CmMYC2-CmMYBML1 module orchestrates the resistance to herbivory by synchronously regulating the trichome development and constitutive terpene biosynthesis in Chrysanthemum.

Trichomes are specialized epidermal outgrowths covering the aerial parts of most terrestrial plants. There is a large species variability in occurrence of different types of trichomes such that the molecular regulatory mechanism underlying the formation and the biological function of trichomes in most plant species remain unexplored. Here, we used Chrysanthemum morifolium as a model plant to explore the regulatory network in trichome formation and terpenoid synthesis and unravel the physical and chemical roles of trichomes in constitutive defense against herbivore feeding. By analyzing the trichome-related genes from transcriptome database of the trichomes-removed leaves and intact leaves, we identified CmMYC2 to positively regulate both development of T-shaped and glandular trichomes as well as the content of terpenoids stored in glandular trichomes. Furthermore, we found that the role of CmMYC2 in trichome formation and terpene synthesis was mediated by interaction with CmMYBML1. Our results reveal a sophisticated molecular mechanism wherein the CmMYC2-CmMYBML1 feedback inhibition loop regulates the formation of trichomes (non-glandular and glandular) and terpene biosynthesis, collectively contributing to the enhanced resistance to Spodoptera litura larvae feeding. Our findings provide new insights into the novel regulatory network by which the plant synchronously regulates trichome density for the physical and chemical defense against herbivory.

Simultaneous Impact of Rhizobacteria Inoculation and Leaf-Chewing Insect Herbivory on Essential Oil Production and VOC Emissions in Ocimum basilicum.

Inoculation with rhizobacteria and feeding by herbivores, two types of abiotic stress, have been shown to increase the production of secondary metabolites in plants as part of the defense response. This study explored the simultaneous effects of inoculation with Bacillus amyloliquefaciens GB03 (a PGPR species) and herbivory by third-instar Spodoptera frugiperda larvae on essential oil (EO) yield and volatile organic compound (VOC) emissions in Ocimum basilicum plants. The density of glandular trichomes was also examined, given that they are linked to EO production and VOC emission. Herbivory increased EO content, but inoculation on its own did not. When combined, however, the two treatments led to a 10-fold rise in EO content with respect to non-inoculated plants. VOC emissions did not significantly differ between inoculated and non-inoculated plants, but they doubled in plants chewed by the larvae with respect to their undamaged counterparts. Interestingly, no changes were observed in VOC emissions when the treatments were tested together. In short, the two biotic stressors elicited differing plant defense responses, mainly when EO was concerned. PGPR did not stimulate EO production, while herbivory significantly enhanced it and increased VOC emissions. The combined treatment acted synergistically, and in this case, PGPR inoculation may have had a priming effect that amplified plant response to herbivory. Peltate trichome density was higher in inoculated plants, those damaged by larvae, and those subjected to the combination of both treatments. The findings highlight the intricate nature of plant defense mechanisms against various stressors and hint at a potential strategy to produce essential oil through the combined application of the two stressors tested here.

QTL mapping and stability analysis of trichome density in zucchini (Cucurbita pepo L.).

