Identification of CEP peptides encoded by the tobacco (Nicotiana tabacum) genome and characterization of their roles in osmotic and salt stress responses.

Members of the CEP (C-terminally Encoded Peptide) gene family have been shown to be involved in various developmental processes and stress responses in plants. In order to understand the roles of CEP peptides in stress response, a comprehensive bioinformatics approach was employed to identify NtCEP genes in tobacco (Nicotiana tabacum L.) and to analyze their potential roles in stress responses. Totally 21 NtCEP proteins were identified and categorized into two subgroups based on their CEP domains. Expression changes of the NtCEP genes in response to various abiotic stresses were analyzed via qRT-PCR and the results showed that a number of NtCEPs were significant up-regulated under drought, salinity, or temperature stress conditions. Furthermore, application of synthesized peptides derived from NtCEP5, NtCEP13, NtCEP14, and NtCEP17 enhanced plant tolerance to different salt stress treatments. NtCEP5, NtCEP9 and NtCEP14, and NtCEP17 peptides were able to promote osmotic tolerance of tobacco plants. The results from this study suggest that NtCEP peptides may serve as important signaling molecules in tobacco's response to abiotic stresses.

Phosphorylation of Thr-225 and Ser-262 on ERD7 Promotes Age-Dependent and Stress-Induced Leaf Senescence through the Regulation of Hydrogen Peroxide Accumulation in Arabidopsis thaliana.

As the final stage of leaf development, leaf senescence is affected by a variety of internal and external signals including age and environmental stresses. Although significant progress has been made in elucidating the mechanisms of age-dependent leaf senescence, it is not clear how stress conditions induce a similar process. Here, we report the roles of a stress-responsive and senescence-induced gene, ERD7 (EARLY RESPONSIVE TO DEHYDRATION 7), in regulating both age-dependent and stress-induced leaf senescence in Arabidopsis. The results showed that the leaves of erd7 mutant exhibited a significant delay in both age-dependent and stress-induced senescence, while transgenic plants overexpressing the gene exhibited an obvious accelerated leaf senescence. Furthermore, based on the results of LC-MS/MS and PRM quantitative analyses, we selected two phosphorylation sites, Thr-225 and Ser-262, which have a higher abundance during senescence, and demonstrated that they play a key role in the function of ERD7 in regulating senescence. Transgenic plants overexpressing the phospho-mimetic mutant of the activation segment residues ERD7T225D and ERD7T262D exhibited a significantly early senescence, while the inactivation segment ERD7T225A and ERD7T262A displayed a delayed senescence. Moreover, we found that ERD7 regulates ROS accumulation by enhancing the expression of AtrbohD and AtrbohF, which is dependent on the critical residues, i.e., Thr-225 and Ser-262. Our findings suggest that ERD7 is a positive regulator of senescence, which might function as a crosstalk hub between age-dependent and stress-induced leaf senescence.

Transcription factors NtNAC028 and NtNAC080 form heterodimers to regulate jasmonic acid biosynthesis during leaf senescence in Nicotiana tabacum.

Plant senescence, as a highly integrated developmental stage, involves functional degeneration and nutrient redistribution. NAM/ATAF1/CUC (NAC) transcription factors orchestrate various senescence-related signals and mediate the fine-tuning underlying plant senescence. Previous data revealed that knockout of either NtNAC028 or NtNAC080 leads to delayed leaf senescence in tobacco (Nicotiana tabacum), which implies that NtNAC028 and NtNAC080 play respective roles in the regulation of leaf senescence, although they share 91.87% identity with each other. However, the mechanism underlying NtNAC028- and NtNAC080-regulated leaf senescence remains obscure. Here, we determined that NtNAC028 and NtNAC080 activate a putative jasmonic acid (JA) biosynthetic gene, NtLOX3, and enhance the JA level in vivo. We found that NtNAC028 and NtNAC080 interact with each other and themselves through their NA-terminal region. Remarkably, only the dimerization between NtNAC028 and NtNAC080 stimulated the transcriptional activation activity, but not the DNA binding activity of this heterodimer on NtLOX3. Metabolome analysis indicated that overexpression of either NtNAC028 or NtNAC080 augments both biosynthesis and degradation of nicotine in the senescent stages. Thus, we conclude that NtNAC028 cooperates with NtNAC080 and forms a heterodimer to enhance NtLOX3 expression and JA biosynthesis to trigger the onset of leaf senescence and impact secondary metabolism in tobacco.

