Subcellular Localization Analysis Research Articles

Hydroxylase-oriented mining and functional characterization of camptothecin 10-hydroxylase from Camptotheca acuminata Decne

The regiospecific C-10 hydroxylation of camptothecin (CPT) is challenging from a chemical perspective. This step bridges the natural-sourced CPT to an important industrial material, 10-hydroxycamptothecin (10HCPT). 10HCPT is naturally generated in a medicinal plant, Camptotheca acuminata Decne. Functionally active hydroxylase responsible for regiospecific C-10 hydroxylation of CPT is undoubtedly existed in this plant. Therefore, the multi-omics database of C. acuminata was utilized to perform hydroxylase-oriented mining and screening. Three CYP81 genes were identified and two of them, including CYP81BQ18 and CYP81B256 catalyzed hydroxylation of CPT at the C-10 position. CYP81B256 and CYP81BN24 displayed quercetin C-5’ hydroxylase activity. CYP81B256 exhibited better catalytic performance for CPT than CYP81BQ18 and CPT10H in vitro. Furthermore, CYP81BQ18 and CYP81B256 displayed CPT 10-hydroxylase activity in tobacco. Functional verification in planta suggested that the newly observed CYP81BQ18 and CYP81B256 were involved in 10HCPT biosynthesis in C. acuminata. Subcellular localization analysis revealed that these CYP81 genes were located on the endoplasmic reticulum. Evolutionary analysis indicated that CYP81B256 and CYP81BN24 probably evolved from a same ancestral gene via gene duplication, while CYP81BQ18 evolved independently and acted as a specie-specific hydroxylase. CYP81B256 acted as an evolutionary intermediate gene bridging the alkaloid and flavonoid metabolism in C. acuminata. This research provides valuable insights into the function and evolutionary origin of CYP81s in the regiospecific hydroxylation step for 10HCPT biosynthesis.

Genome-Wide Identification of GRAS Gene Family in Cunninghamia lanceolata and Expression Pattern Analysis of ClDELLA Protein Under Abiotic Stresses

The Chinese fir (Cunninghamia lanceolata) is a significant species utilized in afforestation efforts in southern China. It is distinguished by its rapid growth and adaptability to diverse environmental conditions. The GRAS gene family comprises a group of plant-specific transcription factors that play a pivotal role in plant growth and development, response to adversity, and hormone regulatory networks. However, the exploration of the GRAS family in gymnosperm Chinese fir has not yet begun. In this study, a total of 43 GRAS genes were identified in the whole genome of Chinese fir, and a phylogenetic analysis classified them into nine distinct subfamilies. Gene structure analysis revealed that the majority of ClGRAS genes lacked introns. It is notable that among these proteins, both ClGAI and ClGRA possess distinctive DELLA structural domains. Cis-acting element analysis revealed that nearly all ClGRAS genes contained light-responsive elements, while hormone-responsive elements, environmental-responsive elements (low-temperature- or defense-responsive elements), and meristem-organization-related elements were also identified. Based on transcriptome data and RT-qPCR expression patterns, we analyzed the expression of ClGAI and ClRGA genes across different developmental stages, hormones, and three abiotic stresses. Subcellular localization analysis demonstrated that ClGAI and ClRGA were localized to the nucleus. Transcriptional activation assays showed that both genes have self-activating activity. In conclusion, the results of this study indicate that the ClGRAS gene family is involved in the response of Chinese fir to environmental stress. Further research on the ClDELLA genes provides valuable information for exploring the potential regulatory network of DELLA proteins in Chinese fir.

Global Profiling of Protein Phosphorylation, Acetylation, and β-Hydroxybutyrylation in Nannochloropsis oceanica.

Protein post-translational modifications (PTMs) regulate protein functions but remain poorly characterized in Nannochloropsis. This study examined three PTMs: lysine acetylation (Kac), lysine β-hydroxybutyrylation (Kbhb), and phosphorylation. Using LC-MS/MS, we identified 4571 Kac sites, 7812 Kbhb sites, and 6237 phosphorylation sites across 2455, 3109, and 2786 proteins, respectively. Subcellular localization analysis revealed significant overlaps between Kac and Kbhb proteins, primarily in the chloroplast, cytosol, and nucleus, while phosphorylated proteins were predominantly located in the nucleus and chloroplast. Motif analysis highlighted specific amino acid enrichments around modification sites, with several motifs conserved. Additionally, 529 proteins harbored all three PTMs, underscoring the potential regulatory interplay. Kac, Kbhb, and phosphorylated proteins were particularly abundant in glycolysis, the TCA cycle, carbon fixation, and lipid metabolism pathways, influencing energy production and lipid accumulation. Based on previous transcriptome data under nutrient-limited conditions, these frequently modified key enzymes appear to be vital components in the response to abiotic stress. The presence of histone modifications related to Kac and Kbhb might also point to the epigenetic regulation in gene expression and stress adaptation. This comprehensive PTM landscape in N. oceanica provides a foundation of valuable insights into future metabolic engineering and biotechnological applications.

