Characterisation of the MLP genes in peach postharvest cold storage and the regulatory role of PpMLP10 in the chilling stress response.

The major latex proteins/ripening-related proteins are a subfamily of the Bet v 1 protein superfamily and are commonly involved in plant development and responses to various stresses. However, the functions of MLPs in the postharvest cold storage of fruits remain uninvestigated. Herein, we identified 30 MLP genes in the peach (Prunus persica) genome that were clustered into three subgroups. Chromosomal location analysis revealed that the PpMLP genes were unevenly distributed on five of the eight peach chromosomes. Synteny analysis of the MLP genes between peach and seven other plant species (five dicotyledons and two monocotyledons) explored their evolutionary characteristics. Furthermore, the PpMLP promoters contained cis-elements for multiple hormones and stress responses. Gene expression analysis revealed that PpMLPs participated in chilling stress responses. Ectopic expression of PpMLP10 in Arabidopsis improved chilling stress tolerance by decreasing membrane damage and maintaining membrane stability. Additional research confirmed that PpWRKY2 participates in PpMLP10-mediated chilling stress by binding to its promoter. Collectively, these results suggest the role of PpMLP10 in enhancing chilling stress tolerance, which is significant for decreasing chilling injury during the postharvest cold storage of peaches.

Overexpression of major latex protein 423 (NtMLP423) enhances the chilling stress tolerance in Nicotiana tabacum.

Chilling stress impedes plant growth and hinders crop development and productivity. In this study, we identified the major latex protein (MLP) in tobacco (NtMLP423) and examined its roles in chilling resistance. NtMLP423 expression was considerably upregulated in response to chilling stress. NtMLP423 function was assessed and compared in plants with overexpression and antisense characteristics. Under chilling stress, plants with overexpression characteristics grew better than wild-type and antisense plants. NtMLP423 overexpression reduced membrane lipid damage, increased antioxidant enzyme activity, and reduced reactive oxygen species (ROS) accumulation under chilling stress. Here, we screened for the first time the upstream transcription factor NtMYB108, which regulates NtMLP423 expression under chilling stress. The NtMYB108 transcription factor directly binds to the NtMLP423 promoter and improves NtMLP423 resistance to chilling stress. Subjecting NtMYB018 to virus-induced gene silencing reduced chilling stress tolerance. Overall, NtMLP423 overexpression enhances chilling stress tolerance, while its suppression has the opposite effect.

CRISPR/Cas9 Gene Editing of NtAITRs, a Family of Transcription Repressor Genes, Leads to Enhanced Drought Tolerance in Tobacco.

Tobacco is a cash crop throughout the world, and its growth and development are affected by abiotic stresses including drought stress; therefore, drought-tolerant breeding may help to improve tobacco yield and quality under drought stress conditions. Considering that the plant hormone ABA (abscisic acid) is able to regulate plant responses to abiotic stresses via activating ABA response genes, the characterization of ABA response genes may enable the identification of genes that can be used for molecular breeding to improve drought tolerance in tobacco. We report here the identification of NtAITRs (Nicotiana tabacum ABA-induced transcription repressors) as a family of novel regulators of drought tolerance in tobacco. Bioinformatics analysis shows that there are a total of eight NtAITR genes in tobacco, and all the NtAITRs have a partially conserved LxLxL motif at their C-terminus. RT-PCR results show that the expression levels of at least some NtAITRs were increased in response to ABA and drought treatments, and NtAITRs, when recruited to the Gal4 promoter via a fused GD (Gal4 DNA-binding domain), were able to repress transcription activator LD-VP activated expression of the LexA-Gal4-GUS reporter gene. Roles of NtAITRs in regulating drought tolerance in tobacco were analyzed by generating CRISPR/Cas9 gene-edited mutants. A total of three Cas9-free ntaitr12356 quintuple mutants were obtained, and drought treatment assays show that drought tolerance was increased in the ntaitr12356 quintuple mutants. On the other hand, results of seed germination and seedling greening assays show that ABA sensitivity was increased in the ntaitr12356 quintuple mutants, and the expression levels of some ABA signaling key regulator genes were altered in the ntaitr12356-c3 mutant. Taken together, our results suggest that NtAITRs are ABA-responsive genes, and that NtAITRs function as transcription repressors and negatively regulate drought tolerance in tobacco, possibly by affecting plant ABA response via affecting the expression of ABA signaling key regulator genes.

De novo transcriptome assembly of transgenic tobacco (Nicotiana tabacum NC89) with early senescence characteristic.

The enzyme, α-farnesene synthase (AFS), which synthesizes α-farnesene, is the final enzyme in α-farnesene synthesis pathway. We overexpressed the α-farnesene synthase gene (previously cloned in our lab from apple peel) and ectopically expressed it in tobacco (Nicotiana tabacum NC89). Then, the transgenic plants showed an accelerated developmental process and bloomed about 7 weeks earlier than the control plants. We anticipate that de novo transcriptomic analyses of N. tabacum may provide useful information on isoprenoid biosynthesis, growth, and development. We generated 318,925,338 bp sequencing data using Illumina paired-end sequencing from the cDNA library of the apical buds of transgenic line and the wild-type line. We annotated and functionally classified the unigenes in a nucleotide and protein database. Differentially expressed unigenes may be involved in carbohydrate metabolism, nitrogen metabolism, transporter activity, hormone signal transduction, antioxidant systems and transcription regulator activity particularly related to senescence. Moreover, we analyzed eight genes related to terpenoid biosynthesis using qRT-PCR to study the changes in growth and development patterns in the transgenic plants. Our study shows that transgenic plants show premature senescence. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00953-z.

The Nicotiana tabacum L. major latex protein-like protein 423 (NtMLP423) positively regulates drought tolerance by ABA-dependent pathway.

BACKGROUND: Drought stress is an environmental factor that limits plant growth and reproduction. Little research has been conducted to investigate the MLP gene in tobacco. Here, NtMLP423 was isolated and identified, and its role in drought stress was studied. RESULTS: Overexpression of NtMLP423 improved tolerance to drought stress in tobacco, as determined by physiological analyses of water loss efficiency, reactive oxygen species levels, malondialdehyde content, and levels of osmotic regulatory substances. Overexpression of NtMLP423 in transgenic plants led to greater sensitivity to abscisic acid (ABA)-mediated seed germination and ABA-induced stomatal closure. NtMLP423 also regulated drought tolerance by increasing the levels of ABA under conditions of drought stress. Our study showed that the transcription level of ABA synthetic genes also increased. Overexpression of NtMLP423 reduced membrane damage and ROS accumulation and increased the expression of stress-related genes under drought stress. We also found that NtWRKY71 regulated the transcription of NtMLP423 to improve drought tolerance. CONCLUSIONS: Our results indicated that NtMLP423-overexpressing increased drought tolerance in tobacco via the ABA pathway.