The promoter sequence of (-)-limonene synthase in Mentha Canadensis and its strong activity in the glandular trichome and in the stomatal guard cells

Mentha canadensis L. is an important spice crop and medicinal herb with high economic value. The plant is covered with peltate glandular trichomes, which are responsible for the biosynthesis and secretion of volatile oils. Plant non-specific lipid transfer proteins (nsLTPs) belong to a complex multigenic family involved in various plant physiological processes. Here, we cloned and identified a non-specific lipid transfer protein gene (McLTPII.9) from M. canadensis, which may positively regulate peltate glandular trichome density and monoterpene metabolism. McLTPII.9 was expressed in most M. canadensis tissues. The GUS signal driven by the McLTPII.9 promoter in transgenic Nicotiana tabacum was observed in stems, leaves, and roots; it was also expressed in trichomes. McLTPII.9 was associated with the plasma membrane. Overexpression of McLTPII.9 in peppermint (Mentha piperita. L) significantly increased the peltate glandular trichome density and total volatile compound content compared with wild-type peppermint; it also altered the volatile oil composition. In McLTPII.9-overexpressing (OE) peppermint, the expression levels of several monoterpenoid synthase genes and glandular trichome development-related transcription factors-such as limonene synthase (LS), limonene-3-hydroxylase (L3OH), geranyl diphosphate synthase (GPPS), HD-ZIP3, and MIXTA-exhibited varying degrees of alteration. McLTPII.9 overexpression resulted in both a change in expression of genes for terpenoid biosynthetic pathways which corresponded with an altered terpenoid profile in OE plants. In addition, peltate glandular trichome density was altered in the OE plants as well as the expression of genes for transcription factors that were shown to be involved in trichome development in plants.

Plant glandular trichomes (GTs) are characterised by their ability to synthesise and store abundant secondary metabolites of significant biological importance. The medicinal plant Artemisia argyi exhibits a dense covering of GTs, yet the precise metabolic compositions and the developmental molecular dynamics of A. argyi GTs remain insufficiently characterised. Using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), we identified 969 differentially accumulated metabolites (DAMs) between GTs and non-glandular trichomes (NGTs) of A. argyi. GTs were significantly enriched in diverse metabolites, including terpenoids, flavonoids and fatty acyls, among which sesquiterpenoids were the most abundant terpenoid subclass in DAMs. To elucidate the molecular basis of GT secondary metabolite biosynthesis and development, we generated a single-cell transcriptomic atlas of A. argyi leaves, annotating cell populations of mesophyll cells (MCs), epidermal cells (ECs), vascular cells (VCs), stomatal guard cells (GCs) and GTs. Pseudotime trajectory analysis uncovered the continuous developmental trajectory of GTs and identified several candidate transcription factors (TFs) potentially involved in GT development. Leveraging the single-cell atlas, we constructed cell-type-specific co-expression networks for sesquiterpene biosynthesis genes. Within the GT-specific expression module, we identified β-caryophyllene synthase AarTPS77, while the EC-specific AarTPS52 was found to function as β-farnesene synthase. Furthermore, we functionally characterised AarTPS95 and AarTPS96, which catalyse the formation of germacrene A and 12 additional sesquiterpenoids. These findings provide insights into the molecular basis of trichome development and secondary metabolite accumulation in A. argyi, laying the foundation for improving the quality of medicinal materials.