Genome-Wide Studies of FH Family Members in Soybean (Glycine max) and Their Responses under Abiotic Stresses.

Formins or formin homology 2 (FH2) proteins, evolutionarily conserved multi-domain proteins in eukaryotes, serve as pivotal actin organizers, orchestrating the structure and dynamics of the actin cytoskeleton. However, a comprehensive investigation into the formin family and their plausible involvement in abiotic stress remains undocumented in soybean (Glycine max). In the current study, 34 soybean FH (GmFH)family members were discerned, their genomic distribution spanning the twenty chromosomes in a non-uniform pattern. Evolutionary analysis of the FH gene family across plant species delineated five discernible groups (Group I to V) and displayed a closer evolutionary relationship within Glycine soja, Glycine max, and Arabidopsis thaliana. Analysis of the gene structure of GmFH unveiled variable sequence lengths and substantial diversity in conserved motifs. Structural prediction in the promoter regions of GmFH gene suggested a large set of cis-acting elements associated with hormone signaling, plant growth and development, and stress responses. The investigation of the syntenic relationship revealed a greater convergence of GmFH genes with dicots, indicating a close evolutionary affinity. Transcriptome data unveiled distinctive expression patterns of several GmFH genes across diverse plant tissues and developmental stages, underscoring a spatiotemporal regulatory framework governing the transcriptional dynamics of GmFH gene. Gene expression and qRT-PCR analysis identified many GmFH genes with a dynamic pattern in response to abiotic stresses, revealing their potential roles in regulating plant stress adaptation. Additionally, protein interaction analysis highlighted an intricate web of interactions among diverse GmFH proteins. These findings collectively underscore a novel biological function of GmFH proteins in facilitating stress adaptation in soybeans.

Small secreted peptides (SSPs) in tomato and their potential roles in drought stress response.

Tomato (Solanum lycopersicum) is one of the most important vegetable crops in the world and abiotic stresses often cause serious problems in tomato production. It is thus important to identify new regulators in stress response and to devise new approaches to promote stress tolerance in tomato. Previous studies have shown that small secreted peptides (SSPs) are important signal molecules regulating plant growth and stress response by mediating intercellular communication. However, little is known about tomato SSPs, especially their roles in responding to abiotic stresses. Here we report the identification of 1,050 putative SSPs in the tomato genome, 557 of which were classified into 38 known SSP families based on their conserved domains. GO and transcriptome analyses revealed that a large proportion of SlSSPs might be involved in abiotic stress response. Further analysis indicated that stress response related cis-elements were present on the SlCEP promotors and a number of SlCEPs were significantly upregulated by drought treatments. Among the drought-inducible SlCEPs, SlCEP10 and SlCEP11b were selected for further analysis via exogenous application of synthetic peptides. The results showed that treatments with both SlCEP10 and SlCEP11b peptides enhanced tomato drought stress tolerance, indicating the potential roles of SlSSPs in abiotic stress response.

Senescence-related receptor kinase 1 functions downstream of WRKY53 in regulating leaf senescence in Arabidopsis.

Receptor-like kinases (RLKs) are the most important class of cell surface receptors, and play crucial roles in plant development and stress responses. However, few studies have been reported about the biofunctions of RLKs in leaf senescence. Here, we characterized a novel Arabidopsis RLK-encoding gene, SENESCENCE-RELATED RECEPTOR KINASE 1 (SENRK1), which was significantly down-regulated during leaf senescence. Notably, the loss-of-function senrk1 mutants displayed an early leaf senescence phenotype, while overexpression of SENRK1 significantly delayed leaf senescence, indicating that SENRK1 negatively regulates age-dependent leaf senescence in Arabidopsis. Furthermore, the senescence-promoting transcription factor WRKY53 repressed the expression of SENRK1. While the wrky53 mutant showed a delayed senescence phenotype as previously reported, the wrky53 senrk1-1 double mutant exhibited precocious leaf senescence, suggesting that SENRK1 functions downstream of WRKY53 in regulating age-dependent leaf senescence in Arabidopsis.

