Summary: Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT‐specific R2R3‐MYB gene, MsMYB, from comparative RNA‐Seq data of spearmint and functionally characterized it. Analysis of MsMYB‐RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB‐overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene‐ and diterpene‐derived metabolite production. In addition, we found that MsMYB binds to cis‐elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3‐MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.

Terpenes are organic compounds and play important roles in plant growth and development as well as in mediating interactions of plants with the environment. Terpene synthases (TPSs) are the key enzymes responsible for the biosynthesis of terpenes. Although some species were employed for the genome-wide identification and characterization of the TPS family, limited information is available regarding the evolution, expansion, and retention mechanisms occurring in this gene family. We performed a genome-wide identification of the TPS family members in 50 sequenced genomes. Additionally, we also characterized the TPS family from aromatic spearmint and basil plants using RNA-Seq data. No TPSs were identified in algae genomes but the remaining plant species encoded various numbers of the family members ranging from 2 to 79 full-length TPSs. Some species showed lineage-specific expansion of certain subfamilies, which might have contributed toward species or ecotype divergence or environmental adaptation. A large-scale family expansion was observed mainly in dicot and monocot plants, which was accompanied by frequent domain loss. Both tandem and segmental duplication significantly contributed toward family expansion and expression divergence and played important roles in the survival of these expanded genes. Our data provide new insight into the TPS family expansion and evolution and suggest that TPSs might have originated from isoprenyl diphosphate synthase genes.

Summary: In many aromatic plants including spearmint (Mentha spicata), the sites of secondary metabolite production are tiny specialized structures called peltate glandular trichomes (PGT). Having high commercial values, these secondary metabolites are exploited largely as flavours, fragrances and pharmaceuticals. But, knowledge about transcription factors (TFs) that regulate secondary metabolism in PGT remains elusive. Understanding the role of TFs in secondary metabolism pathway will aid in metabolic engineering for increased yield of secondary metabolites and also the development of new production techniques for valuable metabolites. Here, we isolated and functionally characterized a novel MsYABBY5 gene that is preferentially expressed in PGT of spearmint. We generated transgenic plants in which MsYABBY5 was either overexpressed or silenced using RNA interference (RNAi). Analysis of the transgenic lines showed that the reduced expression of MsYABBY5 led to increased levels of terpenes and that overexpression decreased terpene levels. Additionally, ectopic expression of MsYABBY5 in Ocimum basilicum and Nicotiana sylvestris decreased secondary metabolite production in them, suggesting that the encoded transcription factor is probably a repressor of secondary metabolism.

Increased exposure to environmental stresses due to climate change have adversely affected plant growth and productivity. Upon stress, plants activate a signaling cascade, involving multiple molecules like H₂O_2, and plant hormones such as salicylic acid (SA) leading to resistance or stress adaptation. However, the temporal ordering and composition of the resulting cascade remains largely unknown. In this study we developed a nanosensor for SA and multiplexed it with H₂O₂ nanosensor for simultaneous monitoring of stress-induced H₂O₂ and SA signals when Brassica rapa subsp. Chinensis (Pak choi) plants were subjected to distinct stress treatments, namely light, heat, pathogen stress and mechanical wounding. Nanosensors reported distinct dynamics and temporal wave characteristics of H₂O₂ and SA generation for each stress. Based on these temporal insights, we have formulated a biochemical kinetic model that suggests the early H₂O₂ waveform encodes information specific to each stress type. These results demonstrate that sensor multiplexing can reveal stress signaling mechanisms in plants, aiding in developing climate-resilient crops and pre-symptomatic stress diagnoses.

Plant glandular trichomes are chemical factories with specialized metabolic capabilities to produce diverse compounds. Aromatic mint plants produce valuable essential oil in specialised glandular trichomes known as peltate glandular trichomes (PGT). Here, we performed next generation transcriptome sequencing of different tissues of Mentha spicata (spearmint) to identify differentially expressed transcripts specific to PGT. Our results provide a comprehensive overview of PGT’s dynamic metabolic activities which will help towards pathway engineering. Spearmint RNAs from 3 different tissues: PGT, leaf and leaf stripped of PGTs (leaf-PGT) were sequenced by Illumina paired end sequencing. The sequences were assembled de novo into 40,587 non-redundant unigenes; spanning a total of 101 Mb. Functions could be assigned to 27,025 (67%) unigenes and among these 3,919 unigenes were differentially expressed in PGT relative to leaf - PGT. Lack of photosynthetic transcripts in PGT transcriptome indicated the high levels of purity of isolated PGT, as mint PGT are non-photosynthetic. A significant number of these unigenes remained unannotated or encoded hypothetical proteins. We found 16 terpene synthases (TPS), 18 cytochrome P450s, 5 lipid transfer proteins and several transcription factors that were preferentially expressed in PGT. Among the 16 TPSs, two were characterized biochemically and found to be sesquiterpene synthases. The extensive transcriptome data set renders a complete description of genes differentially expressed in spearmint PGT. This will facilitate the metabolic engineering of mint terpene pathway to increase yield and also enable the development of strategies for sustainable production of novel or altered valuable compounds in mint.

The bacterium Xanthomonas campestris pv. campestris (Xcc) causes black rot disease in cruciferous crops, resulting in severe yield loss worldwide. The excessive use of chemical pesticides in agriculture to control diseases has raised significant concern about the impact on the environment and human health. Nanoparticles have recently gained significant attention in agriculture owing to their promising application in plant disease control, increasing soil fertility and nutrient availability. In the current study, we synthesized thymol-loaded chitosan nanoparticles (TCNPs) and assessed their antibacterial activity against Xcc. The synthesis of TCNPs was confirmed by using ultraviolet–visible spectroscopy. Fourier-transform infrared spectroscopy, transmission electron microscopy, and scanning electron microscopy analysis revealed the functional groups, size, and shape of TCNPs, with sizes ranging from 54 to 250 nm, respectively. The antibacterial activity of TCNPs against Xcc was investigated in vitro by liquid broth, cell viability, and live dead staining assay, and all of them demonstrated the antibacterial activity of TCNPs. Furthermore, TCNPs were found to directly inhibit the growth of Xcc by suppressing the growth of biofilm formation and the production of exopolysaccharides and xanthomonadin. The ultrastructure studies revealed membrane damage in TCNP-treated Xcc cells, causing a release of intracellular contents. Headspace/gas chromatography (GC)–mass spectrometry (MS) analysis showed changes in the volatile profile of Xcc cells treated with TCNPs. Increased amounts of carbonyl components (mainly ketones) and production of new volatile metabolites were observed in Xcc cells incubated with TCNPs. Overall, this study reveals TCNPs as a promising antibacterial candidate against Xcc.