Artemisia annua ZFP8L regulates glandular trichome development.

Trichomes are known to be important biofactories that contribute to the production of secondary metabolites, such as terpenoids. C2H2-zinc finger proteins (C2H2-ZFPs) are vital transcription factors of plants' trichome development. However, little is known about the function of Artemisia annua C2H2-ZFPs in trichome development. To explore the roles of this gene family in trichome development, two C2H2-ZFP transcription factors, named AaZFP8L and AaGIS3, were identified; both are hormonally regulated in A. annua. Overexpression of AaZFP8L in tobacco led to a significant increase in the density and length of glandular trichomes, and improved terpenoid content. In contrast, AaGIS3 was found to positively regulate non-glandular trichome initiation and elongation, which reduces terpenoid accumulation. In addition, ABA contents significantly increased in AaZFP8L-overexpressing tobacco lines and AaZFP8L also can directly bind the promoter of the ABA biosynthesis genes. This study lays the foundation for further investigating A. annua C2H2-ZFPs in trichome development and terpenoid accumulation.

The impact of mechanical stress on anatomy, morphology, and gene expression in Urtica dioica L.

MAIN CONCLUSION: Mechanical stress induces distinct anatomical, molecular, and morphological changes in Urtica dioica, affecting trichome development, gene expression, and leaf morphology under controlled conditions The experiments were performed on common nettle, a widely known plant characterized by high variability of leaf morphology and responsiveness to mechanical touch. A specially constructed experimental device was used to study the impact of mechanical stress on Urtica dioica plants under strictly controlled parameters of the mechanical stimulus (touching) and environment in the growth chamber. The general anatomical structure of the plants that were touched was similar to that of control plants, but the shape of the internodes' cross section was different. Stress-treated plants showed a distinct four-ribbed structure. However, as the internodes progressed, the shape gradually approached a rectangular form. The epidermis of control plants included stinging, glandular and simple setulose trichomes, but plants that were touched had no stinging trichomes, and setulose trichomes accumulated more callose. Cell wall lignification occurred in the older internodes of the control plants compared to stress-treated ones. Gene analysis revealed upregulation of the expression of the UdTCH1 gene in touched plants compared to control plants. Conversely, the expression of UdERF4 and UdTCH4 was downregulated in stressed plants. These data indicate that the nettle's response to mechanical stress reaches the level of regulatory networks of gene expression. Image analysis revealed reduced leaf area, increased asymmetry and altered contours in touched leaves, especially in advanced growth stages, compared to control plants. Our results indicate that mechanical stress triggers various anatomical, molecular, and morphological changes in nettle; however, further interdisciplinary research is needed to better understand the underlying physiological mechanisms.

Defects in Glabrous 3 (GL3) functionality underlie the absence of trichomes in Brassica napus.

Previously, expression of the Arabidopsis thaliana GLABRA3 (GL3) induced trichome formation in Brassica napus. GL3 orthologues were examined from glabrous (B. oleracea), semi-glabrous (B. napus), moderately hirsute (B. rapa), and very hirsute (B. villosa) Brassica species. Ectopic expression of BnGL3, BrGL3 alleles, or BvGL3 induced trichome formation in glabrous B. napus with the effect on trichome number commensurate with density in the original accessions. Chimeric GL3 proteins in which the B. napus amino terminal region, which interacts with MYB proteins, or the middle region, which interacts with the WD40 protein TTG1, was exchanged with corresponding regions from A. thaliana were as stimulatory to trichome production as AtGL3. Exchange of the carboxy-terminal region containing a bHLH domain and an ACT domain did not alter the trichome stimulatory activity, although modeling of the ACT domain identified differences that could affect GL3 dimerization. B. napus A- and C-genomes orthologues differed in their abilities to form homo- and heterodimers. Modeling of the amino-terminal region revealed a conserved domain that may represent the MYB factor binding pocket. This region interacted with the MYB factors GL1, CPC, and TRY, as well as with JAZ8, which is involved in jasmonic acid-mediated regulation of MYC-like transcription factors. Protein interaction studies indicated that GL1 interaction with GL3 from B. napus and A. thaliana may underlie the difference in their respective abilities to induce trichome formation.

