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Plant Class III peroxidases have diverse roles in controlling root hair growth, anther development, and abiotic and biotic stress responses. However, their abiotic stress response mechanism in rice remains elusive. Here, we identified a peroxidase precursor gene, OsPRX83 , and investigated its role in enhancing osmotic stress tolerance in rice. We used OsPRX83 overexpression and CRISPR-Cas9-generated mutant lines to elucidate OsPRX83 's function and expression patterns under stress conditions. The expression of OsPRX83 was induced by H 2 O 2 , PEG, NaCl, and ABA treatments. Using qRT-PCR, RNA sequencing, and physiological assays, we demonstrated that overexpression of OsPRX83 enhanced the osmotic and oxidative stress tolerance as compared to the wild-type and mutant seedlings, as evident from the higher survival rates, enhanced peroxidase (POD) and ascorbate peroxidase (APX) activities, and increased ABA sensitivity compared with mutants and wild-type plants. Transcriptome analysis further supported the involvement of OsPRX83 in the ROS scavenging, by modulating the expression of OsDREB1B , OsDREB1E , OsDREB1F , OsDREB1G in response to osmotic treatment. In summary, our study suggests that OsPRX83 plays a pivotal role in enhancing stress tolerance in rice through ABA-dependent pathways and ROS scavenging. Therefore, this study elucidates the function of a novel abiotic stress response gene in rice, thereby may contribute to a new genetic engineering resource for engineering drought-resistant rice varieties.

The rice small protein OsS1Fa1, a homolog of spinach S1Fa, plays a significant role in drought tolerance, attributed to its transmembrane domain. In this study, we aim to further elucidate the potential roles of OsS1Fa1 in cold and biotic stresses as an inner nuclear membrane protein. Fluorescence analysis confirmed the localization of OsS1Fa1 to the inner nuclear membrane. Utilizing the bimolecular fluorescence complementation (BiFC) and bacterial infiltration assays with OsS1Fa1 and the inner nuclear membrane protein OsSUN1 (Rice Sad1 and UNC84 (SUN) domain containing 1 (SUN1)), we observed fluorescence detection within the inner nuclear membrane, indicating a direct interaction and colocalization between OsS1Fa1 and OsSUN1. Expression analysis revealed that overexpression of OsS1Fa1 induced the expression of various genes associated with cold and defense responses, including COLD-REGULATED 15A ( COR15A ), PATHOGENESIS-RELATED PROTEIN 1 ( PR1 ), and PLANT DEFENSIN 1.2 ( PDF1.2 ). Our findings collectively indicate that OsS1Fa1 plays crucial roles in both abiotic and biotic stress tolerance as an inner nuclear membrane protein.

A transgenic protein is frequently expressed as different homologous variants in genetically modified crops due to differential processing of targeting peptides or optimization of activity and specificity. The aim of this study was to develop a science-based approach for risk assessment of homologous protein variants using dicamba mono-oxygenase (DMO) as a case study. In this study, DMO expressed in the next-generation dicamba-tolerant maize, sugar beet and soybean crops exhibited up to 27 amino acid sequence differences in the N-terminus. Structure modeling using AlphaFold, ESMFold and OpenFold demonstrates that these small N-terminal extensions lack an ordered secondary structure and do not disrupt the DMO functional structure. Three DMO variants were demonstrated to have equivalent immunoreactivity and functional activity ranging from 214 to 331 nmol/min/mg. Repeated toxicity studies using each DMO variant found no test substance-related adverse effects. These results support that homologous protein variants, which have demonstrated physicochemical and functional equivalence, can leverage existing safety data from one variant without requiring additional de novo safety assessments.

The rising prevalence of viral-induced diseases, particularly those caused by certain strains, poses a substantial risk to the genetic diversity of Solanaceae crops and the overall safety of horticultural produce. According to the "gene-for-gene" hypothesis, resistance proteins are capable of selectively identifying nontoxic effectors produced by pathogens, as they are under purview of the host's immune defenses. The sensitivity and responsiveness of Solanaceae plants to viral attacks play a crucial role in shaping the outcomes of their interactions with viruses. Pathogenic organisms, devise an array of infection tactics aimed at circumventing or neutralizing the host's immune defenses to facilitate effective invasion. The invasion often accomplishes by suppressing or disrupting the host's defensive mechanisms or immune signals, which are integral to the infection strategies of such invading pathogens. This comprehensive review delves into the myriad approaches that pathogenic viruses employ to infiltrate and overcome the sophisticated immune system of tomatoes. Furthermore, the review explores the possibility of utilizing these viral strategies to bolster the resilience of horticultural crops, presenting a hopeful direction for forthcoming progress in plant health and agricultural stability.

One of the main signal transduction pathways that modulate plant growth and stress responses, including drought, is the action of phytohormones. Recent advances in omics approaches have facilitated the exploration of plant genomes. However, the molecular mechanisms underlying the response in the crown of barley, which plays an essential role in plant performance under stress conditions and regeneration after stress treatment, remain largely unclear. The objective of the present study was the elucidation of drought-induced molecular reactions in the crowns of different barley phytohormone mutants. We verified the hypothesis that defects of gibberellins, brassinosteroids, and strigolactones action affect the transcriptomic, proteomic, and hormonal response of barley crown to the transitory drought influencing plant development under stress. Moreover, we assumed that due to the strong connection between strigolactones and branching the hvdwarf14.d mutant, with dysfunctional receptor of strigolactones, manifests the most abundant alternations in crowns and phenotype under drought. Finally, we expected to identify components underlying the core response to drought which are independent of the genetic background. Large-scale analyses were conducted using gibberellins-biosynthesis, brassinosteroids-signaling, and strigolactones-signaling mutants, as well as reference genotypes. Detailed phenotypic evaluation was also conducted. The obtained results clearly demonstrated that hormonal disorders caused by mutations in the HvGA20ox2 , HvBRI1 , and HvD14 genes affected the multifaceted reaction of crowns to drought, although the expression of these genes was not induced by stress. The study further detected not only genes and proteins that were involved in the drought response and reacted specifically in mutants compared to the reaction of reference genotypes and vice versa , but also the candidates that may underlie the genotype-universal stress response. Furthermore, candidate genes involved in phytohormonal interactions during the drought response were identified. We also found that the interplay between hormones, especially gibberellins and auxins, as well as strigolactones and cytokinins may be associated with the regulation of branching in crowns exposed to drought. Overall, the present study provides novel insights into the molecular drought-induced responses that occur in barley crowns.

Lilium pumilum DC ( L. pumilum DC) plays an important role in the rational utilization of salinized soil. To explore the molecular mechanism of salt-tolerant L. pumilum , the LpMYB4 was cloned. LpMYB4 close relationship with Bambusa emeiensis and Zea mays MYB4 throughout the phylogenetic tree construction. LpMYB4 protein was found to be localized in the nucleus. Prokaryotic and eukaryotic bacterial solution resistance experiments proved that the exogenous introduction of LpMYB4 made the overexpression strains obtain better survival ability under saline-alkaline stress. Compared with wild-type plants, tobacco plants overexpressing LpMYB4 had better growth and lower leaf wilting and lodging, the content of chlorophyll was higher, the content of hydrogen peroxide and superoxide anion was lower, the activity of peroxidase and superoxide dismutase was higher and the relative conductivity was lower under saline-alkaline stress. The analysis of seed germination and seedling resistance of transgenic plants under salt stress showed that LpMYB4 transgenic seeds were more tolerant to salt stress during germination and growth. Yeast two-hybrid and two-luciferase complementation experiments showed that LpMYB4 interacted with yeast two-hybrid and LpGPX6. The analysis of the role of LpMYB4 in improving plant saline-alkali resistance is helpful to the transformation of plant germplasm resources and has great significance for agriculture and sustainable development.