Several cis-elements including Myb-binding motifs together confer glandular trichome specificity as revealed from heterologous expression and analysis of menthol biosynthesis pathway gene promoters. Glandular Trichomes (GTs) are result of division of epidermal cells that produce diverse metabolites. Species of mint family are important for their essential oil containing many high-value terpenoids, biosynthesized and stored in these GTs. Hence, GTs constitute attractive targets for metabolic engineering and GT-specific promoters are important. In this investigation, the upstream regions of the Mentha × piperita menthol biosynthetic pathway genes (-)-limonene synthase, (-)-P450 limonene-3- hydroxylase, (-)-trans-isopiperitenol dehydrogenase, (-)-Isopiperitenone reductase, ( +)-Pulegone reductase, (-)-Menthone reductase/ (-)-Menthol dehydrogenase and a branched pathway gene ( +)-menthofuran synthase were isolated and characterized. These fragments, fused to β-glucuronidase (GUS) reporter gene of pBI101 binary vector, are able to drive high level gene expression in transgenic tobacco trichomes with strong signals in GTs, except for (-)-Isopiperitenone reductase. The GT-enriched tissue from transformed plants were analysed for GUS enzyme activity and RNA expression which correlates the GUS staining. To characterize the cis-elements responsible for GT-specific expression, a series of 5' deletion constructs for MpPLS and MpPMFS were cloned and analysed in stable transgenic tobacco lines. The specificity of trichome expression was located to - 797 to- 598bp sequence for (-)-limonene synthase and- 629 to - 530bp for ( +)-menthofuran synthase promoters containing specific Myb-binding motifs in addition to other unique motifs described for developmental regulation without any defined pattern. All other pathway promoters also recruits specific but different Myb factors as indicated by this analysis.

Stomatal movements are associated with pH changes in guard cells. Several authors have demonstrated that cytosolic alkalinization preceded the rise in ROS or Ca2+of guard cells. In contrast, a few reports suggest that the increase in cytosolic pH follows the elevated ROS or Ca2+, suggesting that cytosolic pH rise may not always be an early event. The components, such as ROS, Ca2+, and Ca2+-dependent protein kinases, converge to modulate ion channels, promote ion efflux from guard cells and promote stomatal closure. We propose a hypothetical model to integrate the event of pH rise with other signalling components and explain the argument that cytosolic alkalization can occur downstream or upstream of ROS or Ca2+-rise. Changes in guard cell pH can occur when ATPases are modulated. Stomatal closure and guard cell pH rise are compromised in mutants deficient in vacuolar H+-ATPase (V-ATPase), pointing out the role of V-ATPase. My talk attempts to consider arguments for and against the significance of cytosolic alkalinization in guard cells. Stomatal guard cells are promising model systems for further research into this intriguing topic of cytosolic pH change during stomatal closure.

The p-menthane monoterpenes of the Mentha species are biosynthesized from geranyl pyrophosphate via the monocyclic olefin 4S-limonene. A monoterpene cyclase was isolated from both Mentha x piperita (peppermint) and Mentha spicata (spearmint) that catalyzes the cyclization of geranyl pyrophosphate to 4S-limonene. This enzyme, 4S-limonene synthase, was purified to apparent homogeneity by dye ligand, anion exchange, and hydrophobic interaction chromatography. Since the monoterpenes of Mentha are synthesized and secreted in modified epidermal hairs called glandular trichomes, an extract of isolated glandular trichome cells was used as the source of this enzyme. A combination of gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that purified 4S-limonene synthase had a native molecular weight of 56,000 and was monomeric. The principal product of the enzyme was enantiomerically pure (-)-4S-limonene, and a catalytic constant of 0.3/s was determined. The basic properties of 4S-limonene synthase from both M. x piperita and M. spicata are identical and, in general, are similar to those of other monoterpene, sesquiterpene, and diterpene cyclases isolated from microorganisms and higher plants.