Mutations in CsWOX4 Regulate Leaf Vein Pattern and Leaf Size in Cucumber (Cucumis sativus L.)

The leaf morphology is an important agronomic trait in crop production. Our study identified a maple leaf type (mlt) cucumber mutant and located the regulatory gene for leaf shape changes through BSA results. Hybrid F1 and F2 populations were generated by F1 self-crossing, and the candidate mlt genes were identified within the 2.8 Mb region of chromosome 2 using map cloning. Through the sequencing and expression analysis of genes within the bulk segregant analysis (BSA) region, we identified the target gene for leaf shape regulation as CsWOX4 (CsaV3_2G026510). The change from base C to T in the original sequence led to frameshift mutations and the premature termination of translation, resulting in shortened encoded proteins and conserved WUSCHEL (WUS) box sequence loss. The specific expression analysis of the CsWOX4/Cswox4 genes in the roots, stems, leaves and other tissue types of wild-type (WT) and mutant plants revealed that CsWOX4 was higher in the root, but Cswox4 (mutant gene) was significantly higher in the leaf. Subcellular localization analysis revealed that CsWOX4 was localized in the nucleus. RNA-seq analysis revealed that the differentially expressed genes were mainly enriched in the mitochondrial cell cycle phase transition, nucleosome and microtubule binding pathways. Simultaneously, the quantitative analysis of the expression trends of 25 typical genes regulating the leaf types revealed the significant upregulation of CsPIN3. In our study, we found that the conserved domain of CsWOX4 was missing in the mutant, and the transcriptome data revealed that the expression of some genes, such as CsPIN3, changed simultaneously, thereby jointly regulating changes in the cucumber leaf type.

Integrative Proteome and Metabolomics Analyses of Cryptococcus neoformans Responses to Melanin Substrates Niger seed and L-DOPA.

Melanin, as a pivotal antioxidant pigment, plays a critical role in the pathogenicity of Cryptococcus neoformans. However, the underlying signaling pathways responsible for melanin biosynthesis in C. neoformans are not yet fully elucidated. In this study, proteome and metabolomics analyses were conducted to investigate the response of C. neoformans to melanin substrate Niger seed or L-DOPA. The proteome analysis identified significant differential expression of proteins in cells treated with Niger seed compared to L-DOPA, with distinct functional enrichment patterns observed. Subcellular localization analysis showed unique protein distribution in cells treated with each substrate. Metabolomics analysis revealed distinct metabolic profiles in response to Niger seed or L-DOPA, with notable differences in metabolite regulation between the two treatments. KEGG classification highlighted specific metabolic pathways affected by each substrate. Overall, this study provides valuable insights into the complex regulatory mechanisms underlying C. neoformans response to melanin substrates.

Genome-Wide Analysis of Heat Shock Protein Family and Identification of Their Functions in Rice Quality and Yield

Heat shock proteins (Hsps), acting as molecular chaperones, play a pivotal role in plant responses to environmental stress. In this study, we found a total of 192 genes encoding Hsps, which are distributed across all 12 chromosomes, with higher concentrations on chromosomes 1, 2, 3, and 5. These Hsps can be divided into six subfamilies (sHsp, Hsp40, Hsp60, Hsp70, Hsp90, and Hsp100) based on molecular weight and homology. Expression pattern data indicated that these Hsp genes can be categorized into three groups: generally high expression in almost all tissues, high tissue-specific expression, and low expression in all tissues. Further analysis of 15 representative genes found that the expression of 14 Hsp genes was upregulated by high temperatures. Subcellular localization analysis revealed seven proteins localized to the endoplasmic reticulum, while others localized to the mitochondria, chloroplasts, and nucleus. We successfully obtained the knockout mutants of above 15 Hsps by the CRISPR/Cas9 gene editing system. Under natural high-temperature conditions, the mutants of eight Hsps showed reduced yield mainly due to the seed setting rate or grain weight. Moreover, the rice quality of most of these mutants also changed, including increased grain chalkiness, decreased amylose content, and elevated total protein content, and the expressions of starch metabolism-related genes in the endosperm of these mutants were disturbed compared to the wild type under natural high-temperature conditions. In conclusion, our study provided new insights into the HSP gene family and found that it plays an important role in the formation of rice quality and yield.