Trichomes provide an excellent model for studying cell differentiation and proliferation. The aboveground tissues of plants with long dense trichomes (LDTs) can cause skin itching in people working in a zucchini field, in which management, pollination, and fruit harvesting are difficult. In this study, an F2 population was constructed with the LDT inbred line "16" and the sparse micro trichome (SMT) inbred line "63" for QTL analysis of type I and II trichome density. Two QTLs were identified on chromosomes 3 and 15 using the QTL-seq method. Additionally, 191 InDel markers were developed on 20 chromosomes, a genetic map was constructed for QTL mapping, and three QTLs were identified on chromosomes 3, 6, and 15. Two QTLs, CpTD3.1 and CpTD15.1, were identified in both QTL-seq and genetic map-based QTL analyses, and CpTD15.1 was the major-effect QTL. The stability of CpTD3.1 and CpTD15.1 was confirmed using data from F2 plants under different environmental conditions. The major-effect QTL CpTD15.1 was located between markers chr15-4991349 and chr15-5766791, with a physical distance of 775.44 kb, and explained 12.71%-29.37% of the phenotypic variation observed in the three environments. CpTD3.1 was located between markers chr3-218350 and chr3-2891236, in a region with a physical distance of 2,672.89 kb, and explained 5.00%-10.64% of the phenotypic variation observed in the three environments. The functional annotations of the genes within the CpTD15.1 region were predicted, and five genes encoding transcription factors regulating trichome development were selected. Cp4.1LG15g04400 encoded zinc finger protein (ZFP) and harbored nonsynonymous SNPs in the conserved ring finger domain between the two parental lines. There were significant differences in Cp4.1LG15g04400 expression between "16" and "63", and a similar pattern was found between germplasm resources of LDT lines and SMT lines. It was presumed that Cp4.1LG15g04400 might regulate trichome density in zucchini. These results lay a foundation for better understanding the density of multicellular nonglandular trichomes and the regulatory mechanism of trichome density in zucchini.

Metabolic engineering of a 1,8-cineole synthase enhances aphid repellence and increases trichome density in transgenic tobacco (Nicotiana tabacum L.).

BACKGROUND: The green peach aphid (Myzus persicae Sulzer) is a harmful agricultural pest that causes severe crop damage by directly feeding or indirectly vectoring viruses. 1,8-cineole synthase (CINS) is a multiproduct enzyme that synthesizes monoterpenes, with 1,8-cineole dominating the volatile organic compound profile. However, the relationship between aphid preference and CINS remains elusive. RESULTS: Here, we present evidence that SoCINS, a protein from garden sage (Salvia officinalis), enhanced aphid repellence and increased trichome density in transgenic tobacco. Our results demonstrated that overexpression of SoCINS (SoCINS-OE) led to the emission of 1,8-cineole at a level of up to 181.5 ng per g fresh leaf. Subcellular localization assay showed that SoCINS localized to chloroplasts. A Y-tube olfactometer assay and free-choice assays revealed that SoCINS-OE plants had a repellent effect on aphids, without incurring developmental or fecundity-related penalties. Intriguingly, the SoCINS-OE plants displayed an altered trichome morphology, showing increases in trichome density and in the relative proportion of glandular trichomes, as well as enlarged glandular cells. We also found that SoCINS-OE plants had significantly higher jasmonic acid (JA) levels than wild-type plants. Furthermore, application of 1,8-cineole elicited increased JA content and trichome density. CONCLUSION: Our results demonstrate that SoCINS-OE plants have a repellent effect on aphids, and suggest an apparent link between 1,8-cineole, JA and trichome density. This study presents a viable and sustainable approach for aphid management by engineering the expression of 1,8-cineole synthase gene in plants, and underscores the potential usefulness of monoterpene synthase for pest control. © 2023 Society of Chemical Industry.

Morphological characterization of intraspecific variation for trichome traits in tomato (Solanum lycopersicum).

Trichomes, the hairlike protuberances in plants, have been well known to act as the first line of defense against herbivores, and abiotic stresses, along with other structural defenses such as spines, thorns, and waxes. We previously reported the tremendous variation in trichome traits among different wild and cultivated Solanum species and demonstrated that trichomes types and density are traditionally miscalculated and often misnamed. However, intraspecific variation in trichome traits is poorly understood, although this has implications for stress tolerance and resistance breeding programs in economically important crop species and can also mediate ecological interactions at multiple trophic levels in their wild congeners. In this study, using tomato as a model, we characterized the trichomes from 10 commonly grown varieties using a minimal sample prep desktop scanning electron microscopy, and followed up with estimating their dimensions across the varieties and trichome types. We hypothesized that although trichome number may vary, the varieties will have similar trichome types, based on current literature. Our results show that there is significant variation for trichome number as well as dimensions of trichome types among these varieties. Furthermore, when we separately analyzed the number and dimensions of commonly found glandular and non-glandular trichomes, the results were consistent with broad assessment of trichomes, showing consistent variation among varieties, suggesting that trichome studies should not be limited to basic classification into glandular and non-glandular, and should accommodate the sub-types and their dimensions.