Changes in volatile profile and related gene expression during senescence of tobacco leaves.

BACKGROUND: Volatile organic compounds are critical for food flavor and play important roles in plant-plant interactions and plants' communications with the environment. Tobacco is well-studied for secondary metabolism and most of the typical flavor substances in tobacco leaves are generated at the mature stage of leaf development. However, the changes in volatiles during leaf senescence are rarely studied. RESULTS: The volatile composition of tobacco leaves at different stages of senescence was characterized for the first time. Comparative volatile profiling of tobacco leaves at different stages was performed using solid-phase microextraction coupled with gas chromatography/mass spectrometry. In total, 45 volatile compounds were identified and quantified, including terpenoids, green leaf volatiles (GLVs), phenylpropanoids, Maillard reaction products, esters, and alkanes. Most of the volatile compounds showed differential accumulation during leaf senescence. Some terpenoids, including neophytadiene, ß-springene, and 6-methyl-5-hepten-2-one, increased significantly with the progress of leaf senescence. Hexanal and phenylacetaldehyde also showed increased accumulation in leaves during senescence. The results from gene expression profiling indicated that genes involved in metabolism of terpenoids, phenylpropanoids, and GLVs were differentially expressed during leaf yellowing. CONCLUSION: Dynamic changes in volatile compounds during tobacco leaf senescence are observed and the integration of gene-metabolites datasets offers important readouts for the genetic control of volatile production during the process of leaf senescence. © 2023 Society of Chemical Industry.

Corrigendum: Small secreted peptides encoded on the wheat (Triticum aestivum L.) genome and their potential roles in stress responses.

[This corrects the article DOI: 10.3389/fpls.2022.1000297.].

Small secreted peptides encoded on the wheat (triticum aestivum L.) genome and their potential roles in stress responses.

Small secreted peptides (SSPs) are important signals for cell-to-cell communication in plant, involved in a variety of growth and developmental processes, as well as responses to stresses. While a large number of SSPs have been identified and characterized in various plant species, little is known about SSPs in wheat, one of the most important cereal crops. In this study, 4,981 putative SSPs were identified on the wheat genome, among which 1,790 TaSSPs were grouped into 38 known SSP families. The result also suggested that a large number of the putaitive wheat SSPs, Cys-rich peptides in particular, remained to be characterized. Several TaSSP genes were found to encode multiple SSP domains, including CLE, HEVEIN and HAIRPININ domains, and two potentially novel TaSSP family DYY and CRP8CI were identified manually among unpredicted TaSSPs. Analysis on the transcriptomic data showed that a great proportion of TaSSPs were expressed in response to abiotic stresses. Exogenous application of the TaCEPID peptide encoded by TraesCS1D02G130700 enhanced the tolerance of wheat plants to drought and salinity, suggesting porential roles of SSPs in regulating stress responses in wheat.

Characterization of NAC transcription factor NtNAC028 as a regulator of leaf senescence and stress responses.

NAC proteins constitute one of the largest transcription factor families and are involved in regulation of plant development and stress responses. Our previous transcriptome analyses of tobacco revealed a significant increase in the expression of NtNAC028 during leaf yellowing. In this study, we found that NtNAC028 was rapidly upregulated in response to high salinity, dehydration, and abscisic acid (ABA) stresses, suggesting a vital role of this gene in abiotic stress response. NtNAC028 loss-of-function tobacco plants generated via CRISPR-Cas9 showed delayed leaf senescence and increased tolerance to drought and salt stresses. Meanwhile NtNAC028 overexpression led to precocious leaf senescence and hypersensitivity to abiotic stresses in Arabidopsis, indicating that NtNAC028 functions as a positive regulator of natural leaf senescence and a negative regulator of stress tolerance. Furthermore, NtNAC028-overexpressing Arabidopsis plants showed lower antioxidant enzyme activities, higher reactive oxygen species (ROS), and H2O2 accumulation under high salinity, resulted in more severe oxidative damage after salt stress treatments. On the other hand, NtNAC028 mutation in tobacco resulted in upregulated expression of ROS-scavenging and abiotic stress-related genes, higher antioxidant enzyme activities, and enhanced tolerance against abiotic stresses, suggesting that NtNAC028 might act as a vital regulator for plant stress response likely by mediating ROS scavenging ability. Collectively, our results indicated that the NtNAC028 plays a key regulatory role in leaf senescence and response to multiple abiotic stresses.