A cotton endoreduplication gene, GaTOP6B, regulates trichome branching development.

Trichomes are specialized epidermal structures that protect plants from biotic and abiotic stresses by synthesizing, storing, and secreting defensive compounds. This study investigates the role of the Gossypium arboreum DNA topoisomerase VI subunit B gene (GaTOP6B) in trichome development and branching. Sequence alignment revealed a high similarity between GaTOP6B and AtTOP6B, suggesting a conserved function in trichome regulation. Although AtTOP6B acts as a positive regulator of trichome development, functional analyses showed contrasting effects: Virus-induced gene silencing (VIGS) of GaTOP6B in cotton increased trichome density, while its overexpression in Arabidopsis decreased trichome density but enhanced branching. This demonstrates that GaTOP6B negatively regulates trichome number, indicating species-specific roles in trichome initiation and branching between cotton and Arabidopsis. Overexpression of the GaTOP6B promotes jasmonic acid synthesis, which in turn inhibits the G1/S or G2/M transitions, stalling the cell cycle. On the other hand, it suppresses brassinolide synthesis and signaling while promoting cytokinin degradation, further inhibiting mitosis. These hormonal interactions facilitate the transition of cells from the mitotic cycle to the endoreduplication cycle. As the level of endoreduplication increases, trichomes develop an increased number of branches. These findings highlight GaTOP6B's critical role as a regulator of trichome development, providing new genetic targets for improving cotton varieties in terms of enhanced adaptability and resilience.

Mutations in RABE1C suppress the spirrig mutant phenotype.

The plant BEACH-domain protein SPIRRIG (SPI) is involved in regulating cell morphogenesis and salt stress responses in Arabidopsis thaliana, Arabis alpina, and Marchantia polymorpha and was reported to function in the context of two unrelated cellular processes: vesicular trafficking and P-body mediated RNA metabolism. To further explore the molecular function of SPI, we isolated a second-site mutant, specifically rescuing the spi mutant trichome phenotype. The molecular analysis of the corresponding gene revealed a dominant negative mutation in RABE1C, a ras-related small GTP-binding protein that localizes to Golgi. Taken together, our data identified the genetic interaction between RABE1C and SPI, which is beneficial for further dissecting the function of SPI in vesicle trafficking-associated cell morphogenesis.

Trichomes and unique gene expression confer insect herbivory resistance in Vitis labrusca grapevines.

BACKGROUND: Grapevine (Vitis) is one of the world's most valuable fruit crops, but insect herbivory can decrease yields. Understanding insect herbivory resistance is critical to mitigating these losses. Vitis labrusca, a wild North American grapevine species, has been leveraged in breeding programs to generate hybrid grapevines with enhanced abiotic and biotic stress resistance, rendering it a valuable genetic resource for sustainable viticulture. This study assessed the resistance of V. labrusca acc. 'GREM4' and Vitis vinifera cv. 'PN40024' grapevines to Popillia japonica (Japanese beetle) herbivory and identified morphological and genetic adaptations underlying this putative resistance. RESULTS: 'GREM4' displayed greater resistance to beetle herbivory compared to 'PN40024' in both choice and no-choice herbivory assays spanning periods of 30 min to 19 h. 'GREM4' had significantly higher average leaf trichome densities than 'PN40024' and beetles preferred to feed on the side of leaves with fewer trichomes. When leaves from each species that specifically did not differ in trichome densities were fed on by beetles, significantly less leaf area was damaged in 'GREM4' (3.29mm2) compared to 'PN40024' (9.80mm2), suggesting additional factors beyond trichomes contributed to insect herbivory resistance in 'GREM4'. Comparative transcriptomic analyses revealed 'GREM4' exhibited greater constitutive (0 h) expression of defense response and secondary metabolite biosynthesis genes compared to 'PN40024', indicative of heightened constitutive defenses. Upon herbivory, 'GREM4' displayed a greater number of differentially expressed genes (690) compared to 'PN40024' (502), suggesting a broader response. Genes up-regulated in 'GREM4' were enriched in terpene biosynthesis, flavonoid biosynthesis, phytohormone signaling, and disease defense-related functions, likely contributing to heighted insect herbivory defense, while genes differentially expressed in 'PN40024' under herbivory were enriched in xyloglucan, cell wall formation, and calcium ion binding. The majority of genes implicated in insect herbivory defense were orthologs with specific expression patterns in 'GREM4' and 'PN40024', but some paralogous and genome-specific genes also likely contributed to conferring resistance. CONCLUSIONS: Our findings suggest that 'GREM4' insect herbivory resistance was attributed to a combination of factors, including trichomes and unique constitutive and inducible expression of genes implicated in terpene, flavonoid, and phenylpropanoid biosynthesis, as well as pathogen defense.

