of the IR/SC boundary variations. Although most species of Fagopyrum show the same IRb/SC boundary with species of Polygonaceae, only a few species show different IRa/SC boundaries. The phylogenomic analyses of Fagopyrum supported the cymosum and urophyllum groups and resolved the systematic position of subclades within the urophyllum group. Moreover, the repeat sequence types and numbers were found different between groups of Fagopyrum. The plastome sequence identity showed significant differences between intra-group and inter-group. Conclusions The deletions of intergenic regions cause a short length of Fagopyrum plastomes, which may be the main reason for plastome size diversity in Polygonaceae species. The phylogenomic reconstruction combined with the characteristics comparison of plastomes supports grouping within Fagopyrum. The outcome of these genome resources may facilitate the taxonomy, germplasm resources identification as well as plant breeding of Fagopyrum.

Actin cytoskeleton plays a critical role in a myriad of physiological processes, including plant growth and development, and in response to abiotic and biotic stresses. The highly dynamic actin filaments in plant cells depend on the actin-binding proteins. Here, we analyzed the phenotype of cap1-1 mutants in Arabidopsis and found that CAP1 acts as an important regulator over the life cycle. Plants homozygous for cap1 alleles show a reduction seed germination and morphogenetic disruption of seedlings, exhibiting one leaf or three leaves. We also found that cap1-1 mutants had longer life cycle than the wild-type plants, while there was the indistinguishable flower time, these results indicating that CAP1 prolongs the reproductive growth but not vegetable growth. Loss of CAP1 leads to severely drop in production, due to the disruption of pollen tube elongation. Additionally, RNA-seq and qRT-PCR suggest that CAP1 is involved in biotic and abiotic stresses. Taken together, cap1-1 plants show disruption in plant growth and development, suggesting that CAP1 as actin-binding proteins is required for the balance of the dynamic actin filaments in plant cells.

Actin depolymerization factors (ADFs), as actin-binding proteins, act a crucial role in plant development and growth, as well as in response to abiotic and biotic stresses. Here, we found that CARK3 plays a role in regulating hypocotyl development and links a cross-talk between actin filament and drought stress through interaction with ADF4. By using bimolecular fluorescence complementation (BiFC) and GST pull-down, we confirmed that CARK3 interacts with ADF4 in vivo and in vitro. Next, we generated and characterized double mutant adf4cark3-4 and OE-ADF4:cark3-4. The hypocotyl elongation assay indicated that the cark3-4 mutant seedlings were slightly longer hypocotyls when compared with the wild type plants (WT), while CARK3 overexpressing seedlings had no difference with WT. In addition, overexpression of ADF4 significantly inhibited long hypocotyls of cark3-4 mutants. Surprisingly, we found that overexpression of ADF4 markedly enhance drought resistance in soil when compared with WT. On the other hand, drought tolerance analysis showed that overexpression of CARK3 could rescue adf4 drought susceptibility. Taken together, our results suggest that CARK3 acts as a regulator in hypocotyl elongation and drought tolerance likely via regulating ADF4 phosphorylation.

14-3-3 proteins, a family of conserved molecules in eukaryotes, target a number of protein clients through their ability to recognize well-defined phosphorylated motifs. ADF4, as one of Actin-Depolymerizing Factor (ADF) family of proteins, is involved in plant development, and response to biotic and abiotic stresses. Here, we show that 14-3-3κ specially interacted with ADF4 in vitro and in vivo. The 14-3-3κ×adf4 double mutant displayed less F-actin bundle and shorter hypocotyl compared with adf4 mutant, indicating that 14-3-3κ acts upstream of ADF4 to mediate the hypocotyl growth in the dark-grown seedlings. Under the osmotic stress, 14-3-3κ mutants displayed less survival rate than wild-type plants. The adf4 mutants exhibited markedly enhanced survival rate under osmotic treatment, while ADF4-overexpressing plants displayed the opposite results, indicating that ADF4 plays a negative role in response to osmotic stress in Arabidopsis. The interaction between ADF4 and 14-3-3κ inhibited the association of ADF4 with actin filament. Moreover, the in vitro phosphorylation assay demonstrates that the phosphorylation of ADF4 by CASEIN KINASE1-LIKE PROTEIN2 (CKL2) was enhanced by binding 14-3-3κ. Collectively, our data infer a fundamental role for the interaction between 14-3-3κ and ADF4 in regulating hypocotyl growth and osmotic tolerance of plants.