The R2R3-MYB-SG7 transcription factor CaMYB39 orchestrates surface phenylpropanoid metabolism and pathogen resistance in chickpea.

Flavonoids are important plant pigments and defense compounds; understanding the transcriptional regulation of flavonoid biosynthesis may enable engineering crops with improved nutrition and stress tolerance. Here, we characterize R2R3-MYB domain subgroup 7 transcription factor CaMYB39, which regulates flavonol biosynthesis primarily in chickpea trichomes. CaMYB39 overexpression in chickpea was accompanied by a change in flux availability for the phenylpropanoid pathway, particularly flavonol biosynthesis. Lines overexpressing CaMYB39 showed higher isoflavonoid levels, suggesting its role in regulating isoflavonoid pathway. CaMYB39 transactivates the transcription of early flavonoid biosynthetic genes (EBG). FLAVONOL SYNTHASE2, an EBG, encodes an enzyme with higher substrate specificity for dihydrokaempferol than other dihydroflavonols explaining the preferential accumulation of kaempferol derivatives as prominent flavonols in chickpea. Interestingly, CaMYB39 overexpression increased trichome density and enhanced the accumulation of diverse flavonol derivatives in trichome-rich tissues. Moreover, CaMYB39 overexpression reduced reactive oxygen species levels and induced defense gene expression which aids in partially blocking the penetration efficiency of the fungal pathogen, Ascochyta rabiei, resulting in lesser symptoms, thus establishing its role against deadly Ascochyta blight (AB) disease. Overall, our study reports an instance where R2R3-MYB-SG7 member, CaMYB39, besides regulating flavonol biosynthesis, modulates diverse pathways like general phenylpropanoid, isoflavonoid, trichome density, and defense against necrotrophic fungal infection in chickpea.

Fine Mapping of Leaf Trichome Density Revealed a 747-kb Region on Chromosome 1 in Cold-Hardy Hybrid Wine Grape Populations.

Segregation for leaf trichome density was observed in a cold-hardy hybrid grape population GE1025 (N = ∼125, MN1264 × MN1246) that was previously used to detect a quantitative trait locus (QTL) underlying foliar phylloxera resistance on chromosome 14. Our hypothesis was that high trichome density was associated with resistance to phylloxera. Existing literature found trichome density QTL on chromosomes 1 and 15 using a hybrid grape population of "Horizon" × Illinois 547-1 and suggested a few candidate genes. To validate the reported QTL and our hypothesis, interval mapping was conducted in GE1025 with previous genotyping-by-sequencing (GBS) single nucleotide polymorphism (SNP) genotype data and phenotypic scores collected using a 0-6 trichome density scale at several leaf positions. Evaluations were done on replicated forced dormant cuttings in 2 years and on field-grown leaves in 1 year. There was no strong relationship between trichome density and phylloxera resistance except for a Pearson's correlation (r) of about -0.2 between a few trichome density traits and phylloxera severity traits at 2 and 3 weeks after infestation. Two genetic regions were repeatedly detected for multiple trichome density traits: from 10 to 20.7 Mbp (∼10 Mbp) on chromosome 1 for ribbon and simple density traits and from 2.4 to 8.9 Mbp on chromosome 10 for ribbon density traits, explaining 12.1-48.2 and 12.6-27.5% of phenotypic variation, respectively. To fine map, we genotyped a larger population, GE1783 (N = ∼1,023, MN1264 × MN1246), with conserved rhAmpSeq haplotype markers across multiple Vitis species and phenotyped 233 selected potential recombinants. Evaluations were conducted on field-grown leaves in a single year. The QTL for ribbon trichome density on adaxial vein and adaxial leaf and simple density on abaxial vein was fine mapped to 12.63-13.38 Mbp (747 kb) on chromosome 1. We found variations of MN1264 and MN1246 at candidate genes NAC transcription factor 29, EF-hand protein, and MYB140 in this region and three other surrounding candidate genes proposed previously. Even though no strong relationship between foliar phylloxera resistance and trichome density was found, this study validated and fine mapped a major QTL for trichome density using a cold-hardy hybrid grape population and shed light on a few candidate genes that have implications for different breeding programs.