Integrated multi-omics analysis reveals genes involved in flavonoid biosynthesis and trichome development of Artemisia argyi.

Artemisia argyi is an herbaceous plant of the genus Artemisia. Its young and mature leaves are used as food and medicine, respectively. Glandular trichomes (GTs) are distributed on the leaf surface in A. argyi and are generally considered the location of flavonoid biosynthesis and accumulation. However, the mechanism of flavonoid biosynthesis and accumulation in A. argyi remains unclear. In this study, the coregulatory genes involved in flavonoid biosynthesis and trichome development in this species were screened and evaluated, and the biosynthetic pathways for key flavonoids in A. argyi were uncovered. AaMYB1 and AaYABBY1 were screened using weighted gene co-expression network analysis, and both genes were then genetically transformed into Nicotiana tabacum L. cv. K326 (tobacco). Simultaneously, AaYABBY1 was also genetically transformed into Arabidopsis thaliana. The total flavonoid and rutin contents were increased in tobacco plants overexpressing AaMYB1 and AaYABBY1, and the expression levels of genes participating in the flavonoid synthesis pathway, such as PAL, FLS, and F3H, were significantly up-regulated in plants overexpressing these genes. These results indicated that AaMYB1 and AaYABBY1 promote flavonoid biosynthesis in tobacco. Furthermore, compared to that in the wild-type, the trichome density was significantly increased in tobacco and A. thaliana plants overexpressing AaYABBY1. These results confirm that AaYABBY1 might be involved in regulating trichome formation in A. argyi. This indicates the potential genes involved in and provides new insights into the development of trichome cellular factories based on the "development-metabolism" interaction network and the cultivation of high-quality A. argyi.

Comparative transcriptome analysis reveals genes involved in trichome development and metabolism in tobacco.

BACKGROUND: The glandular trichomes of tobacco (Nicotiana tabacum) can efficiently produce secondary metabolites. They act as natural bioreactors, and their natural products function to protect plants against insect-pests and pathogens and are also components of industrial chemicals. To clarify the molecular mechanisms of tobacco glandular trichome development and secondary metabolic regulation, glandular trichomes and glandless trichomes, as well as other different developmental tissues, were used for RNA sequencing and analysis. RESULTS: By comparing glandless and glandular trichomes with other tissues, we obtained differentially expressed genes. They were obviously enriched in KEGG pathways, such as cutin, suberine, and wax biosynthesis, flavonoid and isoflavonoid biosynthesis, terpenoid biosynthesis, and plant-pathogen interaction. In particular, the expression levels of genes related to the terpenoid, flavonoid, and wax biosynthesis pathway mainly showed down-regulation in glandless trichomes, implying that they lack the capability to synthesize certain exudate compounds. Among the differentially expressed genes, 234 transcription factors were found, including AP2-ERFs, MYBs, bHLHs, WRKYs, Homeoboxes (HD-ZIP), and C2H2-ZFs. These transcription factor and genes that highly expressed in trichomes or specially expressed in GT or GLT. Following the overexpression of R2R3-MYB transcription factor Nitab4.5_0011760g0030.1 in tobacco, an increase in the number of branched glandular trichomes was observed. CONCLUSIONS: Our data provide comprehensive gene expression information at the transcriptional level and an understanding of the regulatory pathways involved in glandular trichome development and secondary metabolism.

