HD-Zip proteins modify floral structures for self-pollination in tomato.

Cleistogamy is a type of self-pollination that relies on the formation of a stigma-enclosing floral structure. We identify three homeodomain-leucine zipper IV (HD-Zip IV) genes that coordinately promote the formation of interlocking trichomes at the anther margin to unite neighboring anthers, generating a closed anther cone and cleistogamy (flower morphology necessitating strict self-pollination). These HD-Zip IV genes also control style length by regulating the transition from cell division to endoreduplication. The expression of these HD-Zip IV genes and their downstream gene, Style 2.1, was sequentially modified to shape the cleistogamy morphology during tomato evolution and domestication. Our results provide insights into the molecular basis of cleistogamy in modern tomato and suggest targets for improving fruit set and preventing pollen contamination in genetically modified crops.

Transcriptome profiling reveals key genes in regulation of the tepal trichome development in Lilium pumilum D.C.

KEY MESSAGE: A number of potential genes and pathways involved in tepal trichome development were identified in a natural lily mutant by transcriptome analysis and were confirmed with trichome and trichomeless species. Trichome is a specialized structure found on the surface of the plant with an important function in survival against abiotic and biotic stress. It is also an important economic trait in crop breeding. Extensive research has investigated the foliar trichome in model plants (Arabidopsis and tomato). However, the developmental mechanism of tepal trichome remains elusive. Lilium pumilum is an edible ornamental bulb and a good breeding parent possessing cold and salt-alkali resistance. Here, we found a natural mutant of Lilium pumilum grown on a highland whose tepals are covered by trichomes. Our data indicate that trichomes of the mutant are multicellular and branchless. Notably, stomata are also developed on the tepal of the mutant as well, suggesting there may be a correlation between trichome and stomata regulation. Furthermore, we isolated 27 differentially expressed genes (DEGs) by comparing the transcriptome profiling between the natural mutant and the wild type. These 27 genes belong to 4 groups: epidermal cell cycle and division, trichome morphogenesis, stress response, and transcription factors. Quantitative real-time PCR in Lilium pumilum (natural mutant and the wild type) and other lily species (Lilium leichtlinii var. maximowiczii/trichome; Lilium davidii var. willmottiae/, trichomeless) confirmed the validation of RNA-seq data and identified several trichome-related genes.

Transcriptome profiling of Capsicum annuum using Illumina- and PacBio SMRT-based RNA-Seq for in-depth understanding of genes involved in trichome formation.

Trichomes, specialized epidermal cells located in aerial parts of plants, play indispensable roles in resisting abiotic and biotic stresses. However, the regulatory genes essential for multicellular trichrome development in Capsicum annuum L. (pepper) remain unclear. In this study, the transcript profiles of peppers GZZY-23 (hairy) and PI246331 (hairless) were investigated to gain insights into the genes responsible for the formation of multicellular trichomes. A total of 40,079 genes, including 4743 novel genes and 13,568 differentially expressed genes (DEGs), were obtained. Functional enrichment analysis revealed that the most noticeable pathways were transcription factor activity, sequence-specific DNA binding, and plant hormone signal transduction, which might be critical for multicellular trichome formation in hairy plants. We screened 11 DEGs related to trichome development; 151 DEGs involved in plant hormone signal transduction; 312 DEGs belonging to the MYB, bHLH, HD-Zip, and zinc finger transcription factor families; and 1629 DEGs predicted as plant resistance genes (PRGs). Most of these DEGs were highly expressed in GZZY-23 or trichomes. Several homologs of trichome regulators, such as SlCycB2, SlCycB3, and H, were considerably upregulated in GZZY-23, especially in the trichomes. The transcriptomic data generated in this study provide a basis for future characterization of trichome formation in pepper.

ARMADILLO REPEAT ONLY proteins confine Rho GTPase signalling to polar growth sites.

Polar growth requires the precise tuning of Rho GTPase signalling at distinct plasma membrane domains. The activity of Rho of plant (ROP) GTPases is regulated by the opposing action of guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). Whereas plant-specific ROPGEFs have been shown to be embedded in higher-level regulatory mechanisms involving membrane-bound receptor-like kinases, the regulation of GAPs has remained enigmatic. Here, we show that three Arabidopsis ARMADILLO REPEAT ONLY (ARO) proteins are essential for the stabilization of growth sites in root hair cells and trichomes. AROs interact with ROP1 enhancer GAPs (RENGAPs) and bind to the plasma membrane via a conserved polybasic region at the ARO amino terminus. The ectopic spreading of ROP2 in aro2/3/4 mutant root hair cells and the preferential interaction of AROs with active ROPs and anionic phospholipids suggests that AROs recruit RENGAPs into complexes with ROPs to confine ROP signalling to distinct membrane regions.

Influence of auto-exhaust pollution on trichome micromorphology of Olea europea L. leaves.

This study aimed to determine the influence of auto-exhaust pollution on trichome structure of Olea europea L. leaves. For this purpose, olive leaves were collected from two areas one of which is urban and the other is rural. Leaf surface sections in leaves were obtained to examine the influence of auto-exhaust pollution on trichome structure of olive. Width-length thicknesses in peltate trichome were measured using a micrometric ocular. To investigate the density and the percentages of damaged-nondamaged peltate trichomes among the groups, the upper epidermis of leaves was photographed with SEM. According to the results, the values of width and length thicknesses of peltate trichome were lower in urban area when compared to rural site. The density and percentages of nondamaged peltate structure were lower in urban area whereas damaged peltate structures were increased. Auto-exhaust pollution decreased the amount of nondamaged peltate trichome structure and caused damaged peltate structures in olive leaves. The plant can be more sensitive to drought because of decreased amount of peltate trichomes and also increased damaged peltate trichome structures in urban area.