Soybean is one of the important oil crops and a major source of protein and lipids. Drought can cause severe soybean yields. Dehydrin protein (DHN) is a subfamily of LEA proteins that play an important role in plant responses to abiotic stresses. In this study, the soybean GmDHN9 gene was cloned and induced under a variety of abiotic stresses. Results showed that the GmDHN9 gene response was more pronounced under drought induction. Subcellular localization results indicated that the protein was localized in the cytoplasm. The role of transgenic Arabidopsis plants in drought stress response was further studied. Under drought stress, the germination rate, root length, chlorophyll, proline, relative water content, and antioxidant enzyme content of transgenic Arabidopsis thaliana transgenic genes were higher than those of wild-type plants, and transgenic plants contained less O 2 - , H 2 O 2 and MDA contents. In short, the GmDHN9 gene can regulate the homeostasis of ROS and enhance the drought resistance of plants.

The CLAVATA pathway plays a key role in the regulation of multicellular shoot and root meristems in flowering plants. In Arabidopsis, CLAVATA 3-like signaling peptides (CLEs) act via receptor-like kinases CLAVATA 1 and CRINKLY 4 (CR4). In the moss Physcomitrium patens , PpCLAVATA and PpCR4 were previously studied independently and shown to play conserved roles in the regulation of cell proliferation and differentiation. The plant calpain DEFECTIVE KERNEL 1 (DEK1) has been identified as another key regulator of cell division and cell fate in vascular plants and bryophytes. The functional interaction between CLAVATA, CR4, and DEK1 remains unknown. Here, we show that P. patens crinkly4 and dek1 mutants respond differently to CLE peptide treatments suggesting their distinct roles in the CLAVATA pathway. Reduced CLAVATA-mediated suppression of leafy shoot growth in Δcr4 mutants indicates that PpCR4 is involved in CLV3p perception, most likely as a receptor. The CLV3p strongly suppressed leaf vein development in Δcr4 mutants, suggesting that other receptors are involved in these processes and indicating a potential role of PpCR4 in organ sensitization to CLEs.

Potassium (K + ) plays a role in enzyme activation, membrane transport, and osmotic regulation processes. An increase in potassium content can significantly improve the elasticity and combustibility of tobacco and reduce the content of harmful substances. Here, we report that the expression analysis of Nt GF14e , a 14-3-3 gene, increased markedly after low-potassium treatment (LK). Then, chlorophyll content, POD activity and potassium content, were significantly increased in overexpression of Nt GF14e transgenic tobacco lines compared with those in the wild type plants. The net K + efflux rates were severely lower in the transgenic plants than in the wild type under LK stress. Furthermore, transcriptome analysis identified 5708 upregulated genes and 2787 downregulated genes between Nt GF14e overexpressing transgenic tobacco plants. The expression levels of some potassium-related genes were increased, such as CBL-interacting protein kinase 2 ( CIPK2 ), Nt CIPK23 , Nt CIPK25 , H + -ATPase isoform 2 a ( AHA2a ), Nt AHA4a , Stelar K + outward rectifier 1( SKOR1 ), and high affinity K + transporter 5 ( HAK5 ). The result of yeast two-hybrid and luciferase complementation imaging experiments suggested Nt GF14e could interact with CIPK2. Overall, these findings indicate that NtGF14e plays a vital roles in improving tobacco LK tolerance and enhancing potassium nutrition signaling pathways in tobacco plants.

The purpose of this study was to analyze the role of transcription factor in Desmodium styracifolium , proving that the DsWRKY6 transcription factor was related to the plant phenotypes of Desmodium styracifolium - cv. 'GuangYaoDa1' and it could be used in molecular-assisted breeding. 'GuangYaoDa1' was used as the material and its DNA was the template to clone DsWRKY6, the transgenic Arabidopsis thaliana line was constructed by agrobacterium tumefaciens‑mediated transformation. Transgenic Arabidopsis thaliana was cultivated to study phenotype and physiological and biochemical indexes. Phenotypic observation showed that DsWRKY6 transgenic Arabidopsis thaliana had a faster growth rate while compared with the control group, they had longer lengths of main stem, lateral branches of cauline leaves, and root, but a lower number of cauline leaves and lateral branches of cauline leaves. And it also showed that their flowering and fruiting periods were advanced. The results of physiological and biochemical indexes showed that the relative expressions of DsWRKY6 increased and the abscisic acid content significantly increased in DsWRKY6 transgenic Arabidopsis thaliana compared with the control group. According to the above results, DsWRKY6 could regulate the advancing of flowering and fruiting periods caused by the improvement of abscisic acid content, and expression of the DsWRKY6 transcription factor might be the cause of the upright growth of 'GuangYaoDa1'.

Elevated temperatures critically impact crop growth, development, and yield, with photosynthesis being the most temperature-sensitive physiological process in plants. This study focused on assessing the photosynthetic response and genetic adaptation of two different heat-resistant jujube varieties 'Junzao' (J) and 'Fucuimi' (F), to high-temperature stress (42°C Day/30°C Night). Comparative analyses of leaf photosynthetic indices, microstructural changes, and transcriptome sequencing were conducted. Results indicated superior high-temperature adaptability in F, evidenced by alterations in leaf stomatal behavior - particularly in J, where defense cells exhibited significant water loss, shrinkage, and reduced stomatal opening, alongside a marked increase in stomatal density. Through transcriptome sequencing 13,884 differentially expressed genes (DEGs) were identified, significantly enriched in pathways related to plant-pathogen interactions, amino acid biosynthesis, starch and sucrose metabolism, and carbohydrate metabolism. Key findings include the identification of photosynthetic pathway related DEGs and HSFA1s as central regulators of thermal morphogenesis and heat stress response. Revealing their upregulation in F and downregulation in J. The results indicate that these genes play a crucial role in improving heat tolerance in F. This study unveils critical photosynthetic genes involved in heat stress, providing a theoretical foundation for comprehending the molecular mechanisms underlying jujube heat tolerance.

During plant growth and development, the YABBY gene plays a crucial role in the morphological structure, hormone signaling, stress resistance, crop breeding, and agricultural production of plant lateral organs, leaves, flowers, and fruits. Astragalus mongholicus is a perennial herbaceous plant in the legume family, widely used worldwide due to its high medicinal and edible value. However, there have been no reports of the YABBY gene family in A. mongholicus . This study used bioinformatics methods, combined with databases and analysis websites, to systematically analyze the AmYABBY gene family in the entire genome of A. mongholicus and verified its expression patterns in different tissues of A. mongholicus through transcriptome data and qRT-PCR experiments. A total of seven AmYABBY genes were identified, which can be divided into five subfamilies and distributed on three chromosomes. Two pairs of AmYABBY genes may be involved in fragment duplication on three chromosomes. All AmYABBY proteins have a zinc finger YABBY domain, and members of the same group have similar motif composition and intron - exon structure. In the promoter region of the genes, light-responsive and MeJa-response cis -elements are dominant. AmYABBY is highly expressed in stems and leaves, especially AmYABBY1 , AmYABBY2 , and AmYABBY3 , which play important roles in the growth and development of stems and leaves. The AmYABBY gene family regulates the growth and development of A. mongholicus . In summary, this study provides a theoretical basis for in-depth research on the function of the AmYABBY gene and new insights into the molecular response mechanism of the growth and development of the traditional Chinese medicine A. mongholicus .

The hairy root induction system was used to efficiently investigate gene expression and function in plant root. Cucumber is a significant vegetable crop worldwide, with shallow roots, few lateral roots, and weak root systems, resulting in low nutrient absorption and utilization efficiency. Identifying essential genes related to root development and nutrient absorption is an effective way to improve the growth and development of cucumbers. However, genetic mechanisms underlying cucumber root development have not been explored. Here, we report a novel, rapid, effective hairy root transformation system. Compared to the in vitro cotyledon transformation method, this method shortened the time needed to obtain transgenic roots by 13 days. Furthermore, we combined this root transformation method with CRISPR/Cas9 technology and validated our system by exploring the expression and function of CsMYB36 , a pivotal gene associated with root development and nutrient uptake. The hairy root transformation system established in this study provides a powerful method for rapidly identifying essential genes related to root development in cucumber and other horticultural crop species. This advancement holds promise for expediting research on root biology and molecular breeding strategies, contributing to the broader understanding and improvements crop growth and development.