Trading acyls and swapping sugars: metabolic innovations in Solanum trichomes.

Solanaceae (nightshade family) species synthesize a remarkable array of clade- and tissue-specific specialized metabolites. Protective acylsugars, one such class of structurally diverse metabolites, are produced by ACYLSUGAR ACYLTRANSFERASE (ASAT) enzymes from sugars and acyl-coenzyme A esters. Published research has revealed trichome acylsugars composed of glucose and sucrose cores in species across the family. In addition, acylsugars have been analyzed across a small fraction of the >1,200 species in the phenotypically megadiverse Solanum genus, with a handful containing inositol and glycosylated inositol cores. The current study sampled several dozen species across subclades of Solanum to get a more detailed view of acylsugar chemodiversity. In depth characterization of acylsugars from the clade II species brinjal eggplant (Solanum melongena) led to the identification of eight unusual structures with inositol or inositol glycoside cores and hydroxyacyl chains. Liquid chromatography-mass spectrometry analysis of 31 additional species in the Solanum genus revealed striking acylsugar diversity, with some traits restricted to specific clades and species. Acylinositols and inositol-based acyldisaccharides were detected throughout much of the genus. In contrast, acylglucoses and acylsucroses were more restricted in distribution. Analysis of tissue-specific transcriptomes and interspecific acylsugar acetylation differences led to the identification of the brinjal eggplant ASAT 3-LIKE 1 (SmASAT3-L1; SMEL4.1_12g015780) enzyme. This enzyme is distinct from previously characterized acylsugar acetyltransferases, which are in the ASAT4 clade, and appears to be a functionally divergent ASAT3. This study provides a foundation for investigating the evolution and function of diverse Solanum acylsugar structures and harnessing this diversity in breeding and synthetic biology.

Characterization and expression analysis of GLABRA3 (GL3) genes in cotton: insights into trichome development and hormonal regulation.

BACKGROUND: GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) genes encode a typical helix-loop-helix (bHLH) transcription factors that primarily regulate trichome branching and root hair development, DNA endoreduplication, trichoblast size, and stomatal formation. The functions of GL3 genes in cotton crop have been poorly characterized. In this study, we performed comprehensive genome-wide scans for GL3 and EGL3 homologs to enhance our comprehension of their potential roles in trichome and fiber development in cotton crop. METHODS AND RESULTS: Our findings paraded that Gossypium hirsutum and G. barbadense have 6 GL3s each, unevenly distributed on 4 chromosomes whereas, G. arboreum, and G. raimondii have 3 GL3s each, unevenly distributed on 2 chromosomes. Gh_A08G2088 and Gb_A09G2187, despite having the same bHLH domain as the other GL3 genes, were excluded due to remarkable short sequences and limited number of motifs, indicating a lack of potential functional activity. The phylogenetic analysis categorized remaining 16 GL3s into three subfamilies (Group I-III) closely related to A. thaliana. The 16 GL3s have complete bHLH domain, encompassing 590-631 amino acids, with molecular weights (MWs) ranging from 65.92 to 71.36 kDa. Within each subfamily GL3s depicted shared similar gene structures and motifs, indicating conserved characteristics within respective groups. Promoter region analysis revealed 27 cis-acting elements, these elements were responsive to salicylic acid, abscisic acid (ABA), methyl jasmonate (MeJA), and gibberellin. The expression of GL3 genes was analyzed across 12 tissues in both G. barbadense and G. hirsutum using the publicly available RNA-seq data. Among GL3s, Gb_D11G0219, Gb_D11G0214, and Gb_D08G2182, were identified as relatively highly expressed across different tissues, consequently selected for hormone treatment and expression validation in G. barbadense. RT-qPCR results demonstrated significant alterations in the expression levels of Gb_D11G0219 and Gb_D11G0214 following MeJA, GA, and ABA treatment. Subcellular localization prediction revealed that most GL3 proteins were predominantly expressed in the nucleus, while a few were localized in the cytoplasm and chloroplasts. CONCLUSIONS: In summary, this study lays the foundation for subsequent functional validation of GL3 genes by identifying hormonal regulation patterns and probable sites of action in cotton trichome formation and fiber development. The results stipulate a rationale to elucidate the roles and regulatory mechanisms of GL3 genes in the intricate process of cotton fibre and trichome development.