Stomatal guard cells are encased in strong, flexible cell walls that underpin their dynamic responses to signaling cues, but how these walls are assembled with these properties is incompletely understood. Here, we explored how the properties of guard cell walls change during stomatal maturation in Arabidopsis thaliana and result in stomatal complexes that open and close efficiently. We established milestones for stomatal maturation, when the stomatal pore and guard cells enlarge with distinct kinetics, and found that although guard cell walls thicken during maturation, they become more mechanically anisotropic and achieve stomatal opening with smaller changes in turgor pressure and less energy input. We also found that cellulose is required for normal stomatal maturation and that both cellulose and pectins are critical for mechanical anisotropy and efficient stomatal opening in mature guard cells. Based on their molecular architectures, we developed a multi-scale model that recapitulates the biomechanics of wild type and mutant guard cell walls. Finally, we used cell ablation and automated cell segmentation in both Arabidopsis and grass stomatal complexes to find that cells adjacent to guard cells constrain their motions in unexpected ways. Together, these data functionally connect the molecular composition and structure of guard cell walls to their biomechanical properties and the efficiency of stomatal dynamics.

Limitation of water loss and control of gas exchange is accomplished in plant leaves via stomatal guard cells. Stomata open in response to light when an increase in guard cell turgor is triggered by ions and water influx across the plasma membrane. Recent evidence demonstrating the existence of ATP-binding cassette proteins in plants led us to analyze the effect of compounds known for their ability to modulate ATP-sensitive potassium channels (K-ATP) in animal cells. By using epidermal strip bioassays and whole-cell patch-clamp experiments with Vicia faba guard cell protoplasts, we describe a pharmacological profile that is specific for the outward K+ channel and very similar to the one described for ATP-sensitive potassium channels in mammalian cells. Tolbutamide and glibenclamide induced stomatal opening in bioassays and in patch-clamp experiments, a specific inhibition of the outward K+ channel by these compounds was observed. Conversely, application of potassium channel openers such as cromakalim or RP49356 triggered stomatal closure. An apparent competition between sulfonylureas and potassium channel openers occurred in bioassays, and outward potassium currents, previously inhibited by glibenclamide, were partially recovered after application of cromakalim. By using an expressed sequence tag clone from an Arabidopsis thaliana homologue of the sulfonylurea receptor, a 7-kb transcript was detected by Northern blot analysis in guard cells and other tissues. Beside the molecular evidence recently obtained for the expression of ATP-binding cassette protein transcripts in plants, these results give pharmacological support to the presence of a sulfonylurea-receptor-like protein in the guard-cell plasma membrane tightly involved in the outward potassium channel regulation during stomatal movements.

Manganese-induced changes in glandular trichomes density and essential oils production of Mentha aquatica L. at different growth stages

Glandular trichomes are specialized anatomical structures that accumulate secretions with important biological roles in plant-environment interactions. These secretions also have commercial uses in the flavor, fragrance, and pharmaceutical industries. The capitate-stalked glandular trichomes of Cannabis sativa (cannabis), situated on the surfaces of the bracts of the female flowers, are the primary site for the biosynthesis and storage of resins rich in cannabinoids and terpenoids. In this study, we profiled nine commercial cannabis strains with purportedly different attributes, such as taste, color, smell, and genetic origin. Glandular trichomes were isolated from each of these strains, and cell type-specific transcriptome data sets were acquired. Cannabinoids and terpenoids were quantified in flower buds. Statistical analyses indicated that these data sets enable the high-resolution differentiation of strains by providing complementary information. Integrative analyses revealed a coexpression network of genes involved in the biosynthesis of both cannabinoids and terpenoids from imported precursors. Terpene synthase genes involved in the biosynthesis of the major monoterpenes and sesquiterpenes routinely assayed by cannabis testing laboratories were identified and functionally evaluated. In addition to cloning variants of previously characterized genes, specifically CsTPS14CT [(-)-limonene synthase] and CsTPS15CT (β-myrcene synthase), we functionally evaluated genes that encode enzymes with activities not previously described in cannabis, namely CsTPS18VF and CsTPS19BL (nerolidol/linalool synthases), CsTPS16CC (germacrene B synthase), and CsTPS20CT (hedycaryol synthase). This study lays the groundwork for developing a better understanding of the complex chemistry and biochemistry underlying resin accumulation across commercial cannabis strains.