Systematic Analysis of Tobacco CrRLK1L Family Genes and Functional Identification of NtCrRLK1L47 in Environmental Stresses.

The Catharanthus roseus RLK1-like (CrRLK1L) family is involved in the regulation of plant reproduction, growth and development, cell wall integrity sensing, as well as responses to both biotic and abiotic stress conditions. Extraordinary progress has been made in elucidating the CrRLK1L family receptor kinases-mediated signaling pathway, while limited research addressed the functions of CrRLK1L proteins in tobacco. In this study, we identified and analyzed 48 NtCrRLK1L members from the tobacco genome. The newly identified NtCrRLK1L members were divided into seven groups together with the Arabidopsis CrRLK1L members. The syntenic analysis revealed that four pairs of NtCrRLK1L genes were predicted to have arisen from segmental duplication events. Expression profiling showed that the NtCrRLK1L genes were expressed in various tissues, and most NtCrRLK1L genes were induced by salt and drought stress conditions. Notably, NtCrRLK1L47 was upregulated under drought and salinity stresses, and the NtCrRLK1L47-GFP fusion protein was located in the cell membrane. Furthermore, overexpression of the NtCrRLK1L47 gene enhanced the salt tolerance in tobacco seedlings.

The INFLORESCENCE DEFICIENT IN ABSCISSION-LIKE6 Peptide Functions as a Positive Modulator of Leaf Senescence in Arabidopsis thaliana.

Leaf senescence is a highly coordinated process and has a significant impact on agriculture. Plant peptides are known to act as important cell-to-cell communication signals that are involved in multiple biological processes such as development and stress responses. However, very limited number of peptides has been reported to be associated with leaf senescence. Here, we report the characterization of the INFLORESCENCE DEFICIENT IN ABSCISSION-LIKE6 (IDL6) peptide as a regulator of leaf senescence. The expression of IDL6 was up-regulated in senescing leaves. Exogenous application of synthetic IDL6 peptides accelerated the process of leaf senescence. The idl6 mutant plants showed delayed natural leaf senescence as well as senescence included by darkness, indicating a regulatory role of IDL6 peptides in leaf senescence. The role of IDL6 as a positive regulator of leaf senescence was further supported by the results of overexpression analysis and complementation test. Transcriptome analysis revealed differential expression of phytohormone-responsive genes in idl6 mutant plants. Further analysis indicated that altered expression of IDL6 led to changes in leaf senescence phenotypes induced by ABA and ethylene treatments. The results from this study suggest that the IDL6 peptide positively regulates leaf senescence in Arabidopsis thaliana.

Establishment and Comparative Analysis of Enzyme-Linked Immunoassay and Time-Resolved Fluoroimmunoassay for the Determination of Trace Quinclorac in Environment.

As a common herbicide in farmland, there has been wide concern over quinclorac residue because of its potential risks to the environment and human health. For the detection and monitoring of quinclorac residue in the environment, enzyme-linked immunoassay (ELISA) and time-resolved fluoroimmunoassay (TRFIA) were established. The half-maximal inhibition concentrations (IC50) of ELISA and TRFIA were 0.169 mg/L and 0.087 mg/L with a linear range (IC20−IC80) of 0.020−1.389 mg/L and 0.004−1.861 mg/L, respectively. Compared with ELISA, the limit of detection (LOD, IC20) and IC50 of TRFIA improved approximately 5-fold and 2-fold. The cross-reaction rates for the quinclorac analogs were less than 2%. The average recoveries of quinclorac in river water, paddy water, paddy soil, and brown rice samples were 77.3−106.1%, with RSDs of 1.7−12.5%. More importantly, the results of the two methods were consistent with that of the referenced method of UPLC-MS/MS (R2 > 0.98). ELISA and TRFIA both showed good detection performance and could meet the requirements of the quantitative determination of quinclorac. Therefore, the proposed ELISA and TRFIA could be applied to the rapid and sensitive detection and monitoring of quinclorac residue in the environment.

NtNAC053, A Novel NAC Transcription Factor, Confers Drought and Salt Tolerances in Tobacco.