Eggplant NAC domain transcription factor SmNST1 as an activator promotes secondary cell wall thickening.

NAC-domain transcription factors (TFs) are plant-specific transcriptional regulators playing crucial roles in plant secondary cell wall (SCW) biosynthesis. SCW is important for plant growth and development, maintaining plant morphology, providing rigid support, ensuring material transportation and participating in plant stress responses as a protective barrier. However, the molecular mechanisms underlying SCW in eggplant have not been thoroughly explored. In this study, the NAC domain TFs SmNST1 and SmNST2 were cloned from the eggplant line 'Sanyue qie'. SmNST1 and SmNST2 expression levels were the highest in the roots and stems. Subcellular localization analysis showed that they were localized in the cell membrane and nucleus. Their overexpression in transgenic tobacco showed that SmNST1 promotes SCW thickening. The expression of a set of SCW biosynthetic genes for cellulose, xylan and lignin, which regulate SCW formation, was increased in transgenic tobacco. Bimolecular fluorescence and luciferase complementation assays showed that SmNST1 interacted with SmNST2 in vivo. Yeast one-hybrid, electrophoretic mobility shift assay (EMSA) and Dual-luciferase reporter assays showed that SmMYB26 directly bound to the SmNST1 promoter and acted as an activator. SmNST1 and SmNST2 interact with the SmMYB108 promoter and repress SmMYB108 expression. Altogether, we showed that SmNST1 positively regulates SCW formation, improving our understanding of SCW biosynthesis transcriptional regulation.

Analysis of bZIP transcription factors in Rhododendron simsii and functional study of RsbZIP6 in regulating anthocyanin biosynthesis

The basic leucine zipper (bZIP) transcription factors play a critical role in various plant biological processes, including anthocyanin biosynthesis. This study focuses on Rhododendron simsii, a notable ornamental species with insufficiently explored bZIP transcription factors. We identified 66 bZIP transcription factors in the R. simsii genome and conducted comprehensive bioinformatics analyses to determine their gene localization, phylogenetic relationships, grouping, gene/protein structure, duplication events, synteny, and expression profiles. Our analysis identified RsbZIP6, a homolog of HY5 known to influence anthocyanin biosynthesis in many plants, as a potential regulator of this pathway. We cloned the complete coding sequence of RsbZIP6, which encodes a 170-amino acid protein spanning 510 bp. Subcellular localization analysis verified the nuclear presence of the RsbZIP6 protein. RT-qPCR analysis revealed the highest expression of RsbZIP6 in petals, which correlated with anthocyanin accumulation. Transgenic experiments indicated that overexpressing RsbZIP6 in Arabidopsis enhanced anthocyanin accumulation by upregulating genes involved in anthocyanin biosynthesis (4CL, CHS, CHI, DFR, F3H, F3’H, ANS and UF3GT). Our findings enhance understanding of the bZIP transcription factor family in R. simsii and underscore the vital role of RsbZIP6 in anthocyanin biosynthesis, providing insights for future genetic enhancement strategies.

Betula platyphylla glucosyltransferase BpGT14;6 is essential for cell wall development and stress response