Constitutive and Inducible Resistance to Thrips Do Not Correlate With Differences in Trichome Density or Enzymatic-Related Defenses in Chrysanthemum.

Western flower thrips (WFT), Frankliniella occidentalis, is a serious insect pest of Chrysanthemum [Chrysanthemum × morifolium Ramat. (Asteraceae)]. Here we have investigated whether genotypic variation in constitutive and inducible resistance to WFT correlates with phenotypic differences in leaf trichome density and the activity of the defense-related enzyme polyphenol oxidase (PPO) in chrysanthemum. Non-glandular and glandular leaf trichome densities significantly varied among ninety-five chrysanthemum cultivars. Additional analyses in a subset of these cultivars, differing in leaf trichome density, revealed significant variation in PPO activities and resistance to WFT as well. Constitutive levels of trichome densities and PPO activity, however, did not correlate with chrysanthemum resistance to WFT. Further tests showed that exogenous application of the phytohormone jasmonic acid (JA) increased non-glandular trichome densities, PPO activity and chrysanthemum resistance to WFT, and that these effects were cultivar dependent. In addition, no tradeoff between constitutive and inducible resistance to WFT was observed. JA-mediated induction of WFT resistance, however, did not correlate with changes in leaf trichome densities nor PPO activity levels. Taken together, our results suggest that chrysanthemum can display both high levels of constitutive and inducible resistance to WFT, and that leaf trichome density and PPO activity may not play a relevant role in chrysanthemum defenses against WFT.

Immobilized atmospheric particulate matter on leaves of 96 urban plant species.

Plants provide many ecosystem services in urban environments, including improving ambient air quality. Leaves of plants permit the deposition of particulate matter (PM) and, depending on their leaf traits, PM may be immobilized within the epicuticular wax (EW) layer, on trichomes, on hyphae of fungi, or inside stomatal cavities. In this study, leaves of 96 perennial urban plant species consisting of 45 deciduous broadleaf/needle-like trees, 32 deciduous broadleaf shrubs, 12 evergreen needle/scale-like trees, 5 evergreen broadleaf trees, and 2 climber species were investigated in June and September 2016 to determine the effectiveness of distinct leaf surfaces in PM immobilization after leaf washing treatment. The leaf surfaces were washed vigorously using a vortex shaker. The magnetizable component of accumulated and immobilized PM on the leaf surfaces was estimated using saturation isothermal remanent magnetization (SIRM) of the unwashed and washed leaves, respectively. In June, the washed leaf SIRM of deciduous (broadleaf/needle-like) tree and shrub species (n = 77) ranged between 0.1 and 13.9 µA. In September, the washed leaf SIRM of all investigated plant species (n = 96) ranged between 1.2 and 35.0 µA. Outcomes of this study indicate that leaves of Buddleja davidii, Viburnum lantana, and Sorbus intermedia showed the highest washed leaf SIRM and thus were the most effective in immobilizing PM on their leaf surfaces while leaves of Populus alba, Robinia pseudoacacia, and Abies fraseri with lowest washed leaf SIRM were the least effective. On average, more than half (i.e., 60%) of the magnetic signal still remained after vigorous washing but a large variation exists between species (9-96%). The leaf SIRM of washed leaves of deciduous broadleaf tree and shrub species was significantly higher compared to leaves of evergreen needle/scale-like species. Evidently, the magnetic signal of unwashed leaves was higher than washed ones and higher in September than in June. Leaf traits significantly influenced the magnetic signal of both washed and unwashed leaves: leaves with a high trichome density or high leaf wettability showed a higher unwashed and washed leaf SIRM compared to leaves with no trichomes or low leaf wettability. The effect of epicuticular wax structure types on leaf SIRM was indicated to be only marginally significant. Moreover, also the immobilized fraction of PM was significantly affected by trichome density and leaf wettability, thus substantiating that plant species with high trichome density and/or leaf wettability not only accumulate more PM but are also less prone to PM re-suspension than other species. In general, the results also indicate that leaf SIRM of unwashed leaves can be a good indicator to determine the effectiveness of a plant species in PM immobilization. Plant species effective in immobilizing PM on their leaf surfaces may likely improve ambient air quality when planted in urban environments. However, it is vital that leaves of these plant species (i.e., with high PM immobilization abilities) are carefully recycled as they may be polluted.