The NAC (NAM, ATAF1/2, and CUC2) family acts as one of the largest families of the transcription factor in the plant kingdom and was revealed to function as the important regulators in various environmental stresses. However, a few studies were reported about the biofunctions of the NAC transcription factor in tobacco. In the current study, we characterized a novel NAC transcription factor encoding the gene NtNAC053 in tobacco, which was significantly up-regulated when exposed to salt and drought treatments. The results of cis-acting elements analysis suggested that the promoter region of NtNAC053 gene possesses a number of stress-responsive elements, and this gene could be induced by exogenous abscisic acid (ABA) treatment. Moreover, the NtNAC053-GFP fusion protein was localized in the cell nucleus and possessed a transactivation domain in its C-terminal, implying that NtNAC053 may undertake as a transcriptional activator in tobacco. Notably, the overexpression of NtNAC053 in tobacco resulted in hypersensitivity to ABA treatment. Furthermore, these overexpression lines showed significantly enhanced tolerances to drought and salt stresses. Under salt and drought stresses, these overexpression lines possessed higher superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities. Interestingly, the expressions of putative stress-related genes, including NtCOR15A, NtRAB18, NtDREB1A, NtERF5, NtKAT2, and NtERD11, were up-regulated in these overexpression lines when subjected to salt and drought stresses. The clues provided in our study suggested that the NtNAC053 gene encodes a novel NAC transcription factor and could confer the drought and salt stress tolerances by inspiring the downstream stress-responsive genes and antioxidant system in tobacco.

CLE14 functions as a "brake signal" to suppress age-dependent and stress-induced leaf senescence by promoting JUB1-mediated ROS scavenging in Arabidopsis.

Leaf senescence is an important developmental process in the plant life cycle and has a significant impact on agriculture. When facing harsh environmental conditions, monocarpic plants often initiate early leaf senescence as an adaptive mechanism to ensure a complete life cycle. Upon initiation, the senescence process is fine-tuned through the coordination of both positive and negative regulators. Here, we report that the small secreted peptide CLAVATA3/ESR-RELATED 14 (CLE14) functions in the suppression of leaf senescence by regulating ROS homeostasis in Arabidopsis. Expression of the CLE14-encoding gene in leaves was significantly induced by age, high salinity, abscisic acid (ABA), salicylic acid, and jasmonic acid. CLE14 knockout plants displayed accelerated progression of both natural and salinity-induced leaf senescence, whereas increased CLE14 expression or treatments with synthetic CLE14 peptides delayed senescence. CLE14 peptide treatments also delayed ABA-induced senescence in detached leaves. Further analysis showed that overexpression of CLE14 led to reduced ROS levels in leaves, where higher expression of ROS scavenging genes was detected. Moreover, CLE14 signaling resulted in transcriptional activation of JUB1, a NAC family transcription factor previously identified as a negative regulator of senescence. Notably, the delay of leaf senescence, reduction in H2O2 level, and activation of ROS scavenging genes by CLE14 peptides were dependent on JUB1. Collectively, these results suggest that the small peptide CLE14 serves as a novel "brake signal" to regulate age-dependent and stress-induced leaf senescence through JUB1-mediated ROS scavenging.

Identification and Analysis of bZIP Family Genes in Potato and Their Potential Roles in Stress Responses.

The bZIP proteins comprise one of the largest transcription factor families and play important roles in plant growth and development, senescence, metabolic reactions, and stress responses. In this study, 49 bZIP transcription factor-encoding genes (StbZIP genes) on the potato genome were identified and analyzed. The 49 StbZIP genes, which are located on 12 chromosomes of the potato genome, were divided into 11 subgroups together with their Arabidopsis homologs based on the results of phylogenetic analysis. Gene structure and protein motif analysis revealed that members from the same subgroup often possessed similar exon/intron structures and motif organizations, further supporting the results of the phylogenetic analysis. Syntenic analysis indicated the existence of gene duplication events, which might play an important role in the expansion of the bZIP gene family in potato. Expressions of the StbZIP genes were analyzed in a variety of tissues via RNA-Seq data, suggesting functional diversity. Several StbZIP genes were found to be induced by different stress conditions. For example, the expression of StbZIP25, the close homolog of AtbZIP36/ABF2, was significantly upregulated by salt stress treatments. The StbZIP25 protein was found to be located in the nucleus and function as a transcriptional activator. Overexpression of StbZIP25 enhanced salt tolerance in Arabidopsis. The results from this study imply potential roles of the bZIP family genes in the stress response of potato.