We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicals, and (b) H2O2. Both methyl viologen and H2O2 inhibited stomatal opening and promoted stomatal closure. At concentrations [less than or equal to]10-5 M, the effects of methyl viologen and H2O2 on stomatal behavior were reversible and were abolished by 2 mM EGTA or 10 [mu]M verapamil. In addition, at 10-5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by EGTA or verapamil, methyl viologen and H2O2 caused an increase in guard cell cytosolic free Ca2+ ([Ca2+]i), which was abolished in the presence of EGTA. Therefore, at low concentrations of methyl viologen and H2O2, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatal aperture and the associated increases in [Ca2+]i. This suggests that in this concentration range the effects of the treatments are Ca2+-dependent and are mediated by changes in [Ca2+]i. In contrast, at concentrations of methyl viologan and H2O2 > 10-5 M, EGTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H2O2 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+]i and the possible disruption of guard-cell Ca2+ homeostasis are discussed in relation to the processes of Ca2+-based signal transduction in stomatal guard cells and the control of stomatal aperture.

Stomata are vital for controlling gas exchange and water vapor release, which significantly affect photosynthesis and transpiration. Characterizing stomatal traits such as size, density, and distribution is essential for adaptation to the environment. While microscopy is widely used for this purpose, manual analysis is labor-intensive and time-consuming that limit large scale studies. To overcome this, we introduce an automated, high-throughput method that leverages YOLOv8, an advanced deep learning model, for more accurate and efficient stomatal trait measurement. Our approach provides a comprehensive analysis of stomatal morphology by examining both stomatal pores and guard cells. A key finding is the introduction of stomatal angles as a novel phenotyping trait, which can offer deeper insights into stomatal function. We developed a model using a carefully annotated dataset that accurately segments and analyzes stomatal guard cells from high-resolution images. Additionally, our study introduces a new opening ratio metric, calculated from the areas of the guard cells and the stomatal pore, providing a valuable morphological descriptor for future physiological research. This scalable system significantly enhances the precision and efficiency of large-scale plant phenotyping, offering a new tool to advance research in plant physiology.

Study on the Relationship Between the Ploidy Level of Microspore-Derived Plants and the Number of Chloroplast in Stomatal Guard Cells in Brassica oleracea

Within the plant kingdom the stomatal guard cell is presented as a model system of inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3]‐mediated signal transduction. Despite this it is only recently that the phosphoinositide components of animal signal transduction pathways have been identified in stomatal guard cells. Interestingly, stomatal guard cells contain both 3‐ and 4‐phosphorylated phosphatidylinositols though their relative contributions to signalling remain undefined. An appraisal of the routes of synthesis and rates of turnover of these phosphatidylinositols would appear timely as the in vivo biosynthesis of these components is a much neglected facet of the phosphoinositide‐mediated signalling paradigm as purported to apply to plants.A non‐equilibrium [32P]Pi labelling strategy and enzymic and chemical dissection of labelled phosphatidylinositols have been used to address not only the route of synthesis but also the rates of turnover of phosphatidylinositols in stomatal guard cells of Commelina communis L.The specific activity of the ATP pool of isolated guard cells was found to increase over a 4 h period when labelled from [32P]Pi. In separate experiments, isolated guard cells were labelled over a 40–240 min period, their lipids extracted, deacylated and resolved by HPLC. Glycerophosphoinositol phosphate (GroPInsP) and glycerophosphoinositol bisphosphate (GroPInsP2) peaks were desalted and enzymically cleaved with alkaline phosphatase and human erythrocyte ghosts, respectively. The monoester phosphate in phosphatidylinositol 4‐monophosphate (PtdIns4P) accounted for 90–97% of the [32P]Pi label while the 4‐ and 5‐monoester phosphates of phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] accounted for typically 39% and 61% respectively. Therefore, the evidence is consistent with synthesis of PtdIns(4,5)P2 by successive 4‐ and 5‐phosphorylation of phosphatidylinositol (PtdIns). This study therefore represents the first report of the pathway of the synthesis of 4‐ and 5‐phosphorylated phosphatidylinositols in a single defined hormone‐responsive plant cell type.The monoester phosphate in phosphatidylinositol 3‐monophosphate (PtdIns3P) accounted for 83–95% of the 32P label. It was not possible, however, to determine the route of synthesis of phosphatidylinositol 3,4‐bisphosphate [PtdIns(3,4)P2] owing to the rapid attainment of equilibrium between the 3‐ and 4‐monoester phosphates of PtdIns(3,4)P2, each containing approximately 50% of the label at just 40 min of labelling. Turnover of PtdIns3P was quicker than that of PtdIns4P. Similarly, turnover of PtdIns(3,4)P2 was quicker than that of PtdIns(4,5)P2, and in mass terms PtdIns(3,4)P2 appeared to predominate over PtdIns(4,5)P2. By analogy with animal systems, in which signalling molecules such as PtdIns(4,5)P2 show considerable basal turnover, the evidence presented is consistent with signalling roles for PtdIns3P and PtdIns(3,4)P2 in addition to those previously indicated for PtdIns(4,5)P2 in stomatal guard cells.