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins in metazoans that mediate the regulation of various physiological responses to discrete ligands through heterotrimeric G protein subunits. The existence of GPCRs in plant is contentious, but their comparable crucial role in various signaling pathways necessitates the identification of novel remote GPCR-like proteins that essentially interact with the plant G protein α subunit and facilitate the transduction of various stimuli. In this study, we identified three putative GPCR-like proteins (OsGPCRLPs) (LOC_Os06g09930.1, LOC_Os04g36630.1, and LOC_Os01g54784.1) in the rice proteome using a stringent bioinformatics workflow. The identified OsGPCRLPs exhibited a canonical GPCR 'type I' 7TM topology, patterns, and biologically significant sites for membrane anchorage and desensitization. Cluster-based interactome mapping revealed that the identified proteins interact with the G protein α subunit which is a characteristic feature of GPCRs. Computational results showing the interaction of identified GPCR-like proteins with G protein α subunit and its further validation by the membrane yeast-two-hybrid assay strongly suggest the presence of GPCR-like 7TM proteins in the rice proteome. The absence of a regulator of G protein signaling (RGS) box in the C- terminal domain, and the presence of signature motifs of canonical GPCR in the identified OsGPCRLPs strongly suggest that the rice proteome contains GPCR-like proteins that might be involved in signal transduction.

Cotton is an important agricultural crop to many regions across the globe but is sensitive to low-temperature exposure. The activity of the enzyme SENSITIVE TO FREEZING 2 (SFR2) improves cold tolerance of plants and produces trigalactosylsyldiacylglycerol (TGDG), but its role in cold sensitive plants, such as cotton remains unknown. Recently, it was reported that cotton SFR2 produced very little TGDG under normal and cold conditions. Here, we investigate cotton SFR2 activation and TGDG production. Using multiple approaches in the native system and transformation into Arabidopsis thaliana , as well as heterologous yeast expression, we provide evidence that cotton SFR2 activates differently than previously found among other plant species. We conclude with the hypothesis that SFR2 in cotton is not activated in a similar manner regarding acidification or freezing like Arabidopsis and that other regions of SFR2 protein are critical for activation of the enzyme than previously reported.

Salvia miltiorrhiza , known as Danshen, is a traditional Chinese medicinal plant with significant cardiovascular benefits, attributed to its secondary metabolites, particularly tanshinones. Despite their medicinal value, tanshinones occur in low natural abundance, necessitating research to increase their content. This study explores the role of the ARF transcription factor (SmARF1) in tanshinone accumulation in Danshen. Overexpressing SmARF1 in hairy roots significantly increased tanshinone levels. EMSA and Dual-LUC assays revealed that SmMYB36, a transcription factor interacting with SmMAPK3, binds to and regulates the SmARF1 promoter. SmMYB36 alone inhibited the expression of SmARF1 gene, while its interaction with SmMAPK3 enhanced SmARF1 promoter activity. This MAPK3-MYB36-ARF1 module elucidates a complex regulatory mechanism for tanshinone biosynthesis, offering insights for targeted enhancement of tanshinone content through advanced biotechnological approaches.

Rapeseed ( Brassica napus L.) is an important oilseed crop widely cultivated worldwide, and drought is the main environmental factor limiting its yield enhancement and the expansion of planted areas. SIMILAR TO RCD ONE ( SRO ) is a plant-specific small gene family that plays a crucial role in plant growth, development, and responses to abiotic stresses such as drought. However, the functional role of SROs in rapeseed remains poorly understood. In this study, 19 BnaSROs were identified from the rapeseed genome, with 9, 10, 10, 18, and 20 members identified from the genomes of Brassica rapa , Brassica nigra , Brassica oleracea , Brassica juncea , and Brassica carinata , respectively. We then analyzed their sequence characteristics, phylogenetic relationships, gene structures, and conserved domains, and explored the collinearity relationships of the SRO members in Brassica napus and Brassica juncea . Next, we focused on the analysis of tissue expression and stress-responsive expression patterns of rapeseed SRO members and examined their expression profiles under ABA, MeJA and water-deficit drought treatments using qPCR. Transcriptome data analysis and qPCR detection indicated that BnaSROs exhibit multiple stress-responsive expression patterns. BnaSRO1 and BnaSRO11 , which are likely to function through interactions with NAC transcription factors, were screened as major drought-regulated members. Our results provide a solid foundation for functional analysis of the role of the SRO gene family in abiotic stress responses, especially drought stress responses, in rapeseed.

Rainfall, wind and touch, as mechanical forces, were mimicked on 6-week-old soil-grown tomato and potato under controlled conditions. Expression level changes of xyloglucan endotransglucosylase/hydrolase genes ( XTH s) of tomato ( Solanum lycopersicum L. cv. Micro Tom; SlXTH s) and potato ( Solanum tuberosum L. cv. Desirée; StXTH s) were analyzed in response to these mechanical forces. Transcription intensity of every SlXTH s of tomato was altered in response to rainfall, while the expression intensity of 72% and 64% of SlXTH s was modified by wind and touch, respectively. Ninety-one percent of StXTH s (32 out of 35) in potato responded to the rainfall, while 49% and 66% of the StXTH s were responsive to the wind and touch treatments, respectively. As previously demonstrated, all StXTH s were responsive to ultrasound treatment, and all were sensitive to one or more of the environmental mechanical factors examined in the current study. To our best knowledge, this is the first study to demonstrate that these ubiquitous mechanical environmental cues, such as rainfall, wind and touch, influence the transcription of most XTH s examined in both species.

Crabapple is a valuable tree species in gardens due to its captivating array of flower and leaf colors, rendering it a favored choice in landscaping. The economic and ornamental values of Malus crabapple are closely associated with the biosynthesis of anthocyanin, a pigment responsible for its vibrant hues. The intricate regulation of anthocyanin biosynthesis involves the concerted activity of various genes. However, the specific mechanism governing this process in crabapple warrants in-depth exploration. In this study, we explored the inhibitory role of MsMYB62-like in anthocyanin biosynthesis. We identified MsDFR and MsANS as two downstream target genes of MsMYB62-like. These genes encode enzymes integral to the anthocyanin biosynthetic pathway. The findings demonstrate that MsMYB62-like directly binds to the promoters of MsDFR and MsANS , resulting in the downregulation of their expression levels. Additionally, our observations indicate that the plant hormone cytokinins exert a suppressive effect on the expression levels of MsMYB62-like , while concurrently upregulating MsDFR and MsANS . This study reveals that the MsMYB62-like- MsDFR / MsANS module plays an important role in governing anthocyanin levels in Malus crabapple. Notably, the regulatory interplay is modulated by the plant hormone cytokinins.

Global crop yield has been affected by a number of abiotic stresses. Heat, salinity, and drought stress are at the top of the list as serious environmental growth-limiting factors. To enhance crop productivity, molecular approaches have been used to determine the key regulators affecting stress-related phenomena. MYB transcription factors (TF) have been reported as one of the promising defensive proteins against the unfavorable conditions that plants must face. Different roles of MYB TFs have been suggested such as regulation of cellular growth and differentiation, hormonal signaling, mediating abiotic stress responses, etc. To gain significant insights, a comprehensive in-silico analysis of OsMYB TF was carried out in comparison with 21 dicot MYB TFs and 10 monocot MYB TFs. Their chromosomal location, gene structure, protein domain, and motifs were analyzed. The phylogenetic relationship was also studied, which resulted in the classification of proteins into four basic groups: groups A, B, C, and D. The protein motif analysis identified several conserved sequences responsible for cellular activities. The gene structure analysis suggested that proteins that were present in the same class, showed similar intron-exon structures. Promoter analysis revealed major cis-acting elements that were found to be responsible for hormonal signaling and initiating a response to abiotic stress and light-induced mechanisms. The transformation of OsMYB TF into tobacco was carried out using the Agrobacterium -mediated transformation method, to further analyze the expression level of a gene in different plant parts, under stress conditions. To summarize, the current studies shed light on the evolution and role of OsMYB TF in plants. Future investigations should focus on elucidating the functional roles of MYB transcription factors in abiotic stress tolerance through targeted genetic modification and CRISPR/Cas9-mediated genome editing. The application of omics approaches and systems biology will be indispensable in delineating the regulatory networks orchestrated by MYB TFs, facilitating the development of crop genotypes with enhanced resilience to environmental stressors. Rigorous field validation of these genetically engineered or edited crops is imperative to ascertain their utility in promoting sustainable agricultural practices.