AtWAKL10, a Cell Wall Associated Receptor-Like Kinase, Negatively Regulates Leaf Senescence in Arabidopsis thaliana.

Receptor-like kinases (RLKs) constitute a large group of cell surface receptors that play crucial roles in multiple biological processes. However, the function of most RLKs in plants has not been extensively explored, and much less for the class of cell wall associated kinases (WAKs) and WAK-like kinases (WAKLs). In this study, analyses of developmental expression patterns uncovered a putative role of AtWAKL10 in modulating leaf senescence, which was further investigated at physiological and molecular levels. The expression level of AtWAKL10 increased with the developmental progression and was rapidly upregulated in senescing leaf tissues. The promoter of AtWAKL10 contains various defense and hormone responsive elements, and its expression could be significantly induced by exogenous ABA, JA and SA. Moreover, the loss-of-function atwakl10 mutant showed earlier senescence along the course of natural development and accelerated leaf senescence under darkness and hormonal stresses, while plants overexpressing AtWAKL10 showed an opposite trend. Additionally, some defense and senescence related WRKY transcription factors could bind to the promoter of AtWAKL10. In addition, deletion and overexpression of AtWAKL10 caused several specific transcriptional alterations, including genes involved in cell extension, cell wall modification, defense response and senescence related WRKYs, which may be implicated in regulatory mechanisms adopted by AtWAKL10 in controlling leaf senescence. Taken together, these results revealed that AtWAKL10 negatively regulated leaf senescence.

Bioinformatics and Expression Analysis of IDA-Like Genes Reveal Their Potential Functions in Flower Abscission and Stress Response in Tobacco (Nicotiana tabacum L.).

The inflorescence deficient in abscission-like (IDL) genes have been shown to play critical roles in floral organ abscission, lateral root formation and various stress responses in Arabidopsis. The IDL gene family has been characterized in a number of plant species, while limited information is available about IDL genes of tobacco. In the current study, 15 NtIDL members were identified in the tobacco genome, and were classified into six groups together with IDL members from other species. Evolution analysis suggested that the NtIDL members form group VI might have originated from duplication events. Notably, NtIDL06 shared high similarities with AtIDA in the EPIP sequence, and its encoding gene was highly expressed in the abscission zone of flowers at late developmental stages, implying that NtIDL06 might regulate tobacco flower abscission. In addition, the results from cis-elements analysis of promoters and expression after stress treatments suggested that NtIDL members might be involved in various stress responses of tobacco. The results from this study provide information for further functional analysis related to flower abscission and stress responses of NtIDL genes.

Potato NAC Transcription Factor StNAC053 Enhances Salt and Drought Tolerance in Transgenic Arabidopsis.

The NAC (NAM, ATAF1/2, and CUC2) transcription factors comprise one of the largest transcription factor families in plants and play important roles in stress responses. However, little is known about the functions of potato NAC family members. Here we report the cloning of a potato NAC transcription factor gene StNAC053, which was significantly upregulated after salt, drought, and abscisic acid treatments. Furthermore, the StNAC053-GFP fusion protein was found to be located in the nucleus and had a C-terminal transactivation domain, implying that StNAC053 may function as a transcriptional activator in potato. Notably, Arabidopsis plants overexpressing StNAC053 displayed lower seed germination rates compared to wild-type under exogenous ABA treatment. In addition, the StNAC053 overexpression Arabidopsis lines displayed significantly increased tolerance to salt and drought stress treatments. Moreover, the StNAC053-OE lines were found to have higher activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) under multiple stress treatments. Interestingly, the expression levels of several stress-related genes including COR15A,DREB1A, ERD11, RAB18, ERF5, and KAT2, were significantly upregulated in these StNAC053-overexpressing lines. Taken together, overexpression of the stress-inducible StNAC053 gene could enhance the tolerances to both salt and drought stress treatments in Arabidopsis, likely by upregulating stress-related genes.