Zenglin Zhang

Pathogenesis-related 1 (PR1) proteins are important components of plant defense and stress responses and also serve as precursors of CAP-derived peptides (CAPE), a class of small bioactive peptides involved in immune and stress signaling. Despite their potential biological significance, CAPE-producing PR1 genes have not been systematically characterized in tobacco (Nicotiana tabacum). In this study, a genome-wide analysis identified 17 CAPE-producing PR1 genes, designated NtCAPE1 to NtCAPE17, in the tobacco genome. These genes encode proteins containing conserved CAP domains and N-terminal signal peptides, with predicted hydrophilic properties and mainly vacuolar localization, indicating conserved structural features within the family. Phylogenetic analysis, gene structure organization, conserved motif profiling, chromosomal distribution, and synteny analyses revealed both evolutionary conservation and duplication-driven diversification of the NtCAPE family. Promoter cis-element analysis showed enrichment of regulatory elements associated with phytohormone signaling, development, and stress responses. Public transcriptomic datasets revealed dynamic and gene-specific expression patterns under water-deficit and salinity stress, and qRT-PCR analysis further confirmed the stress-responsive expression of selected NtCAPE genes. Functional assays using synthetic mature peptides showed that NtCAPE9 and NtCAPE17 alleviated salinity stress- and osmotic stress-induced leaf yellowing, improved chlorophyll retention, suppressed senescence-associated responses, reduced H₂O₂ accumulation and POD activity, modulated stress-responsive gene expression, and promoted seed germination under salinity and osmotic stress, respectively. These results provide a comprehensive characterization of CAPE-producing PR1 genes in tobacco and identify NtCAPE9 and NtCAPE17 as candidate stress-associated peptides with exogenous activity under salinity and osmotic stress conditions.

Introduction: Aspartic proteases (APs) play crucial roles in plant growth, stress responses, and protein metabolism. However, endogenous APs in Nicotiana benthamiana , a key plant molecular farming platform, pose a significant challenge by degrading valuable recombinant proteins, thereby limiting production yields. Methods: A comprehensive understanding of the AP gene family in N. benthamiana is essential for developing strategies to mitigate this degradation. In this study, multiple bioinformatics approaches were employed to identify the NbAPs gene family in the Nicotiana benthamiana genome. The analysis encompassed the protein characteristics, phylogenetic relationships, gene structures, conserved motifs, gene duplication events, and chromosomal distribution of family members. Additionally, the role of NbAPs members in recombinant protein expression was investigated. Results: We performed a genome-wide analysis and identified 89 Aspartic Protease (NbAP) genes in N. benthamiana . Phylogenetic analysis classified these genes into three subfamilies-typical, nucellin-like, and atypical-revealing both evolutionary conservation and diversification. The NbAP members exhibited diverse gene structures, conserved motifs, and subcellular localizations, with significant enrichment predicted for the vacuole and chloroplasts. Segmental duplication was identified as the primary mechanism of NbAP family expansion. Promoter analysis revealed the presence of cis-elements associated with stress responses, hormone regulation, and developmental processes. Transcriptome and RT-qPCR analyses showed dynamic expression patterns of NbAPs following Agrobacterium infiltration, with genes such as NbAP46 , NbAP47 , or NbAP79 showing sustained upregulation. Functional validation using Virus-Induced Gene Silencing (VIGS) demonstrated that silencing NbAP46 , NbAP47 , or NbAP79 significantly enhanced the accumulation of transiently expressed GFP protein. Discussion: This study provides the first comprehensive genomic identification and characterization of the AP family in N. benthamiana . We highlight the diversity of NbAPs and their potential biological roles, and experimentally validate that specific members negatively influence recombinant protein stability. Silencing these proteases leads to enhanced accumulation of foreign proteins. Overall, our findings establish a foundational resource and identify potential targets for genetic engineering to further optimize N. benthamiana as a superior bioreactor for plant molecular farming.

Plant Molecular Farming (PMF) capitalizes on the unique properties of plants as bioreactors to efficiently produce valuable proteins, pharmaceuticals, and enzymes. This review emphasizes the critical role of transient expression systems, particularly in Nicotiana benthamiana, due to its susceptibility to various pathogens. Viral vector-based transient expression has proven essential during health emergencies like COVID-19, enabling rapid recombinant protein production. The review also evaluates different transient expression platforms and highlights their applications in biopharmaceutical production, education, synthetic biology, and gene editing. Advances in viral vector modification, hydroponics, and Controlled Environment Agriculture (CEA) are presented as transformative innovations enhancing scalability and regulatory compliance. Furthermore, glycoengineering advancements broaden the range of producible biopharmaceuticals, improving global medication access. By exploring these advancements, this review underscores the vast potential of transient expression systems to meet dynamic scientific and market demands, positioning PMF as a vital component in modern biotechnology.