Parthenocarpy, characterized by seedless fruit development without pollination or fertilization, offers the advantage of consistent fruit formation, even under challenging conditions such as high temperatures. It can be induced by regulating auxin homeostasis; PAD1 ( PARENTAL ADVICE-1 ) is an inducer of parthenocarpy in Solanaceae plants. However, precise editing of PAD1 is not well studied in peppers. Here, we report a highly efficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) for CaPAD1 editing in three valuable cultivars of pepper ( Capsicum annuum L.): Dempsey, a gene-editable bell pepper; C15, a transformable commercial inbred line; and Younggo 4, a Korean landrace. To achieve the seedless pepper trait under high temperatures caused by unstable climate change, we designed five single guide RNAs (sgRNAs) targeting the CaPAD1 gene. We evaluated the in vitro on-target activity of the RNP complexes in three cultivars. Subsequently, we introduced five CRISPR/Cas9-RNP complexes into protoplasts isolated from three pepper leaves and compared indel frequencies and patterns through targeted deep sequencing analyses. We selected two sgRNAs, sgRNA2 and sgRNA5, which had high in vivo target efficiencies for the CaPAD1 gene across the three cultivars and were validated as potential off-targets in their genomes. These findings are expected to be valuable tools for developing new seedless pepper cultivars through precise molecular breeding of recalcitrant crops in response to climate change.

The NYN domain gene family consists of genes that encode ribonucleases that are characterized by a newly identified NYN domain. Members of the family were widely distributed in all life kingdoms and play a crucial role in various RNA regulation processes, although the wide genome overview of the NYN domain gene family is not yet available in any species. Rapeseed (Brassica napus L.), a polyploid model species, is an important oilseed crop. Here, the phylogenetic analysis of these BnaNYNs revealed five distinct groups strongly supported by gene structure, conserved domains, and conserved motifs. The survey of the expansion of the gene family showed that the birth of BnaNYNs is explained by various duplication events. Furthermore, tissue-specific expression analysis, protein-protein interaction prediction, and cis-element prediction suggested a role for BnaNYNs in plant growth and development. Interestingly, the data showed that three tandem duplicated BnaNYNs (TDBs) exhibited distinct expression patterns from those other BnaNYNs and had a high similarity in protein sequence level. Furthermore, the analysis of one of these TDBs, BnaNYN57, showed that overexpression of BnaNYN57 in Arabidopsis thaliana and B. napus accelerated plant growth and significantly increased silique length, while RNA interference resulted in the opposite growth pattern. It suggesting a key role for the TDBs in processes related to plant growth and development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

In plants, cytochrome P450s are monooxygenase that play key roles in the synthesis and degradation of intracellular substances. In tobacco, the majority of studies examining the P450 superfamily have concentrated on the CYP82E subfamily, where multiple family members function as demethylases, facilitating the synthesis of nornicotine. In this study, NtCYP82C4, a tobacco P450 superfamily member, was identified from a gene-edited tobacco mutant that nicotine biosynthesis in tobacco leaves is evidently reduced. Compared to the wild-type plants, the knockout of NtCYP82C4 resulted in a significantly lower nicotine content and biomass in tobacco leaves. Transcriptome and metabolome analyses indicated that the knockout of NtCYP82C4 inhibites secondary metabolic processes in tobacco plants, leading to the accumulation of some important precursors in the nicotine synthesis process, including aspartic acid and nicotinic acid, and increases nitrogen partitioning associated with those processes such as amino acid synthesis and utilization. It is speculated that NtCYP82C4 may function as an important catalase downstream of the nicotine synthesis. Currently, most of the steps and enzymes involved in the nicotine biosynthesis process in tobacco have been elucidated. Here, our study deepens the current understanding of nicotine biosynthesis process and provides new enzyme targets for nicotine synthesis in tobacco plants., Competing Interests: Declaration of competing interest The authors declare neither competing interests nor conflict of financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Regulation of protein longevity via the ubiquitin (Ub) - proteasome pathway is fundamental to eukaryotic biology. Ubiquitin E3 ligases (E3s) interact with substrate proteins and provide specificity to the pathway. A small subset of E3s bind to specific exposed N-termini (N-degrons) and promote the ubiquitination of the bound protein. Collectively these E3s, and other N-degron binding proteins, are known as N-recognins. There is considerable functional divergence between fungi, animal, and plant N-recognins. In plants, at least three proteins (PRT1, PRT6, and BIG) participate in the Arg/N-degron pathway. PRT1 has demonstrated E3 ligase activity, whereas PRT6 and BIG are candidate E3s. The Arg/N-degron pathway plays a central role in plant development, germination, and submersion tolerance. The pathway has been manipulated both to improve crop performance and for conditional protein degradation. A more detailed structural and biochemical understanding of the Arg/N-recognins and their substrates is required to fully realise the biotechnological potential of the pathway. This perspective focuses on the structural and molecular details of substrate recognition and ubiquitination in the plant Arg/N-degron pathway. While PRT1 appears to be plant specific, the PRT6 and BIG proteins are similar to UBR1 and UBR4, respectively. Analysis of the cryo-EM structures of Saccharomyces UBR1 suggests that the mode of ubiquitin conjugating enzyme (E2) and substrate recruitment is conserved in PRT6, but regulation of the two N-recognins may be significantly different. The structurally characterised domains from human UBR4 are also likely to be conserved in BIG, however, there are sizeable gaps in our understanding of both proteins., (© 2024 The Author(s).)

The classical acid growth theory suggests that auxin stimulates cell expansion by triggering apoplast acidification via plasma membrane (PM)-localized H + -ATPase. Here, we reconstructed the origin and evolutionary history of auxin-mediated acid growth. Comparative phylogenomic analysis showed that most core components of acid growth originated in Charophyta and then underwent subclass expansion and functional innovation during plant terrestrialization. In Charophyceae algae Chara braunii, we found that PM H + -ATPase has formed a core regulatory module with TMK and PP2C.D, which can be activated by photosynthesis-dependent phosphorylation through light rather than auxin. Despite the lack of canonical auxin receptor TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB), auxin elicits significant internodal elongation and transcriptional reprogramming in C. braunii , implying the existence of an ancient auxin-mediated growth mechanism. We propose that the evolution of acid growth represents a neofunctional adaptation to terrestrial environments, in which PM H + -ATPase in carbon concentrating for photosynthesis was utilized to acidify apoplast for cell expansion, and the core components responsible for acid growth eventually established a regulatory network in land plants by connecting with the TIR1/AFB pathway., Competing Interests: Competing interests statement:The authors declare no competing interest.