Cytosolic ABA Receptor Kinases (CARKs) play a pivotal role in abscisic acid (ABA)-dependent pathway in response to dehydration, but their regulatory mechanism in ABA signaling remains unexplored. In this study, we showed that CARK4/5 of CARK family physically interacted with ABA receptors (RCARs/PYR1/PYLs), including RCAR3, RCAR11-RCAR14, while CARK2/7/11 only interacted with RCAR11-RCAR14, but not RCAR3. It indicates that the members in CARK family function redundantly and differentially in ABA signaling. RCAR12 can form heterodimer with RCAR3 in vitro and in vivo. Moreover, the members of CARK family can form homodimer or heterodimer in a kinase activity dependent manner. ITC (isothermal titration calorimetry) analysis demonstrated that the phosphorylation of RCAR12 by CARK1 enhanced the ABA binding affinity. The phosphor-mimic RCAR12(T105D) significantly displayed ABA-induced inhibition of the phosphatase ABI1 (ABA insensitive 1) activity, leading to upregulation of ABA-responsive genes RD29A and RD29B in cark157:RCAR12(T105D) transgenic plants, which exhibited ABA hypersensitive phenotype. The transcription factor ABI5 (ABA insensitive 5) activates the transcriptions of CARK1 and CARK3 by binding to ABA-response elements (ABREs) of their promoters. Collectively, our data imply that the dimeric CARKs phosphorylate homodimer or heterodimer ABA receptors, leading to monomerization for triggering ABA responses in Arabidopsis.

Abstract Background: Fagopyrum (Polygonaceae) is a small plant lineage comprised of more than fifteen economically and medicinally important species. However, the phylogenetic relationships of the genus are not well explored, and the characteristics of Fagopyrum chloroplast genomes (plastomes) remain poorly understood so far. It restricts the comprehension of species diversity in Fagopyrum. Therefore, a comparative plastome analysis and comprehensive phylogenomic analyses are required to reveal the taxonomic relationship among species of Fagopyrum. Results: In the current study, 12 plastomes were sequenced and assembled from eight species and two varieties of Fagopyrum. In the comparative analysis and phylogenetic analysis, eight previously published plastomes of Fagopyrum were also included. A total of 49 plastomes of other genera in Polygonaceae were retrieved from GenBank and used for comparative analysis with Fagopyrum. The variation of the Fagopyrum plastomes is mainly reflected in the size and boundaries of inverted repeat/single copy (IR/SC) regions. Fagopyrum is a relatively basal taxon in the phylogenomic framework of Polygonaceae comprising a relatively smaller plastome size (158,768–159,985 bp) than another genus of Polygonaceae (158,851–170,232 bp). A few genera of Polygonaceae have nested distribution of the IR/SC boundary variations. Although most species of Fagopyrum show the same IRb/SC boundary with species of Polygonaceae, only a few species show different IRa/SC boundaries. The phylogenomic analyses of Fagopyrum supported the cymosum and urophyllum groups and resolved the systematic position of subclades within the urophyllum group. Moreover, the repeat sequence types and numbers were found different between groups of Fagopyrum. The plastome sequence similarity showed significant differences between intra-group and inter-group.Conclusions: The deletions of intergenic regions cause a short length of Fagopyrum plastomes, which may be the main reason for plastome size diversity in Polygonaceae species. Most of the Polygonaceae plastomes may have experienced gene loss events, resulting in the nested distribution of IR/SC boundary variation. The phylogenomic reconstruction combined with the characteristics comparison of plastomes supports grouping within Fagopyrum. The outcome of these genome resources may facilitate the taxonomy, germplasm resources identification as well as plant breeding of Fagopyrum.

