The level of methylesterification alters the functional properties of pectin, which is believed to influence plant growth and development. However, the mechanisms that regulate demethylesterification remain largely unexplored. Pectin with a high degree of methylesterification is produced in the Golgi apparatus and then transferred to the primary cell wall where it is partially demethylesterified by pectin methylesterases (PMEs). Here, we show that in Arabidopsis (Arabidopsis thaliana) seed mucilage, pectin demethylesterification is negatively regulated by the transcription factor ZINC FINGER FAMILY PROTEIN5 (ZAT5). Plants carrying null mutations in ZAT5 had increased PME activity, decreased pectin methylesterification, and produced seeds with a thinner mucilage layer. We provide evidence that ZAT5 binds to a TGATCA-motif and thereby negatively regulates methylesterification by reducing the expression of PME5, HIGHLY METHYL ESTERIFIED SEEDS (HMS)/PME6, PME12, and PME16. We also demonstrate that ZAT5 physically interacts with BEL1-LIKE HOMEODOMAIN2 (BLH2) and BLH4 transcription factors. BLH2 and BLH4 are known to modulate pectin demethylesterification by directly regulating PME58 expression. The ZAT5–BLH2/4 interaction provides a mechanism to control the degree of pectin methylesterification in seed coat mucilage by modifying each transcription factor’s ability to regulate the expression of target genes encoding PMEs. Taken together, these findings reveal a transcriptional regulatory module comprising ZAT5, BLH2 and BLH4, that functions in modulating the de-methylesterification of homogalacturonan in seed coat mucilage.

2-acetyl-1-pyrroline (2AP) is a highly effective volatile compound that gives fragrance to numerous plant species and food. Mutation(s) in the betaine aldehyde dehydrogenase 2 (BADH2) gene results in the accumulation of 2AP. However, the function of BADH genes in tobacco (Nicotiana tabacum L.) remains poorly understood. In this study, we successfully obtained four betaine aldehyde dehydrogenase (BADH) genes from tobacco. Phylogenetic analysis of the protein sequences showed that two of the four BADH genes were closely related to the wolfberry (Lycium barbarum) BADH gene (LbBADH1), so we named them NtBADH1a and NtBADH1b, respectively. The other two BADH genes were orthologues of the tomato (Solanum lycopersicum) aminoaldehyde dehydrogenase 2 (SlAMADH2) gene, and were named NtBADH2a and NtBADH2b, respectively. Expression analysis revealed that the biological functions of NtBADH1a and NtBADH1b were different from those of genes NtBADH2a and NtBADH2b. We introduced mutations into NtBADH1a, NtBADH1b, NtBADH2a and NtBADH2b in tobacco using the CRISPR/Cas9 system and identified transgenic Ntbadh mutant tobacco lines. Single mutants (Ntbadh1a, Ntbadh1b, Ntbadh2a and Ntbadh2b) and double mutants (Ntbadh1a-Ntbadh1b and Ntbadh2a-Ntbadh2b) harbored deletion or insertion of nucleotides, both of which led to the production of a frameshift, preventing protein accumulation. A popcorn-like scent was noticeable in tobacco leaves from the Ntbadh2a-Ntbadh2b double mutant, but not from any single mutant or the Ntbadh1a-Ntbadh1b double mutant or the wild type. Consistent with this observation, we only detected 2AP in fresh leaves from the Ntbadh2a-Ntbadh2b double mutant. These findings indicate that only the combined inactivation of NtBADH2a and NtBADH2b results in 2AP accumulation in tobacco, which was not related to NtBADH1.

The content of terpenoids in tobacco can alter its resistance to TMV. NtSPS1, a pivotal structural gene in tobacco, is capable to regulate the terpenoid content. In this study, we investigated the effect of NtSPS1 knockout in HD on the content of terpenoids and the anti-TMV activity of this mutant using gene editing, widely targeted metabolomics, network pharmacology, and molecular docking. 48 terpenoids (six up-regulated and five down-regulated) in NtSPS1 knockout tobacco compared with WT leaves. Notably, solanesol was remarkable downregulation which was lowered by fourfold and compounds 1 (log₂FC = 18.2), 8 (log₂FC = 16.7) were significant upregulation between the mutants and wild-type line leaves. The 46 terpenoid’s target network encompassed 150 nodes, 509 edges and their underlying mechanisms in the therapeutic management of TMV are discussed. Furthermore, the network pharmacology and molecular docking revealed that compounds 16, 18, 23, 27, and 36 exhibited significant affinity in their respective interactions. Ultimately, five compounds were assayed for their anti-TMV effects, noteworthily, compounds 36 showed potential anti-TMV activity. Above all, we adopted a multifaceted approach to gain a comprehensive understanding of the terpenoid content and anti-TMV properties in NtSPS1 knockout HD. It enlightens the therapeutic potential of NtSPS1 knockout tobacco and it is helpful to find undescribed anti-TMV activity inhibitors, as well as searching for new anti-TMV candidates from the mutants.

Tobacco was an important cash crop and model plant, and the senescence (yellowing) of its leaves were essential markers of harvesting and secondary metabolite formation. However, the cellular senescence process and its regulatory mechanisms in tobacco leaves remain unknown. Therefore, we constructed single-cell transcriptome profiles using tobacco leaf tissues at the maturation and senescence stages to clarify the molecular mechanism of the process. We obtained 17,100 high-quality cells and sorted them into 15 clusters. Based on the marker genes of the leaf cells, we sorted the 15 clusters into 10 cell types, including palisade mesophyll, spongy mesophyll, epidermis, xylem, phloem, hydathode, companion, bundle sheath, guard, and trichome cells. Comparison of gene expression patterns in mature and senescent leaf cells revealed that senescence-related cells were mainly associated with palisade mesophyll, spongy mesophyll, epidermis, xylem, and phloem cells. Enrichment analysis indicated that the differentially expressed genes in various cells were mainly related to chlorophyll synthesis and photosynthesis. Interestingly, most genes tended to show downregulated expression during senescence, whereas genes in the sphingolipid metabolism pathway were significantly upregulated in the senescent samples. We hypothesized that genes such as SPT may regulate the senescence process of tobacco leaves through the sphingolipid metabolism pathway. To our knowledge, this is the first study to analyze single-cell transcriptomes in aging tobacco leaves. Our findings offer insights for future molecular and cellular studies on tobacco leaf senescence.