Oxidosqualene cyclases (OSCs) are important regulatory enzymes involved in cyclization reactions of 2, 3-oxidosqualene to form triterpenes and sterols. This study presents the identification and characterization of three OSC genes, a β - amyrin synthase (VuβAS), a lupeol synthase (VuLUS) and a cycloartenol synthase (VuCAS) in Vigna unguiculata, an edible leguminous plant with high nutritional and nutraceutical value. Phylogenetic analysis showed that the VuβAS, VuLUS and VuCAS were clustered within the clades of previously characterized β - amyrin synthases, lupeol synthases and cycloartenol synthases. Heterologous expression in Saccharomyces cerevisiae and Gas Chromatography - Mass Spectrometry (GC - MS) analysis in different plant stages confirmed their specific functions. VuβAS showed higher expression in roots from early germinating seedlings to older plants (4-day to 28-day), while VuLUS expression levels were higher in the roots of older plants only (14-day to 28-day). VuCAS expression was increased in all the tissues of 4-day seedlings, with a peak in stem and leaves and a lower accumulation in radicles. These findings revealed the presence and function of OSC genes in V. unguiculata, and future research could lead to the discovery of promising biologically active compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Anthocyanin regulation can be fruitfully explored from a diverse perspective by studying distantly related model organisms. Land plants pioneers faced a huge evolutionary leap, involving substantial physiological and genetic changes. Anthocyanins have evolved alongside these changes, becoming versatile compounds capable of mitigating terrestrial challenges such as drought, salinity, extreme temperatures and high radiation. With the accessibility of whole-genome sequences from ancient plant lineages, deeper insights into the evolution of key metabolic pathways like phenylpropanoids have emerged. Despite understanding the function of anthocyanins under stress, gaps remain in uncovering the precise metabolic and regulatory mechanisms driving their overproduction under stressful conditions. For example, the regulatory effect of reactive oxygen species (ROS) on well-known transcription factors like MYBs is not fully elucidated. This manuscript presents an evolutionary analysis of the anthocyanin biosynthetic pathway to elucidate key genes. CINNAMATE 4-HYDROXYLASE (C4H) and CHALCONE ISOMERASE (CHI2) received particular attention. C4H exposes remarkable differences between aquatic and land plants, while CHI2 demonstrates substantial variation in gene copy number and sequence similarity across species. The role of transcription factors, such as MYB, and the involvement of ROS in the regulation of anthocyanin biosynthesis are discussed. Complementary gene expression analyses under abiotic stress in Arabidopsis thaliana, Selaginella moellendorffii, and Marchantia polymorpha reveal intriguing gene-stress relationships. This study highlights evolutionary trends and the regulatory complexity of anthocyanin production under abiotic stress, providing insights and opening avenues for future research., Competing Interests: Declarations. Conflict of interest: The authors declare no conflict of interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

C-repeat binding factors (CsCBFs) play a pivotal role in regulating cold response in higher plants. Camellia sinensis cv. Baiye 1, a representative albino tea cultivar, has been identified as temperature-sensitive based on long-term observations by tea farmers. However, it remains unclear whether CsCBFs are involved in temperature-mediated albinism and seasonal greening in 'Baiye 1', and the mechanisms by which CBFs regulate cold responses in albino leaves are unknown. In this study, we demonstrate that CsCBF2 suppresses the seasonal greening of albino leaves by inhibiting chlorophyll and carotenoid biosynthesis under cold stress. In tea plantations, the accumulation of chlorophylls and carotenoids in the albino shoots of 'Baiye 1' is closely correlated with the effective accumulated temperature during its seasonal greening process. Weighted Gene Co-expression Network Analysis revealed negative associations between CsCBF expression and chlorophylls, carotenoids, as well as their biosynthetic genes REVEILLE 1 (CsRVE1) and Zeaxanthin epoxidase 1 (CsZEP1) under temperature fluctuations during seasonal greening. Cold-induced upregulation of CsCBF2 expression and decreased chlorophyll and carotenoids under controlled climate conditions. Transient suppression of CsCBF2 by antisense oligodeoxynucleotides elevated expressions of target genes and increased chlorophylls and carotenoids. CBF-binding cis-elements were identified in CsRVE1, Protochlorophyllide oxidoreductase A (CsPORA) and CsZEP1 promoters. Luciferase assays suggested CsCBF2 binding to the CRT/DRE cis-elements and repressing expression of CsRVE1, CsPORA and CsZEP1. These findings highlight CsCBF2 as a key transcriptional repressor involved in the seasonal greening of albino 'Baiye 1' under cold stress by modulating cold responses and inhibiting genes associated with chlorophyll and carotenoid biosynthesis., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Inorganic toxicants like arsenic, copper, lead, nickel and fluoride are notorious agro-pollutants, impeding plant-productivity due to high bioaccumulation. Consumption of such contaminated plant-parts causes irreversible health hazards. We identified a G-protein coupled receptor, serving as melatonin receptor (MelR) in Nicotiana tabacum (NtMelR), that relayed downstream-signaling after binding melatonin, a potent growth regulator and antioxidant. Using inhibitors against G-protein-α and NADPH oxidase (NOX), and by supplementing epidermal strips with exogenous melatonin and H 2 O 2 , we established that NtMelR acted upstream of reactive oxygen species (ROS) production in guard cells. Transgenic lines of N. benthamiana overexpressing NtMelR maintained constitutive melatonin-signaling via MelR, leading to efficient stomatal closure for preventing desiccation during oxidative stress. Melatonin biosynthesis was stimulated in the transgenic lines, exposed to different agro-pollutant stress, providing a steady-abundance of ligand for NtMelR binding and activating the defence machinery, comprising of enzymatic-antioxidants like superoxide dismutase, catalase, peroxidases and glyoxalases. Due to increased antioxidant capacity, the transgenics exhibited less molecular injuries (electrolyte leakage, methylglyoxal accumulation and NOX activity), generated less ROS and bioaccumulated significantly lower levels of toxicants. Unlike the wild-type counterparts, the transgenics maintained high relative water content, photosynthetic efficiency, could flower abundantly and even produce seeds. Overall, we established that overexpression of NtMelR is a single-window strategy to generate multiple-stress tolerant genotypes., Competing Interests: Declaration of competing interest Aryadeep Roychoudhury reports that financial support was provided by Science and Engineering Research Board, Government of India. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Potato tubers accumulate substantial quantities of starch, which serves as their primary energy reserve. As the predominant component of potato tubers, starch strongly influences tuber yield, processing quality, and nutritional attributes. Potato starch is distinguished from other food starches by its unique granule morphology and compositional attributes. It possesses large, oval granules with amylose content ranging from 20 to 33 % and high phosphorus levels, which collectively determine the unique physicochemical characteristics. These physicochemical properties direct the utility of potato starch across diverse food and industrial applications. This review synthesizes current knowledge on the molecular factors controlling potato starch biosynthesis and structure-function relationships. Key topics covered are starch granule morphology, the roles and regulation of major biosynthetic enzymes, transcriptional and hormonal control, genetic engineering strategies, and opportunities to tailor starch functionality. Elucidating the contributions of different enzymes in starch biosynthesis has enabled targeted modification of potato starch composition and properties. However, realizing the full potential of this knowledge faces challenges in optimizing starch quality without compromising plant vigor and yield. Overall, integrating multi-omics datasets with advanced genetic and metabolic engineering tools can facilitate the development of elite cultivars with enhanced starch yield and tailored functionalities., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A critical transcription factor, PacC, modulates the expression of fungal pH signaling. Although PacC-mediated environmental pH has been reported to regulate the growth and pathogenicity of postharvest pathogens, the involvement of PacC in sucrose metabolism, sugar transport, and the pentose phosphate pathway (PPP) in different zones of decayed fruit remains unclear. Our work showed that the inoculation with a PePacC deletion strain of Penicillium expansum (ΔPePacC) accelerated sucrose catabolism and glucose and fructose accumulation in different zones of apple fruit. This was attributed to an increase in sucrose metabolism enzyme activities and up-regulation of the sugar transporter protein-related gene expression. Moreover, ΔPePacC inoculation increased the PPP-related enzyme activities and the levels of nicotinamide adenine dinucleotide phosphate (NADPH) and NADP + . In conclusion, PacC modulates sucrose metabolism, sugar transport, and the PPP in apple fruit by mediating dynamic changes in environmental pH, thereby enhancing fruit disease resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The longan pericarp turns brown dramatically after harvesting, but the mechanism is not well understood. In this work, two peroxidases were purified from longan pericarp and found to be identical to the class III peroxidases PRX53-2 and PRX53-3. In vitro, PRX53-2/3 catalyzed the browning of several pericarp abundant proanthocyanidin and lignin monomers, such as (-)-epicatechin (EC), (+)-catechin (CT) and coniferyl alcohol (ConA). PRX53-2 was upregulated and highly-expressed, while PRX53-3 was expressed at low levels after harvesting; thus, PRX53-2 was considered a browning-related gene. The reaction with both proanthocyanidin and lignin presented a greater degree of brown coloration compared to the single substrate reactions. Several procyanidins isomers, EC-ConA and CT-ConA were detected in the double-substrate reaction. These results not only demonstrate that the effects of PRX53-2 on proanthocyanidin and lignin polymerization may be crucial for longan pericarp browning, but also help in developing new strategies or preservatives to delay pericarp browning., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Methionine (Met) can inhibit plant diseases caused by phytopathogens. However, the effect of Met on gray mold resulted from Botrytis cinerea in tomato is still unclear. This study showed 5 mM Met alleviated disease development of gray mold, enhanced chitinase (CHI) and β-1, 3-glucanase (GNS) activities and the expression of SlCHI, SlGNS, SlPR1 and SlNPR1 in tomatoes, rather than inhibited the growth of B. cinerea directly. Moreover, ethylene biosynthesis and signal transduction before pathogen inoculating were induced by 5 mM Met. Interestingly, Met reduced the nitrosylation levels of ACS4 and ACO6, enhanced the activities of nitric oxide synthase, nitrite reductase (NR) and S-nitrosoglutathione reductase (GSNOR) and the expression of SlNR and SlGSNOR. Tomatoes treated with aminoethoxyvinylglycine and carboxy-PTIO exhibited lower resistance to B. cinerea. These results indicate 5 mM Met promoted ethylene biosynthesis and signal transduction to facilitate NO synthesis and metabolism, enhancing the resistance of tomatoes to B. cinerea., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. No competing interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