Glycosyltransferases (GTs) constitute a diverse family of synthetic polysaccharides with important roles in plant growth and development. This study characterized the GT14 family gene BpGT14;6 of birch (Betula platyphylla Suk.). BpGT14;6 was highly expressed in the xylem and stem of birch plants. Subcellular localization analysis suggested that BpGT14;6 was located in the Golgi apparatus. RNA interference (RNAi) silencing of BpGT14;6 revealed lower lignin, hemicellulose, and pectin contents compared to wild type (WT) plants. Following treatment with abscisic acid (ABA), compared to WT plants, RNAi-BpGT14;6 plants were more sensitive to ABA, suffered more membrane lipid damage, and accumulated more reactive oxygen species. The inhibition of BpGT14;6 expression narrowed the birch xylem and thinned the cell wall, and increased the expression of multiple ABA pathway-related genes in birch under ABA treatment. Compared to WT plants, RNAi-BpGT14;6 plants showed increased tolerance to drought stress. Promoter analysis revealed that BpGT14;6 is involved in hormone regulation and adaptation to adversity. Using the 1 156 bp BpGT14;6 promoter as bait, two potential transcription factors, BpWRKY1 and BpARF2, were identified through Y1H screening that may regulate its expression. EMSA confirmed that BpWRKY1 and BpARF2 can directly bind to the W-BOX and AuxRE cis-acting elements on the BpGT14;6 promoter, respectively. The collective results suggest that BpGT14;6 affects birch xylem and cell wall development by affecting lignin, hemicellulose, and pectin synthesis, and participates in birch adversity adaptation.

Mutational analysis of Phanerochaete chrysosporium´s purine transporter.

We present here a mutational analysis of the purine transporter from Phanerochaete chrysosporium (PhZ), a member of the AzgA-like subfamily within the Nucleobase Ascorbate Transporters family. We identified key residues that determine its substrate specificity and transport efficiency. Thirteen PhZ mutants were generated and heterologously expressed in Aspergillus nidulans. The growth of mutant strains in the presence of purines and toxic analogues and the uptake rate of radiolabelled hypoxanthine were evaluated. Results revealed that ten mutants showed differences in transport compared to the wild-type PhZ: six mutants completely lost function, two exhibited decreased transport activity, and two showed increased hypoxanthine uptake. Subcellular localization and expression level analyses indicated that the differences in transport activity were not due to trafficking issues to the plasma membrane or protein stability. A three-dimensional model of PhZ, constructed with the artificial intelligence-based AlphaFold2 program, suggested that critical residues for transport are located in transmembrane segments and an internal helix. In the latter, the A418 residue was identified as playing a pivotal role in transport efficiency despite being far from the putative substrate binding site, as mutant A418V showed an increased initial uptake efficiency for the transporter´s physiological substrates. We also report that residue L124, which lies in the putative substrate binding site, plays a critical role in substrate transport, emerging as an additional determinant in the transport mechanism of this family of transporters. These findings underscore the importance of specific residues in AzgA-like transporters and enhance our understanding of the intricate mechanisms governing substrate specificity and transport efficiency within this family.

PePalA/B/C are required for virulence and patulin biosynthesis by regulating the PePacC-processing proteolytic activity in Penicillium expansum

Abstract Background The Pal/Rim signaling pathway is crucial for fungal responses to ambient pH conditions. It comprises the transcription factor PacC/Rim101 and six upstream Pal proteins. While the role of PacC has been extensively studied, there is limited information on the functions of the upstream Pal proteins. Results In the genome of Penicillium expansum, genes encoding the endosomal membrane complex (PalA/B/C) were identified. Among these, only the expression of PePalB, not PePalA or PePalC, is pH-dependent. Subcellular localization and functional analyses showed that PePalA/B/C are localized to the cytosol and peripheral punctate structures. Deletion of PePalA/B/C resulted in reduced growth and conidiation of P. expansum across various pH conditions. The virulence of the ΔPePalA/B/C mutant was significantly reduced in pear and apple fruits. Additionally, the mutants exhibited a loss of patulin production under both acidic and alkaline conditions and the down-regulation of genes in the patulin biosynthetic cluster. pH shift experiments further demonstrated that PePalA/B/C are essential for both the PePacC expression and the pH-dependent proteolytic activation of PePacC. Conclusions These findings highlight the significant roles of PePalA/B/C in regulating growth, conidiation, virulence, and patulin production in P. expansum, thereby enhancing our understanding of the regulatory mechanisms underlying the Pal/Rim signaling pathway.

ZmLSD1 Enhances Salt Tolerance by Regulating the Expression of ZmWRKY29 in Maize.