A large presence/absence variation in the promotor of the ClLOG gene determines trichome elongation in watermelon.

KEY MESSAGE: The ClLOG gene encoding a cytokinin riboside 5'-monophosphate phosphoribohydrolase determines trichome length in watermelon, which is associated with its promoter variations. Trichomes, which are differentiated from epidermal cells, are special accessory structures that cover the above-ground organs of plants and possibly contribute to biotic and abiotic stress resistance. Here, a bulked segregant analysis (BSA) of an F2 population with significant variations in trichome length was undertaken. A 1.84-Mb candidate region on chromosome 10 was associated with trichome length. Resequencing and fine-mapping analyses indicated that a 12-kb structural variation in the promoter of Cla97C10G203450 (ClLOG) led to a significant expression difference in this gene in watermelon lines with different trichome lengths. In addition, a virus-induced gene silencing analysis confirmed that ClLOG positively regulated trichome elongation. These findings provide new information and identify a potential target gene for controlling multicellular trichome elongation in watermelon.

Genetic interaction between TTG2 and AtPLC1 reveals a role for phosphoinositide signaling in a co-regulated suite of Arabidopsis epidermal pathways.

The TTG2 transcription factor of Arabidopsis regulates a set of epidermal traits, including the differentiation of leaf trichomes, flavonoid pigment production in cells of the inner testa (or seed coat) layer and mucilage production in specialized cells of the outer testa layer. Despite the fact that TTG2 has been known for over twenty years as an important regulator of multiple developmental pathways, little has been discovered about the downstream mechanisms by which TTG2 co-regulates these epidermal features. In this study, we present evidence of phosphoinositide lipid signaling as a mechanism for the regulation of TTG2-dependent epidermal pathways. Overexpression of the AtPLC1 gene rescues the trichome and seed coat phenotypes of the ttg2-1 mutant plant. Moreover, in the case of seed coat color rescue, AtPLC1 overexpression restored expression of the TTG2 flavonoid pathway target genes, TT12 and TT13/AHA10. Consistent with these observations, a dominant AtPLC1 T-DNA insertion allele (plc1-1D) promotes trichome development in both wild-type and ttg2-3 plants. Also, AtPLC1 promoter:GUS analysis shows expression in trichomes and this expression appears dependent on TTG2. Taken together, the discovery of a genetic interaction between TTG2 and AtPLC1 suggests a role for phosphoinositide signaling in the regulation of trichome development, flavonoid pigment biosynthesis and the differentiation of mucilage-producing cells of the seed coat. This finding provides new avenues for future research at the intersection of the TTG2-dependent developmental pathways and the numerous molecular and cellular phenomena influenced by phospholipid signaling.

Tomato root specialized metabolites evolved through gene duplication and regulatory divergence within a biosynthetic gene cluster.

Tremendous plant metabolic diversity arises from phylogenetically restricted specialized metabolic pathways. Specialized metabolites are synthesized in dedicated cells or tissues, with pathway genes sometimes colocalizing in biosynthetic gene clusters (BGCs). However, the mechanisms by which spatial expression patterns arise and the role of BGCs in pathway evolution remain underappreciated. In this study, we investigated the mechanisms driving acylsugar evolution in the Solanaceae. Previously thought to be restricted to glandular trichomes, acylsugars were recently found in cultivated tomato roots. We demonstrated that acylsugars in cultivated tomato roots and trichomes have different sugar cores, identified root-enriched paralogs of trichome acylsugar pathway genes, and characterized a key paralog required for root acylsugar biosynthesis, SlASAT1-LIKE (SlASAT1-L), which is nested within a previously reported trichome acylsugar BGC. Last, we provided evidence that ASAT1-L arose through duplication of its paralog, ASAT1, and was trichome-expressed before acquiring root-specific expression in the Solanum genus. Our results illuminate the genomic context and molecular mechanisms underpinning metabolic diversity in plants.