High-sensitivity 4D label-free proteomic technology was used to identify protein components related to gluten quality and celiac disease (CD) in strong-gluten wheat cultivar KX 3302 and medium-gluten wheat cultivar BN 207. The highly expressed storage protein components in KX3302 were high-molecular-weight-glutenin-subunits (HMW-GSs), α-gliadin, and globulin, whereas those in BN207 were γ-gliadin, low-molecular-weight-glutenin-subunits (LMW-GSs) and avenin-like proteins. In addition, BN207 had more upregulated metabolic proteins than KX3302. The abundance of storage proteins increased during dough formation. After heat treatment, the upregulated proteins accounted for 57.53 % of the total proteins, but the downregulated storage proteins accounted for 79.34 % of the total storage proteins. In cultivar KX3302, CD proteins mainly included α-gliadin and HMW-GSs, whereas in BN207, they were mainly γ-gliadin and LMW-GSs. Thermal treatment significantly reduces the expression levels of CD-related proteins. These findings provide a new perspective on reducing the content of CD-related proteins in wheat products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Key Message: SlNAC12 enhances salt stress tolerance of transgenic tomato by regulating ion homeostasis, antioxidant activity and flavonoids biosynthesis Soil salinization is a major environmental factor that adversely affects plant growth and development. NAC (NAM, ATAF1/2, and CUC2) is a large family of plant-specific transcription factors that play crucial roles in stress response. Here, we investigated the role of a novel NAC transcription factor, SlNAC12, in conferring salt stress tolerance in tomato (Solanum lycopersicum). Subcellular localization and yeast assays studies revealed that SlNAC12 is localized in the nucleus with weak transcriptional activity. SlNAC12 transcript was induced by salt stress in the leaves and roots of tomato seedlings. Overexpression of SlNAC12 in tomato led to significantly reduced plant height and root length. Transgenic tomato lines overexpressing of SlNAC12 (OE#1 and OE#3) exhibited enhanced tolerance to salinity, as evidenced by reduced the inhibitory effect of growth parameters under salt stress compared to wild type (WT). Overexpression of SlNAC12 in tomato affected Na + and K + homeostasis, leading to reduced Na + /K + ratio, enhanced activity of antioxidant enzymes and decreased reactive oxygen species (ROS) accumulation under salt stress. Furthermore, the transcript levels of several genes involved in flavonoids metabolism and the levels of flavonoids accumulation were increased in SlNAC12-overexpressing tomato lines. Collectively, this study suggests that SlNAC12 transcription factor enhances salt stress tolerance in tomato is correlated with ion homeostasis, antioxidant enzyme systems, and flavonoids accumulation., Competing Interests: Declarations. Conflict of interest: The authors declare no conflict of interest., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Pathogenesis-related proteins (PR), whose expressions are induced by biotic and abiotic stress, play important roles in plant defense. Previous research identified the salt-induced HcPR10 gene in the halophyte Halostachys caspica as a regulator of plant growth and development through interactions with cytokinin. However, the mechanisms by which HcPR10 mediates resistance to abiotic stress remain poorly understood. In this study, we found that the heterologous expression of HcPR10 significantly enhanced salt and drought tolerance in Arabidopsis, likely by increasing the activity of antioxidant enzyme systems, allowing for effective scavenging of reactive oxygen species (ROS) and thus protecting plant cells from oxidative damage. Additionally, the overexpression of HcPR10 also activated the expression of stress-related genes in Arabidopsis. Furthermore, using yeast two-hybrid technology, five proteins (HcLTPG6, HcGPX6, HcUGT73B3, HcLHCB2.2, and HcMSA1) were identified as potential interacting partners for HcPR10, which could positively regulate the salt stress response mediated by HcPR10. Our findings lay the foundation for a better understanding of the molecular mechanisms of HcPR10 in response to abiotic stress and reveal additional candidate genes for improving crop salt tolerance through genetic engineering., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Key Message: All ten dehydrin genes from three Medicago species are responsive to different kinds of abiotic stress, and CAS31 confers transgenic plants salt tolerance by down-regulating HKT1 expression. Dehydrins are protective proteins playing crucial roles in the tolerance of plants to abiotic stresses. However, a full-scale and systemic analysis of total dehydrin genes in Medicago at the genome level is still lacking. In this study, we identified ten dehydrin genes from three Medicago species (M. truncatula, M. ruthenica, and M. sativa), categorizing the coding proteins into four types. Genome collinearity analysis among the three Medicago species revealed six orthologous gene pairs. Promoter regions of dehydrin genes contained various phytohormone- and stress-related cis-elements, and transcriptome analysis showed up-regulation of all ten dehydrin genes under different stress conditions. Transformation of dehydrin gene CAS31 increased the tolerance of transgenic seedlings compared with wild-type seedlings under salt stress. Our study demonstrated that transgenic seedlings maintained the more chlorophyll, accumulated more proline and less hydrogen peroxide and malondialdehyde than wild-type seedlings under salt stress. Further study revealed that CAS31 reduced Na + accumulation by down-regulating HKT1 expression under salt stress. These findings enhance our understanding of the dehydrin gene family in three Medicago species and provide insights into their mechanisms of tolerance., Competing Interests: Declarations. Conflict of interests: The authors declare that they have no known competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Main Conclusion: PpZAT12, a transcription factor differentially expressed in peach varieties with distinct susceptibility tochilling injury (CI), is a potential candidate gene for CI tolerance by regulating several identified gene targets. ZAT (zinc finger of Arabidopsis thaliana) proteins play roles in multiple abiotic stress tolerance in Arabidopsis and other species; however, there are few reports on these transcription factors (TFs) in fruit crops. This study aimed to evaluate PpZAT12, a C2H2 TF up-regulated in peach fruit by a heat treatment applied before postharvest cold storage for reducing chilling injury (CI) symptoms. Here, the expression of PpZAT12 in different tissues and fruits subjected to either postharvest heat or cold treatments, was evaluated in peach varieties with differential susceptibility to develop CI. PpZAT12 increased by cold storage in CI-resistant cultivars ('Elegant Lady' and 'Rojo 2'), while it was not modified in a cultivar susceptible to develop CI ('Flordaking'). Besides, we expressed PpZAT12 in Arabidopsis (35S::PpZAT12) and found that these plants show impaired plant growth and development, rendering small plants with senescence delay and aborted seeds. We applied a proteomic approach to decipher the peptides responding to PpZAT12 in Arabidopsis and found 348 differential expressed proteins (DEPs) relative to the wild type. Besides, comparing the DEPs between Arabidopsis plants expressing PpZAT12 or AtZAT12 (35S::AtZAT12) we found common and specific responses to these TFs. Based on the proteomic information obtained here and published data about AtZAT12, we searched ZAT12-targets in peach allowing the identification of a putative ZAT12 regulon in this species. The identified peach ZAT12-protein targets could underlie the differential susceptibility to CI among different peach varieties and can be used as future targets to improve adaptation to refrigeration in fleshy fruits., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no conflict of interest., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Similar to cellulose synthases (CESAs), cellulose synthase-like D (CSLD) proteins synthesize β-1,4-glucan in plants. CSLDs are important for tip growth and cytokinesis, but it was unknown whether they form membrane complexes in vivo or produce microfibrillar cellulose. We produced viable CESA-deficient mutants of the moss Physcomitrium patens to investigate CSLD function without interfering CESA activity. Microscopy and spectroscopy showed that CESA-deficient mutants synthesize cellulose microfibrils that are indistinguishable from those in vascular plants. Correspondingly, freeze-fracture electron microscopy revealed rosette-shaped particle assemblies in the plasma membrane that are indistinguishable from CESA-containing rosette cellulose synthesis complexes (CSCs). Our data show that proteins other than CESAs, most likely CSLDs, produce cellulose microfibrils in P. patens protonemal filaments. The data suggest that the specialized roles of CSLDs in cytokinesis and tip growth are based on differential expression and different interactions with microtubules and possibly Ca 2+ , rather than structural differences in the microfibrils they produce.