Salt stress significantly impairs plant growth, presenting a challenge to agricultural productivity. Exploring the regulatory mechanisms underlying salt stress responses is critically important. Here, we identified a significant role for the maize LESION-SIMULATING DISEASE transcription factor, ZmLSD1, in enhancing salt stress response. Subcellular localization analysis indicated that ZmLSD1-GFP was localized in the nucleus in the maize protoplast. Overexpressing ZmLSD1 in maize obviously enhanced the tolerance of plants to salt stress. Physiological analysis indicated that overexpressed ZmLSD1 in maize could mitigate the accumulation of H2O2 and MDA content exposed to salt stress. RNA-seq and qPCR-PCR analyses showed that ZmLSD1 positively regulated ZmWRKY29 expression. ChIP-qPCR and EMSA experiments demonstrated that ZmLSD1 could directly bind to the promoter of ZmWRKY29 through the GTAC motif both in vitro and in vivo. Overall, our findings suggest that ZmLSD1 plays a positive role in enhancing the tolerance of maize to salt by affecting ZmWRKY29 expression.

Genome-wide analysis of Triticum aestivum bromodomain gene family and expression analysis under salt stress.

This study identified 82 wheat BRD genes, revealing both conserved evolutionary and functional characteristics across plant species and novel features specific to wheat. GTE8-12 cluster TaBRDs were found as positive response to salt stress. Bromodomain-containing proteins (BRDs) are crucial in histone acetylation "reading" and chromatin remodeling in eukaryotes. Despite some of their members showing importance in various biological processes in plants, our understanding of the BRD family in wheat (Triticum aestivum) remains limited. This study comprehensively analyzes the T. aestivum BRD (TaBRD) family. We identified 82 TaBRD genes in wheat genome encoding hydrophobic proteins with a conserved pocket structure. Phylogenetic analysis classified these genes into 16 distinct clusters, with conserved protein motifs and gene structures within clusters but diverse patterns across clusters. Gene duplication analysis revealed that whole-genome or segmental duplication events were the primary expansion mechanism for the TaBRD family, with purifying selection acting on these genes. Subcellular localization and Gene Ontology (GO) analyses indicated that TaBRD proteins are predominantly nuclear-localized and involved in transcription regulation and RNA metabolism. Promoter analysis and interaction network prediction suggested diverse regulatory mechanisms for TaBRDs. Notably, TaBRDs from the GTE8-12 cluster were enriched with cis-elements responsive to abscisic acid (ABA), methyl jasmonate (MeJA), and light, implying their involvement in physiological functions and abiotic stress responses. Expression analysis confirmed tissue-specific patterns and responsiveness to salinity stress. This comprehensive study enhances our understanding of the BRD family in higher plants and provides a foundation for developing salt-tolerant wheat varieties.

The Sorting and Transport of the Cargo Protein CcSnc1 by the Retromer Complex Regulate the Growth, Development, and Pathogenicity of Corynespora cassiicola.

In eukaryotes, the retromer complex is critical for the transport of cargo proteins from endosomes to the trans-Golgi network (TGN). Despite its importance, there is a lack of research on the retromer-mediated transport of cargo proteins regulating the growth, development, and pathogenicity of filamentous fungi. In the present study, transcriptome analysis showed that the expression levels of the retromer complex (CcVPS35, CcVPS29 and CcVPS26) were significantly elevated during the early stages of Corynespora cassiicola invasion. Gene knockout and complementation analyses further highlighted the critical role of the retromer complex in C. cassiicola infection. Subcellular localization analysis showed that the retromer complex was mainly localized to the vacuolar membrane and partially to endosomes and the TGN. Further research found that the retromer core subunit CcVps35 can interact with the cargo protein CcSnc1. Subcellular localization showed that CcSnc1 is mainly located at the hyphal tip and partially in endosomes and the Golgi apparatus. Deletion of CcVPS35 resulted in the missorting of CcSnc1 into the vacuolar degradation pathway, indicating that the retromer can sort CcSnc1 from endosomes and transport it to the TGN. Additionally, gene knockout and complementation analyses demonstrated that CcSnc1 is critical for the growth, development, and pathogenicity of C. cassiicola. In summary, the vesicular transport pathway involving the retromer complex regulates the sorting and transport of the cargo protein CcSnc1, which is important for the growth, development and pathogenicity of C. cassiicola.