Identification of a cis-element for long glandular trichome-specific gene expression, which is targeted by a HD-ZIP IV protein.

Glandular trichomes are epidermal outgrowths that secret a variety of secondary metabolites, which not only help plants adapt to environmental stresses but also have important commercial value in fragrances, pharmaceuticals, and pesticides. In Nicotiana tabacum, it has been confirmed that a B-type cyclin, CycB2, negatively regulates the formation of long glandular trichomes (LGTs). This study aimed to identify the upstream regulatory gene involved in LGT formation by screening LGT-specific cis-elements within the NtCycB2 promoter. Using GUS as a reporter gene, the tissue-driven ability of NtCycB2 promoter showed that NtCycB2 promoter could drive GUS expression specifically in LGTs. Function analysis of a series of successive 5' truncations and synthetic segments of the NtCycB2 promoter indicated that the 87-bp region from -1221 to -1134 of the NtCycB2 promoter was required for gene expression in LGTs, and the L1-element (5'-AAAATTAATAAGAG-3') located in the 87-bp region contributed to the gene expression in the stalk of LGTs. Further Y1H and LUC assays confirmed that this L1-element exclusively binds to a HD-Zip IV protein, NtHD13. Gene function analysis revealed that NtHD13 positively controlled LGT formation, as overexpression of NtHD13 resulted in a high number of LGTs, whereas knockout of NtHD13 led to a decrease in LGTs. These findings demonstrate that NtHD13 can bind to an L1-element within the NtCycB2 promoter to regulate LGT formation.

A gradient of the HD-Zip regulator Woolly regulates multicellular trichome morphogenesis in tomato.

Homeodomain (HD) proteins regulate embryogenesis in animals such as the fruit fly (Drosophila melanogaster), often in a concentration-dependent manner. HD-leucine zipper (Zip) IV family genes are unique to plants and often function in the L1 epidermal cell layer. However, our understanding of the roles of HD-Zip IV family genes in plant morphogenesis is limited. In this study, we investigated the morphogenesis of tomato (Solanum lycopersicum) multicellular trichomes, a type of micro-organ in plants. We found that a gradient of the HD-Zip IV regulator Woolly (Wo) coordinates spatially polarized cell division and cell expansion in multicellular trichomes. Moreover, we identified a TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR (TCP) transcription factor-encoding gene, SlBRANCHED2a (SlBRC2a), as a key downstream target of Wo that regulates the transition from cell division to cell expansion. High levels of Wo promote cell division in apical trichome cells, whereas in basal trichome cells, Wo mediates a negative feedback loop with SlBRC2a that forces basal cells to enter endoreduplication. The restricted high and low activities of Wo pattern the morphogenesis of tomato multicellular trichomes. These findings provide insights into the functions of HD-Zip IV genes during plant morphogenesis.

Chemical and transcriptomic analyses of leaf trichomes from Cistus creticus subsp. creticus reveal the biosynthetic pathways of certain labdane-type diterpenoids and their acetylated forms.