Background: Chrysanthemum ( Chrysanthemum × morifolium Ramat.) is a particularly important autumn perennial flower for potted plant, flower bed and border, and cut flower with high ornamental value. However, abiotic stress can affect the ornamental quality of Chrysanthemum . NAC (NAM, ATAF1-2, and CUC2) transcription factors (TFs) play an important role in regulating plant growth and development, as well as responding to abiotic stresses., Methods: In this study, the ClBRN1 ( Chrysanthemum lavandulifolium BEARSKIN gene) was isolated from the Chrysanthemum model plant C. lavandulifolium . And analyze the function of the gene through bioinformatics, subcellular localization and overexpression., Results: Bioinformatics analysis showed that the ClBRN1 gene was a member of the NAC TFs family, with a CDS (coding sequence) length of 1,080 bp and encoding 359 amino acids. The subcellular localization results found that the gene was located in the nucleus and cell membrane. Furthermore, the transgenic results in Arabidopsis thaliana showed that the gene significantly reduces plant height while improving salt and low temperature tolerance. Observation of paraffin sections of Arabidopsis stems also revealed that the secondary cell wall of overexpressing Arabidopsis stems was significantly thicker than that of wild-type. The above results indicate that the ClBRN1 gene may play an important role in regulating plant resistance to abiotic stress. This study will provide new insights for molecular breeding of resistant chrysanthemums in the future., Competing Interests: The authors declare that they have no competing interests., (© 2024 Li et al.)

The issue of soil salinization is a global concern that significantly impairs crop productivity, quality, and distribution. Tonoplast Dicarboxylate Transporter (TDT) is a pivotal malic acid transporter localized on the vacuolar membrane, involving in maintaining intracellular pH homeostasis in plants. However, the molecular mechanisms and regulatory pathways underlying plant salt tolerance through TDT remain elusive. In this study, we cloned a gene encoding vacuolar membrane dicarboxylic acid transporter designated as SaTDT from the halophyte Spartina alterniflora. Subsequently, its role in regulating salt stress was investigated. The heterologous expression of SaTDT in Arabidopsis thaliana was observed to enhance the transgenic plants' tolerance to salt stress and alleviate the growth damage caused by this stress. The overexpression of SaTDT can simultaneously enhance plant photosynthetic efficiency by regulating the cellular contents of malic acid and citric acid, or by increasing the activity of MDH and PEPC enzymes. It also regulates and balances energy utilization during carbon assimilation under salt-stressed conditions, thereby establishing an energetic foundation for enhancing plant tolerance to stress. SaTDT also has the capacity to enhance the plant cells' ability in regulating antioxidant enzyme activity or osmotic accumulation, thereby playing a crucial role in maintaining intracellular redox homeostasis. In conclusion, our findings establish a foundation basis for elucidating the regulatory role of the SaTDT gene in S.alterniflora's adaptation to high-salinity habitats., Competing Interests: Declarations. Conflict of interest: The authors declare no conflict of interest., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

In the past decades, cyclic nucleotide-gated ion channels (CNGCs) have been extensively studied in diploid species Arabidopsis thaliana. However, the functional diversification of CNGCs in crop plants, mostly polyploid, remains poorly understood. In allotetraploid Upland cotton (Gossypium hirsutum), GhCNGC31 is one of the multiple orthologs of AtCNGC2, being present in the plasma membrane, capable of interacting with itself and binding to calmodulins and cyclic nucleotides. GhCNGC31 knockdown plants exhibited slight growth inhibition, and became more susceptible to Verticillium dahliae infection, which was associated with the reduced lignin and flavonoid accumulation, impaired ROS (reactive oxygen species) burst, and down-regulation of defense-related genes PR1, JAZ2, LOX2, and RBOH10. RNA-Seq analysis identified 1817 differentially expressed genes from GhCNGC31 knockdown, of which 1184 (65%) were responsive to V. dahliae infection and accounted for 57% among a total of 2065 V. dahliae-responsive genes identified in this study. These GhCNGC31-regulated genes mainly function with cell wall organization and biogenesis, cellular carbohydrate metabolic or biosynthetic process, cellular component macromolecule biosynthetic process, and rhythmic process. They are significantly enriched in the pathways of plant MAPK signaling, plant-pathogen interaction, phenylpropanoid biosynthesis, and plant hormone signal transduction. A set of transcription factors (TFs) and resistance (R) genes are among the GhCNGC31-regulated genes, which are significantly over-represented with the TCP and WRKY TFs families, as well as with the R genes of T (TIR) and TNL (TIR-NB-LRR) classes. Together, our results unraveled a critical role of GhCNGC31 for conferring resistance to Verticillium wilt in cotton., Competing Interests: Declarations. Conflict of interest: No potential conflict of interest was reported by the authors., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Key Message: Mutations in the CsEMS1 gene result in male sterility and reduced wart number and density. Male sterility and fruit wart formation are two significant agronomic characteristics in cucumber (Cucumis sativus), yet knowledge of our underlying genetics is limited. In this study, we identified an EMS-induced male sterility and few small warts mutant (msfsw). Histological observations revealed defects the absence of tapetum, meiotic aberration and impaired microspore formation in the anthers of the mutant. The mutant also exhibits a reduction in both the size and number of fruit spines and fruit tubercules. Genetic analysis revealed that a single recessive gene is responsible for the mutant phenotypes. BSA-Seq and fine genetic mapping mapped the msfsw locus to a 63.7 kb region with four predicted genes. Multiple lines of evidence support CsEMS1(CsaV3_3G016940) as the candidate for the mutant allele which encodes an LRR receptor-like kinase, and a non-synonymous SNP inside the exon of CsEMS1 is the causal polymorphisms for the mutant phenotypes. This function of CsEMS1 in determination of pollen fertility was confirmed with generation and characterization of multiple knockout mutations with CRISPR/Cas9 based gene editing. In the wild-type (WT) plants, CsEMS1 was highly expressed in male flowers. In the mutant, the expression level of CsEMS1, several tapetum identity-related genes, and trichome-related genes were all significantly reduced as compared with the wild-type. Protein-protein interaction assays revealed physical interactions between CsEMS1 and CsTPD1. Quantitation of endogenous phytohormones revealed a reduction in the ethylene precursor ACC in CsEMS1 knockout lines. This work identified an important role of CsEMS1 in anther and pollen development as well as fruit spine/wart development in cucumber., Competing Interests: Declarations. Conflict of interest: The authors declare that there is no conflict of interest., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: Identification of salt-responsive calcineurin B-like protein-interacting protein kinases (CIPKs) in Populus. Calcineurin B-like protein-interacting protein kinases (CIPKs) play vital roles in plant growth and abiotic stress responses. Currently, the regulatory mechanisms underlying these processes mediated by CIPK proteins are not completely understood in woody species. This study provided the first systematic analysis of 31 Populus CIPK genes and investigated their evolutionary relationships, gene structures, motif compositions, and salt stress responses. A total of 11 pairs of paralogous PtCIPK genes were identified, of which three pairs may be resulted from whole genome duplication, and two pairs that may be created by tandem duplications. RT-qPCR analysis revealed that 93.5% (29/31) genes showed altered expression levels in roots after salt treatment. Ectopic expression of PdCIPK21 or PdCIPK31 in Arabidopsis resulted in significant increases of seed germination, root elongation and fresh weight under salt stress conditions. Cytological observation revealed that PdCIPK21/31 overexpression lines showed increased number, lumen area and cell wall thickness of xylem vessels, and higher lignin content in stems compared with the wild type, with decreased sensitivity to long-term salt stress treatment. Our results suggest that PdCIPK21/31 serve as candidate genes for improving wood production and enhancing salt tolerance of tree species., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no conflicts of interest., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Tea contains a variety of flavone C -glycosides, which are important compounds that distinguish tea cultivars and tea categories. However, the biosynthesis pathway of flavone C -glycosides in tea plant remains unknown, and the key enzymes involved have not been characterized. In this study, a liquid chromatography-mass spectrometry method to determine 9 flavone C -glycosides was developed, and the accumulation patterns of 9 flavone C -glycosides in tea plants were examined first. Then, an entry enzyme Cs F2H for flavone C -glycoside biosynthesis was identified, which had four cytochrome P450-specific conserved motifs and was targeted to the endoplasmic reticulum. Correlation analysis indicated that the expression level of CsF2H was positively correlated with all contents of 9 flavone C -glycosides. The recombinant Cs F2H could convert flavanone (naringenin) into the corresponding 2-hydroxyflavonone (2-hydroxynaringenin), rather than into flavone (apigenin). Heterologous coexpression of Cs F2H and Cs CGT1 in yeast revealed that the substrate naringenin could be enzymatically converted to flavone mono- C -glycosides vitexin and isovitexin under the catalytic control of Cs F2H and Cs CGT1 following dehydration. Gene-specific antisense oligonucleotide analysis suggested that suppressing CsF2H significantly reduced the levels of 9 flavone C -glycosides. Together, Cs F2H is the first key enzyme that generates flavone C -glycosides through the 2-hydroxyflavanone biosynthesis pathway in tea plants.