Ontogenetic changes in the targets of natural selection in three plant defenses.

The evolution of plant defenses has traditionally been studied at single plant ontogenetic stages, overlooking the fact that natural selection acts continuously on organisms along their development, and that the adaptive value of phenotypes can change along ontogeny. We exposed 20 replicated genotypes of Turnera velutina to field conditions to evaluate whether the targets of natural selection on different defenses and their adaptative value change across plant development. We found that low chemical defense was favored in seedlings, which seems to be explained by the assimilation efficiency and the ability of the specialist herbivore to sequester cyanogenic glycosides. Whereas trichome density was unfavored in juvenile plants, it increased relative plant fitness in reproductive plants. At this stage we also found a positive correlative gradient between cyanogenic potential and sugar content in extrafloral nectar. We visualize this complex multi-trait combination as an ontogenetic defensive strategy. The inclusion of whole-plant ontogeny as a key source of variation in plant defense revealed that the targets and intensity of selection change along the development of plants, indicating that the influence of natural selection cannot be inferred without the assessment of ontogenetic strategies in the expression of multiple defenses.

Effects of Salt Stress on Plant Growth, Antioxidant Capacity, Glandular Trichome Density, and Volatile Exudates of Schizonepeta tenuifolia Briq.

Salinity is a major abiotic factor affecting plant growth and secondary metabolism. However, no information is available about its effects on Schizonepeta tenuifolia Briq., a traditional Chinese herb. Here, we investigated the changes of plant growth, antioxidant capacity, glandular trichome density, and volatile exudates of S. tenuifolia exposed to salt stress (0, 25, 50, 75, 100 mM NaCl). Results showed that its dry biomass was reduced by salt treatments except 25 mM NaCl. Contents of antioxidants, including phenolics and flavonoids, increased at low (25 mM) or moderate (50 mM) levels, but declined at severe (75 and 100 mM) levels. On leaf surfaces, big peltate and small capitate glandular trichomes (GTs) were found. Salt treatments, especially at moderate and severe concentrations, enhanced the density of total GTs on both leaf sides. The most abundant compound in GT volatile exudates was pulegone. Under salinity, relative contents of this component and other monoterpenes decreased significantly; biosynthesis and accumulation of esters were enhanced, particularly sulfurous acid,2-ethylhexyl hexyl ester, which became the second major compound as salinity increased. In conclusion, salt stress significantly influenced the growth and secondary metabolism of S. tenuifolia, enabling us to study the changes of its pharmacological activities.

Specificity of induction responses in Sinapis alba L.: Plant growth and development.

Plant defenses are expected to be negatively correlated with plant growth, development and reproduction. In a recent study, we investigated the specificity of induction responses of chemical defenses in the Brassicaceae Sinapis alba.1 It was shown that glucosinolate levels and myrosinase activities increased to different degrees after 24-hours-feeding by a specialist or generalist herbivore or mechanical wounding. Here, we present the specific influences of these treatments on organ biomasses which were recorded as a measure of growth. Directly after the treatments, organ biomasses were reduced locally and systemically by herbivore feeding, but not by mechanical wounding compared to control plants. Induction of glucosinolates, which increased in all treatments, is thus not necessarily expressed as cost in terms of reduced growth in S. alba. No significant long-term differences in plant development between herbivore treated and control plants were found. Thus, tissue loss and increased investments in chemical defenses could be compensated over time, but compensation patterns depended on the inducing agent. Furthermore, herbivore treatments resulted in an increased mechanical defense, measured as abaxial trichome densities. Plants respond highly dynamic with regard to defense and growth allocation and due to different inductors.