Labdane-related diterpenoids (LRDs), a subgroup of terpenoids, exhibit structural diversity and significant commercial and pharmacological potential. LRDs share the characteristic decalin-labdanic core structure that derives from the cycloisomerization of geranylgeranyl diphosphate (GGPP). Labdanes derive their name from the oleoresin known as 'Labdanum', 'Ladano', or 'Aladano', used since ancient Greek times. Acetylated labdanes, rarely identified in plants, are associated with enhanced biological activities. Chemical analysis of Cistus creticus subsp. creticus revealed labda-7,13(E)-dien-15-yl acetate and labda-7,13(E)-dien-15-ol as major constituents. In addition, novel labdanes such as cis-abienol, neoabienol, ent-copalol, and one as yet unidentified labdane-type diterpenoid were detected for the first time. These compounds exhibit developmental regulation, with higher accumulation observed in young leaves. Using RNA-sequencing (RNA-seq) analysis of young leaf trichomes, it was possible to identify, clone, and eventually functionally characterize labdane-type diterpenoid synthase (diTPS) genes, encoding proteins responsible for the production of labda-7,13(E)-dien-15-yl diphosphate (endo-7,13-CPP), labda-7,13(E)-dien-15-yl acetate, and labda-13(E)-ene-8α-ol-15-yl acetate. Moreover, the reconstitution of labda-7,13(E)-dien-15-yl acetate and labda-13(E)-ene-8α-ol-15-yl acetate production in yeast is presented. Finally, the accumulation of LRDs in different plant tissues showed a correlation with the expression profiles of the corresponding genes.

Woolly mutation with the Get02 locus overcomes the polygenic nature of trichome-based pest resistance in tomato.

Type-IV glandular trichomes, which only occur in the juvenile developmental phase of the cultivated tomato (Solanum lycopersicum), produce acylsugars that broadly protect against arthropod herbivory. Previously, we introgressed the capacity to retain type-IV trichomes in the adult phase from the wild tomato, Solanum galapagense, into the cultivated species cv. Micro-Tom (MT). The resulting MT-Galapagos enhanced trichome (MT-Get) introgression line contained 5 loci associated with enhancing the density of type-IV trichomes in adult plants. We genetically dissected MT-Get and obtained a subline containing only the locus on Chromosome 2 (MT-Get02). This genotype displayed about half the density of type-IV trichomes compared to the wild progenitor. However, when we stacked the gain-of-function allele of WOOLLY, which encodes a homeodomain leucine zipper IV transcription factor, Get02/Wo exhibited double the number of type-IV trichomes compared to S. galapagense. This discovery corroborates previous reports positioning WOOLLY as a master regulator of trichome development. Acylsugar levels in Get02/Wo were comparable to the wild progenitor, although the composition of acylsugar types differed, especially regarding fewer types with medium-length acyl chains. Agronomical parameters of Get02/Wo, including yield, were comparable to MT. Pest resistance assays showed enhanced protection against silverleaf whitefly (Bemisia tabaci), tobacco hornworm (Manduca sexta), and the fungus Septoria lycopersici. However, resistance levels did not reach those of the wild progenitor, suggesting the specificity of acylsugar types in the pest resistance mechanism. Our findings in trichome-mediated resistance advance the development of robust, naturally resistant tomato varieties, harnessing the potential of natural genetic variation. Moreover, by manipulating only 2 loci, we achieved exceptional results for a highly complex, polygenic trait, such as herbivory resistance in tomato.

Mapping and Detection of Genes Related to Trichome Development in Black Gram (Vigna mungo (L.) Hepper).

Black gram (Vigna mungo (L.) Hepper) is a pulses crop with good digestible protein and a high carbohydrate content, so it is widely consumed as human food and animal feed. Trichomes are large, specialized epidermal cells that confer advantages on plants under biotic and abiotic stresses. Genes regulating the development of trichomes are well characterized in Arabidopsis and tomato. However, little is known about trichome development in black gram. In this study, a high-density map with 5734 bin markers using an F2 population derived from a trichome-bearing and a glabrous cultivar of black gram was constructed, and a major quantitative trait locus (QTL) related to trichomes was identified. Six candidate genes were located in the mapped interval region. Fourteen single-nucleotide polymorphisms (SNPs) or insertion/deletions (indels) were associated with those genes. One indel was located in the coding region of the gene designated as Scaffold_9372_HRSCAF_11447.164. Real-time quantitative PCR (qPCR) analysis demonstrated that only one candidate gene, Scaffold_9372_HRSCAF_11447.166, was differentially expressed in the stem between the two parental lines. These two candidate genes encoded the RNA polymerase-associated protein Rtf1 and Bromodomain adjacent to zinc finger domain protein 1A (BAZ1A). These results provide insights into the regulation of trichome development in black gram. The candidate genes may be useful for creating transgenic plants with improved stress resistance and for developing molecular markers for trichome selection in black gram breeding programs.