As the final stage of leaf development, leaf senescence is affected by a variety of internal and external signals including age and environmental stresses. Although significant progress has been made in elucidating the mechanisms of age-dependent leaf senescence, it is not clear how stress conditions induce a similar process. Here, we report the roles of a stress-responsive and senescence-induced gene, ERD7 (EARLY RESPONSIVE TO DEHYDRATION 7), in regulating both age-dependent and stress-induced leaf senescence in Arabidopsis. The results showed that the leaves of erd7 mutant exhibited a significant delay in both age-dependent and stress-induced senescence, while transgenic plants overexpressing the gene exhibited an obvious accelerated leaf senescence. Furthermore, based on the results of LC-MS/MS and PRM quantitative analyses, we selected two phosphorylation sites, Thr-225 and Ser-262, which have a higher abundance during senescence, and demonstrated that they play a key role in the function of ERD7 in regulating senescence. Transgenic plants overexpressing the phospho-mimetic mutant of the activation segment residues ERD7^T225D and ERD7^T262D exhibited a significantly early senescence, while the inactivation segment ERD7^T225A and ERD7^T262A displayed a delayed senescence. Moreover, we found that ERD7 regulates ROS accumulation by enhancing the expression of AtrbohD and AtrbohF, which is dependent on the critical residues, i.e., Thr-225 and Ser-262. Our findings suggest that ERD7 is a positive regulator of senescence, which might function as a crosstalk hub between age-dependent and stress-induced leaf senescence.

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

Senescence is the final stage of leaf development which is accompanied by highly coordinated and complicated reprogramming of gene expression. Genetic manipulation of leaf senescence in major crops including wheat has been shown to be able to increase stress tolerance and grain yield. NAC(No apical meristem (NAM), ATAF1/2, and cup-shaped cotyledon (CUC)) transcription factors (TFs) play important roles in regulating gene expression changes during leaf senescence and in response to abiotic stresses. Here, we report the characterization of TaSNAC11-4B (Uniprot: A0A1D5XI64), a wheat NAC family member that acts as a functional homolog of AtNAP, a key regulator of leaf senescence in Arabidopsis. The expression of TaSNAC11-4B was up-regulated with the progression of leaf senescence, in response to abscisic acid (ABA) and drought treatments in wheat. Ectopic expression of TaSNAC11-4B in Arabidopsis promoted ROS accumulation and significantly accelerated age-dependent as well as drought- and ABA-induced leaf senescence. Results from transcriptional activity assays indicated that the TaSNAC11-4B protein displayed transcriptional activation activities that are dependent on its C terminus. Furthermore, qRT-PCR and dual-Luciferase assay results suggested that TaSNAC11-4B could positively regulate the expression of AtrbohD and AtrbohF, which encode catalytic subunits of the ROS-producing NADPH oxidase. Further analysis of TaSNAC11-4B in wheat senescence and the potential application of this gene in manipulating leaf senescence with the purpose of yield increase and stress tolerance is discussed.

As the last stage of plant development, leaf senescence has a great impact on plant’s life cycle. Genetic manipulation of leaf senescence has been used as an efficient approach in improving the yield and quality of crop plants. Here we describe an ethyl methane sulfonate (EMS) mutagenesis induced premature leaf senescence mutant yellow leaf 1 (yl1) in common tobacco (Nicotiana tabacum L.). The yl1 plants displayed early leaf yellowing. Physiological parameters and marker genes expression indicated that the yl1 phenotype was caused by premature leaf senescence. Genetic analyses indicated that the yl1 phenotype was controlled by a single recessive gene that was subsequently mapped to a specific interval of tobacco linkage group 11 using simple sequence repeat (SSR) markers. Exogenous plant hormone treatments of leaves showed that the yl1 mutant was more sensitive to ethylene and jasmonic acid than the wild type. No similar tobacco premature leaf senescence mutants have been reported. This study laid a foundation for finding the gene controlling the mutation phenotype and revealing the molecular regulation mechanism of tobacco leaf senescence in the next stage.