R2R3-MYB transcription factor GmMYB68 is involved in the accumulation of soybean isoflavones

We aimed to investigate the regulatory function of the soybean transcription factor R2R3-MYB (GmMYB68) in isoflavone biosynthesis. Through comprehensive subcellular and chromosomal localization analyses, we found that GmMYB68 was predominantly localized to the nucleus and mapped to chromosome Gm04. Notably, SSR markers near this gene significantly correlated with seed isoflavone content. GmMYB68 overexpression markedly increased isoflavone contents, confirming its positive role in regulating isoflavone synthesis. GmMYB68 also played a crucial role in the response of soybean to abiotic stress. Using RNA-seq and yeast one-hybrid techniques, we discovered an intricate interaction between GmMYB68 and key isoflavone biosynthesis genes GmCHS7 and GmCHS8. These findings provide novel insights into the mechanisms underlying isoflavone biosynthesis. Furthermore, using yeast two-hybrid experiments, we identified proteins interacting with GmMYB68, suggesting roles in the synthesis of physiologically active compounds and abiotic stress response. We not only elucidated the regulatory mechanisms of GmMYB68 in isoflavone biosynthesis and abiotic stress response but also constructed a molecular network encompassing GmMYB68, GmCHS7, and GmCHS8. This network provides a theoretical basis for a better understanding of and strategies for improving soybean isoflavone biosynthesis.

The essential function of cathepsin X of the orange-spotted grouper, Epinephelus coioides during SGIV infection

Cathepsin X, a class of cysteine proteases in the lysosome, involved in intracellular protein degradation processes. Numerous reports revealed that many kinds of cysteine proteases played a crucial role in pathogen invasion. To investigate the relationship between cathepsin X of teleost fish and virus infection, EcCX was cloned and characterized in the orange-spotted grouper, Epinephelus coioides. The open reading frame (ORF) of EcCX included 909 nucleotides and encoded a protein consisting of 302 amino acids, which shared 75% and 56% identity with zebrafish and humans, respectively. The protein EcCX mainly consisted of a signal peptide (1–19 aa), a pro-pre-peptide region (20–55 aa), and a mature cysteine protease region (56–302 aa). Subcellular localization analysis showed that EcCX was mainly distributed in the cytoplasm, but EcCX ectoped to the vicinity of apoptotic vesicles in FHM cells during SGIV infection. Following stimulation with SGIV or Poly (dA:dT), there was a notable rise in the expression levels of EcCX. EcCX overexpression facilitated virus infection, upregulated the production of inflammatory factors, and induced the activation of the NF-κB promoter. Furthermore, the overexpression of EcCX also accelerated the process of SGIV-induced apoptosis, potentially by enhancing the promoter activity of P53 and AP-1. Overall, our findings demonstrated a correlation between the function of EcCX and SGIV infection, providing a new understanding of the mechanisms involved in fish virus infection.

Members of WRKY Group III Transcription Factors Are Important in Mite Infestation in Strawberry (Fragaria × ananassa Duch.)

Strawberry is frequently attacked by mites, which directly affects the yield and quality of this fruit species. The WRKY Group III transcription factors (TFs) play an important role in plant tolerance to biotic sources of stress, such as pathogens and insect pests. In this study, six Group III WRKY TFs (FaWRKY25, FaWRKY31, FaWRKY32, FaWRKY43, FaWRKY44, and FaWRKY45) were identified in strawberry. A phylogenetic analysis showed that the six WRKY III TFs were divided into two clades and all had a conserved WRKYGQK domain and the C-X7-C-X23-H-T-C zinc finger motif. An interaction network analysis revealed that FaWRKY44 was co-expressing with FaWRKY25 and FaWRKY45. The expression patterns showed that the WRKY Group III genes responded to plant hormones and mite infestation in strawberry. To further verify the role of FaWRKY25 in plant resistance to mites, we cloned the FaWRKY25 gene and overexpressed it in transgenic plants. An in vivo subcellular localization analysis indicated that the FaWRKY25 protein was localized in the nucleus. Fewer mites were also detected on the wild-type plants than on FaWRKY25-overexpressing transgenic plants, suggesting that FaWRKY25 negatively regulates the resistance of strawberry to mites. The present study advances our understanding on a potential target that mites use to manipulate host plant defenses.

Knockdown of SlYTHDF2 Accelerates Dark-Induced Tomato Leaf Senescence by Affecting the ABA Pathway.