TRANSPARENT TESTA GLABRA2 defines trichome cell shape by modulating actin cytoskeleton in Arabidopsis thaliana.

The Arabidopsis (Arabidopsis thaliana) TRANSPARENT TESTA GLABRA2 (TTG2) gene encodes a WRKY transcription factor that regulates a range of development events like trichome, seed coat, and atrichoblast formation. Loss-of-function of TTG2 was previously shown to reduce or eliminate trichome specification and branching. Here, we report the identification of an allele of TTG2, ttg2-6. In contrast to the ttg2 mutants described before, ttg2-6 displayed unique trichome phenotypes. Some ttg2-6 mutant trichomes were hyper-branched, whereas others were hypo-branched, distorted, or clustered. Further, we found that in addition to specifically activating R3 MYB transcription factor TRIPTYCHON (TRY) to modulate trichome specification, TTG2 also integrated cytoskeletal signaling to regulate trichome morphogenesis. The ttg2-6 trichomes displayed aberrant cortical microtubules (cMTs) and actin filaments (F-actin) configurations. Moreover, genetic and biochemical analyses showed that TTG2 could directly bind to the promoter and regulate the expression of BRICK1 (BRK1), which encodes a subunit of the actin nucleation promoting complex suppressor of cyclic AMP repressor (SCAR)/Wiskott-Aldrich syndrome protein family verprolin homologous protein (WAVE). Collectively, taking advantage of ttg2-6, we uncovered a function for TTG2 in facilitating cMTs and F-actin cytoskeleton-dependent trichome development, providing insight into cellular signaling events downstream of the core transcriptional regulation during trichome development in Arabidopsis.

The GaKAN2, a KANADI transcription factor, modulates stem trichomes in Gossypium arboreum.

KEY MESSAGE: GaKAN2, a member of the KANADI family, was found to be widely expressed in the cotton tissues and regulates trichome development through complex pathways. Cotton trichomes are believed to be the defense barrier against insect pests. Cotton fiber and trichomes are single-cell epidermal extensions with shared regulatory mechanisms. Despite several studies underlying mechanism of trichome development remains elusive. The KANADI is one of the key transcription factors (TFs) family, regulating Arabidopsis trichomes growth. However, the function of KANADI genes in cotton remains unknown. In the current study genome-wide scanning, transcriptomic analysis, gene silencing, subcellular localization, and yeast two-hybrid techniques were employed to decipher the function of KANADI TFs family genes in cotton crop. A total of 7 GaKAN genes were found in the Gossypium arboreum. Transcriptomic data revealed that these genes were significantly expressed in stem and root. Moreover, GaKAN2 was widely expressed in other tissues also. Subsequently, we selected GaKAN2 to validate the function of KANADI genes. Silencing of GaKAN2 resulted in a 24.99% decrease in single-cell trichomes and an 11.33% reduction in internodal distance, indicating its potential role in regulating trichomes and plant growth. RNA-Seq analysis elucidated that GaSuS and GaERS were the downstream genes of GaKAN2. The transcriptional activation and similarity in silencing phenotype between GaKAN2 and GaERS suggested that GaKAN2 regulates trichomes development through GaERS. Moreover, KEGG analysis revealed that a significant number of genes were enriched in the biosynthesis of secondary metabolites and plant hormone signal transduction pathways, thereby suggesting that GaKAN2 regulates the stem trichomes and plant growth. The GFP subcellular localization and yeast transcriptional activation analysis elucidated that GaKAN2 was located in the nucleus and capable of regulating the transcription of downstream genes. This study elucidated the function and characteristics of the KANADI gene family in cotton, providing a fundamental basis for further research on GaKAN2 gene in cotton plant trichomes and plant developmental processes.