Dufulin is a novel immuno-inducer in plants, used to control viral diseases. Salt stress is one of the major abiotic stresses affecting plant growth. OsJAZ5 is a member of the JAZ (JASMONATE ZIM-DOMAIN) protein family, which are key regulators of the JA (jasmonic acid) response. In this study, Dufulin promoted expression of the gene OsJAZ5 in the salt stress response process of rice, and the interaction between Dufulin and OsJAZ5 was confirmed using microscale thermophoresis (MST) and molecular docking studies. We further explored the function of OsJAZ5 protein, using yeast functional complementation studies, physiological and biochemical data of OsJAZ5 -overexpressed plants, and transcriptome data to determine its role in enhancing rice salt tolerance, and found that OsJAZ5 indirectly promotes JA-signaling transduction through its interacting proteins OsMYL1 and OsMYL2. In conclusion, OsJAZ5 is a functional target for Dufulin to achieve enhanced rice salt tolerance, and overexpression of OsJAZ5 can improve rice tolerance to salt stress.

Rebaudioside M2 (RebM2) is characterized as 13-[(2- O -β-d-glucopyranosyl-3- O -β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(2- O -β-d-glucopyranosyl-6- O -β-d-glucopyranosyl-β-d-glucopyranosyl) ester], an isomer of rebaudioside M with a 1 → 6 sugar linkage. The product was found in the biotransformation of rebaudioside D (RebD) catalyzed by a glycosyltransferase from Nicotiana tomentosiformis ( Nt UGT). Herein, guided by consensus engineering and molecular dynamics simulations, a variant Nt UGT F72L/L123P/L157P with enhanced activity and thermostability was obtained. It exhibits a strikingly reduced K m (22.47 mM to 0.15 mM) toward RebD, and the catalytic efficiency was over 5000-fold higher than that of the wildtype. When an Arabidopsis sucrose synthase At SuSy was used for UDP-glucose recycling, Nt UGT F72L/L123P/L157P effectively converted 80 g/L RebD to 90.14 g/L RebM2. In a one-pot three-enzyme reaction involving an engineered glycosyltransferase UGTSL2 N358F , which catalyzed the conversion of RebA into RebD, 78.8 g/L of RebM2 (with a yield of 84.56%) was produced from 70 g/L of RebA, avoiding the use of the naturally rare and poorly soluble RebD as the starting material. This work will provide a promising biocatalyst for RebM2 biosynthesis on a large scale and create an opportunity to accelerate the exploration of the biological activity of RebM2 and its potential as a candidate for superior SG sweeteners.

Farnesene synthase from Artemisia annua (AaFS) catalyzes the reaction from farnesyl pyrophosphate (FPP) to give the sesquiterpene β-farnesene, a key building block for the biosynthesis of vitamin E. However, an insufficient yield of β-farnesene precludes its industrialization. Understanding the mechanism would be essential for attaining β-farnesene in high yield. Guided by structure-based enzyme engineering, we designed several potent variants, among which L326I increased the β-farnesene yield from 450.65 to 3877.42 mg/L. Furthermore, we found that the function of β-farnesene synthase AaFS can be modulated at two positions; W299 is responsible for tuning the enzyme's function to give its isomeric product α-farnesene and Y402 is the key residue for diverting from the linear farnesene products to the monocyclic α-bisabolol product. These findings provide valuable insights into the catalytic mechanism and functional modulation of farnesene synthases and set the basis for rational engineering of farnesene synthases for selective biosynthesis of diverse sesquiterpene natural products.

Methyl jasmonate (MeJA) is a growth regulator that is involved in plant defense and development. Studies have demonstrated its role in pigmentation, particularly in synergy with UV-B in plant tissues. However, its role in pigment metabolism remains incompletely understood. To explore the metabolic synergistic effects, we evaluated pigments and gene expression in mature green apples. MeJA alone had no effect on pigment accumulation, while UV-B increased flavonols up to S2 stage and anthocyanins up to S3 stage. With UV-B > 2 W m -2 , MeJA doubled anthocyanin accumulation compared to UV-B alone but had no synergistic effect on flavonols. MeJA selectively upregulated structural genes and transcription factors involved in the late biosynthetic pathway, transport, and the MYB-bHLH-WD40 complex, inducing anthocyanin hyperaccumulation without affecting early flavonoid biosynthetic genes or MdFLS , which controls flavonol biosynthesis. These results suggest that MeJA relates to downstream gene expression in flavonoid biosynthesis with alternative regulation of the anthocyanin pathway in UV-B-irradiated apples.

Sweet potato ( Ipomoea batatas ) serves as a significant food and economic crop worldwide. However, its production and safety are jeopardized by black rot, a disease caused by Ceratocystis fimbriata . Although polyamines (PAs) are common biological growth factors, their function in the storage of fruits and vegetables remains poorly understood. This study examines the physiological roles of both exogenous and endogenous PAs in C. fimbriata , particularly their metabolism via gene knockout techniques. Additionally, we assessed how exogenous PAs affect sweet potato storage resistance. Our findings reveal that PAs are crucial in managing oxidative and cell wall stress in C. fimbriata . At high concentrations, PAs displayed cytotoxic effects through the upregulation of nitric oxide synthase ( TAH18 ). Furthermore, exogenous PAs significantly enhanced the defense mechanisms of sweet potatoes during storage. The concurrent use of perillaldehyde (PAE), a natural antibacterial compound, additionally decreased the incidence of black rot in sweet potatoes. This study provides a novel strategy and theoretical basis for the prevention and control of fungal diseases in stored fruits and vegetables.