N6-methyladenosine (m6A) is a widespread post-transcriptional modification in eukaryotic mRNAs. Proteins with the YTH structural domain act as m6A-binding proteins by recognizing the m6A modification and regulating mRNA through this recognition. In this study, SlYTHDF2, a prototypical m6A -binding protein gene in the YTH family was expressed in various tissues, and subcellular localization analyses indicated that the SlYTHDF2 protein was localized in the nucleus and cytoplasm. SlYTHDF2 knockout lines were obtained using CRISPR/Cas9 technology and showed the senesced leaves prematurely increased endogenous ABA accumulation compared with the wild type. Moreover, we found that dark promoted leaf senescence in SlYTHDF2 knockout lines and exogenous ABA further accelerated leaf senescence under dark conditions. The qRT-PCR analysis revealed significant alterations in the expression of genes associated with the ABA pathway. Relative to the wild type, the CR-slythdf2 plants exhibited reduced levels of photosynthetic pigments, higher accumulation of reactive oxygen species, and increased damage to cell membranes. Additionally, we discovered that SlYTHDF2 interacts with the chloroplast-binding protein SlRBCS3 through yeast two-hybrid and BiFC experiments. Overall, our data suggest the important role of SlYTHDF2 in regulating tomato leaf senescence.

Isolation and functional determination of inward-rectifier K+ channel PbrAKT1 in pear

ABSTRACT Potassium (K+) is essential to plant growth and development. The absorption and transport of K+ by plant roots occurs mainly through K+ channels or transporters. In this study, an AKT1-type channel named PbrAKT1 was successfully cloned and characterised by a series of detailed procedures, including protein sequence alignment, expression and subcellular localisation analysis, as well as functional verification by heterologous expression function validation in yeast mutant R5421, Arabidopsis akt1 mutant, and Xenopus oocytes. The results showed that PbrAKT1 is preferentially expressed in pear roots and exhibits plasma membrane localisation, but its expression is unaffected by low K+ treatment. PbrAKT1 exhibits the function of an inwardly-rectifying K+ channel, and its activity is autonomously regulated when expressed in Xenopus oocytes. In addition, a series of heterologous expression experiments confirmed that PbrAKT1 plays an indispensable role in the processes of K+ transport, as well as alleviating the low-K+ -sensitive phenotype of yeast mutant R5421 and Arabidopsis akt1 mutant. In summary, PbrAKT1 plays an indispensable role in the processes of K+ transport and provide new information on the molecular mechanism of K+ absorption in pear roots.

Genome-wide identification of the endonuclease family genes implicates potential roles of TaENDO23 in drought-stressed response and grain development in wheat

BackgroundEndonucleases play a crucial role in plant growth and stress response by breaking down nuclear DNA. However, the specific members and biological functions of the endonuclease encoding genes in wheat remain to be determined.ResultsIn this study, we identified a total of 26 TaENDO family genes at the wheat genome-wide level. These genes were located on chromosomes 2 A, 2B, 2D, 3 A, 3B, and 3D and classified into four groups, each sharing similar gene structures and conserved motifs. Furthermore, we identified diverse stress-response and growth-related cis-elements in the promoter of TaENDO genes, which were broadly expressed in different organs, and several TaENDO genes were significantly induced under drought and salt stresses. We further examined the biological function of TaENDO23 gene since it was rapidly induced under drought stress and exhibited high expression in spikes and grains. Subcellular localization analysis revealed that TaENDO23 was localized in the cytoplasm of wheat protoplasts. qRT-PCR results indicated that the expression of TaENDO23 increased under PEG6000 and abscisic acid treatments, but decreased under NaCl treatment. TaENDO23 mainly expressed in leaves and spikes. A kompetitive allele-specific PCR (KASP) marker was developed to identify single nucleotide polymorphisms in TaENDO23 gene in 256 wheat accessions. The alleles with TaENDO23-HapI haplotypes had higher grain weight and size compared to TaENDO23-HapII. The geographical and annual frequency distributions of the two TaENDO23 haplotypes revealed that the elite haplotype TaENDO23-HapI was positively selected in the wheat breeding process.ConclusionWe systematically analyzed the evolutionary relationships, gene structure characteristics, and expression patterns of TaENDO genes in wheat. The expression of TaENDO23, in particular, was induced under drought stress, mainly expressed in the leaves and grains. The KASP marker of TaENDO23 gene successfully distinguished between the wheat accessions, revealing TaENDO23-HapI as the elite haplotype associated with improved grain weight and size. These findings provide insights into the evolution and characteristics of TaENDO genes at the genome-wide level in wheat, laying the foundation for further biological analysis of TaENDO23 gene, especially in response to drought stress and grain development.