CDeT6-19 is an ABA-regulated gene which has been isolated from Craterostigma plantagineum. The CDeT6-19 gene promoter has been fused to the beta-glucuronidase reporter gene (GUS) and used to stably transform Arabidopsis thaliana and Nicotiana tabacum. This construct has been shown to be expressed in stomatal guard cells and often in the adjacent epidermal cells of both species in response to both exogenous ABA and drought stress. These results indicate that the stomatal guard cell is competent to relay an ABA signal to the nucleus. In contrast GUS expression directed by the promoter from a predominantly seed-specific, ABA-regulated gene, Em, or the promoter from the ABA-regulated CDeT27-45 gene is not detectable in the epidermal or guard cells of tobacco or Arabidopsis in response to ABA. The fact that not all ABA-regulated gene promoters are active in stomatal guard cells suggests that effective transduction of the signal is dependent upon particular regions within the gene promoter or that guard cells lack all or part of the specific transduction apparatus required to couple the ABA signal to these promoters. This suggests that there are multiple ABA stimulus response coupling pathways. The identification of a regulatory sequence from an ABA-induced gene which is expressed in stomatal guard cells creates the possibility of examining the role of Ca2+ and other second messengers in ABA-induced gene expression.

Guard cell walls combine exceptional strength and flexibility in order to accommodate the turgor pressure-driven changes in size and shape that underlie the opening and closing of stomatal pores. To investigate the molecular basis of these exceptional qualities, we have used a combination of compositional and functional analyses in three different plant species. We show that comparisons of FTIR spectra from stomatal guard cells and those of other epidermal cells indicate a number of clear differences in cell-wall composition. The most obvious characteristics are that stomatal guard cells are enriched in phenolic esters of pectins. This enrichment is apparent in guard cells from Vicia faba (possessing a type I cell wall) and Commelina communis and Zea mays (having a type II wall). We further show that these common defining elements of guard cell walls have conserved functional roles. As previously reported in C. communis, we show that enzymatic modification of the pectin network in guard cell walls in both V. faba and Z. mays has profound effects on stomatal function. In all three species, incubation of epidermal strips with a combination of pectin methyl esterase and endopolygalacturonase (EPG) caused an increase in stomatal aperture on opening. This effect was not seen when strips were incubated with EPG alone indicating that the methyl-esterified fraction of homogalacturonan is key to this effect. In contrast, arabinanase treatment, and incubation with feruloyl esterase both impeded stomatal opening. It therefore appears that pectins and phenolic esters have a conserved functional role in guard cell walls even in grass species with type II walls, which characteristically are composed of low levels of pectins.