The phytohormones jasmonates (JAs) act as important molecules of elicitors for the chlorophyll degradation and anthocyanin biosynthesis. JAs do usually not act independently but integrate in complex networks linking to other hormonal signaling transduction. Here, the crosstalk was detected between the JAs (jasmonic acid) and abscisic acid (ABA) signaling pathways in the mediation of chlorophyll degradation and anthocyanin biosynthesis. In this study, we found that the ABA receptor mutants, pyr1pyl1pyl2pyl4 (1124) and pyr1pyl1ply2pyl4pyl5pyl8 (112458) showed less level of chlorophyll and anthocyanin than the wild-type plants, while gain-of-function of RCAR13 transgenic lines inhibited chlorophyll degradation and enhanced anthocyanin accumulation after MeJA treatment. The amidohydrolases, including ILL6 and IAR3 and cytochrome P450 (CYP94B3), encoding JA-Ile catabolism were markedly depressed by ABA receptors. While transcripts of the enzymes for activation of anthocyanin biosynthesis pathway were analyzed, the results indicating that JA biosynthetic genes, including allene oxide synthase (AOS), LOX3 and LOX4 were enhanced by the link of JAs and ABA receptors. Moreover, the ABA receptors are also involved in JAs signal transduction through the regulation of COI, JAZ and MYC2 transcripts. These findings elucidate a connection between a core component of the ABA signaling pathway and JA responses.

E3 ubiquitin ligase plays a vital role in the ubiquitin-mediated heat-related protein degradation pathway. Herein, we report that the expression of AtPPRT1, a C3HC4 zinc-finger ubiquitin E3 ligase gene, was induced by heat stress, and the β-glucuronidase (GUS) gene driven by the AtPPRT1 promoter has shown increased activity after basal and acquired thermotolerance. To further explore the function of AtPPRT1 in heat stress response (HSR), we used the atpprt1 mutant and AtPPRT1-overexpressing lines (OE2 and OE10) to expose in heat shock. In this study, the atpprt1 mutant had a lower germination and survival rate than those of Col-0 when suffered from the heat stress, whereas OEs enhanced basal and acquired thermotolerance in Arabidopsis seedlings. When compared to Col-0 and OEs, loss-of-function in AtPPRT1 resulted in lower chlorophyll retention and higher content of reactive oxygen species (ROS) after heat treatment. Moreover, the transcript levels of AtPPRT1 and several heat-related genes (AtZAT12, AtHSP21 and AtHSFA7a) were upregulated to greater extents in OEs and lower extents in atpprt1 compared to Col-0 after heat treated. Hence, we suggest that AtPPRT1 may act as a positive role in regulating the high temperature by mediating the degradation of unknown target proteins.

Salt stress is one of the environmental factors that negatively affect plant growth and development. We have previously reported a putative C3HC4 zinc-finger ubiquitin E3 ligase (AtPPRT1) negatively regulates Abscisic acid (ABA) and drought stress response. According to previous studies, the accumulation of ABA in plants can further regulate the salt stress response. Therefore, in this study, we further analyzed whether AtPPRT1 negatively regulates the salt stress response. The results showed that AtPPRT1 expression was induced by salt stress. Furthermore, under salt stress, the β-glucuronidase (GUS) gene driven by the AtPPRT1 promoter has shown increased activity in the hypocotyl and petioles of Arabidopsis seedlings. Additionally, seedlings of the T-DNA insertion mutant atpprt1 showed significant growth advantage under salt stress, whereas overexpressing AtPPRT1 (OE lines) in Arabidopsis seedlings displayed hypersensitive under salt stress. Etiolated atpprt1 seedlings also demonstrated significantly elongated hypocotyl lengths in salt stress. The elevated or reduced salt tolerance of atpprt1 and AtPPRT1 overexpressing lines was confirmed by the changes in chlorophyll content and 3,3'-Diaminobenzidine (DAB) staining. The above data suggest that AtPPRT1 has a negative effect on salt tolerance in Arabidopsis seedlings.