Histochemical assays of secretory trichomes and the structure and content of mineral nutrients in Rubus idaeus L. leaves.

Leaves of Rubus idaeus are a raw material, ingredients of herbal blend, and a source of antioxidants. There are no data concerning histochemistry of trichomes, and little is known about the leaf structure of this species. The aim of this study was to determine the histochemistry of active compounds and the structure of glandular trichomes, micromorphology, anatomy, and ultrastructure of leaves as well as content of elements. To determine the histochemistry of glandular trichomes, different chemical compounds were used. The leaf structure was analysed using light, scanning, and transmission electron microscopes. The content of elements was determined with atomic absorption spectrometry, and the microanalysis of the epidermis ultrastructure was carried out with a transmission electron microscope equipped with a digital X-ray analyser. In glandular trichomes, polyphenols, terpenes, lipids, proteins, and carbohydrates were identified. The main elements in the ultrastructure of the epidermis were Na, Mo, Se, Ca, and Mg. In dry matter of leaves, K, Mg, Ca, P, and Fe were dominant. Infusions from leaves are safe for health in terms of the Cd and Pb concentrations. Leaves can be a valuable raw material. Non-glandular trichomes prevent clumping of mixed raw materials in herbal mixtures.

Arabidopsis nucleoporin CPR5 controls trichome cell death through the core cell cycle regulator CKI.

The Arabidopsis trichome is a polyploid epidermal cell resulting from multiple rounds of endocycles. The CYCLIN-DEPENDENT KINASE INHIBITOR (CKI) family proteins are core cell cycle regulators that promote the endocycle. CONSTITUTIVE EXPRESSION OF PR GENES 5 (CPR5) is a plant-specific nucleoporin. It has been found that two Arabidopsis CKI, SIAMESE (SIM) and SIAMESE-RELATED 1 (SMR1), function downstream of CPR5 to activate plant effector-triggered cell death. The sim smr1 double mutants form multicellular and clustered trichomes, while the cpr5 mutants produce dead and branchless trichomes. This study explored roles of the CPR5-CKI signalling pathway in trichome cell cycle transition. To examine the underlying mechanism of how cell cycle transition is regulated in plant trichomes, Trypan blue staining, flow cytometry, scanning electron microscopy (SEM) and nuclear DNA measurement were conducted. The native promoter-driven CKI and GUS fusion reporter showed that both SIM and SMR1 proteins were preferentially expressed in trichomes. The cpr5-induced dead and branchless trichomes were fully suppressed by the sim smr1 double mutant, suggesting that SIM and SMR1 function downstream of CPR5 in trichome development. Flow cytometry analysis showed that as compared to the number of 2C (C = DNA content in a haploid nucleus) cells, the number of 4C cells significantly increased, whereas that of polyploidy cells (8C and 16C) dramatically decreased in the cpr5 mutant. The elevated 4C/2C ratio in the cpr5 mutant is consistent with de-repression of pro-endocycle regulators SIM and SMR1. The polyploidy cells (8C and 16C) may be selectively targeted to cell death, which is therefore attributed to the branchless trichomes in the cpr5 mutant. Nuclear DNA content analysis demonstrated that the nuclear DNA content of trichomes in the cpr5 sim mutant was significantly higher than in the sim mutant, indicating that CPR5 is a negative endocycle regulator in trichomes. This study reveals that the CPR5-CKI signalling pathway controls trichome cell cycle transition and excessive endocycles are required for cell death in plant trichomes.

Anatomy and Histochemistry of Leaf and Stem of Brazilian Endemic Species Mollinedia clavigera Tul

Abstract This study aimed to investigate the anatomy and histochemistry of Mollinedia clavigera leaves and stems through photonic microscopy and scanning electron microscopy. Noteworthy features of leaves were: presence of paracytic stomata on both surfaces; simple as well as bifurcate non-glandular trichomes; prismatic calcium oxalate crystals; flat-convex midrib with a central and two dorsal bundles; concave-convex petiole with a single vascular bundle in open archh. Stems were cylindrical and showed prismatic and styloid crystals in the pith. Histochemical analysis detected lipophilic and phenolic compounds, starch grains and lignified elements such as brachysclereids and fibers. These features may assist in future identifications and quality control of M. clavigera, avoid misidentification between other genus members, once species and genus studies are scarce.

Trichomes.

Trichomes serve diverse functions for plants, and are an intriguing developmental model.

Glandular trichomes of the leaves in three Doronicum species (Senecioneae, Asteraceae): morphology, histochemistry, and ultrastructure.

Two types of glandular tichomes (GTs) develop on the leaves in three Doronicum species. The purpose of the work was to establish common and distinctive morphological, anatomical, histochemical, and ultrustructural features of the trichomes. It turned out that differences between types of trichomes are more significant than interspecific ones. For each Doronicum species, differences between GTs of two types include the dimensions, intensity of coloration by histochemical dyes, as well as ultrastructural features of the cells. The GTs of the first type are higher than GTs of the second type. Two to three upper cell layers of the first trichomes develop histochemical staining, whereas in the second ones, only apical cells give a positive histochemical reaction. In all trichomes, polysaccharides, polyphenols, and terpenoids are detected. In the GTs of the first type, polysaccharides are synthesized in larger quantity; in the GTs of the second type, synthesis of the secondary metabolites predominates. Main ultrastructural features of the GTs of the first type include proliferation of RER and an activity of Golgi apparatus denoting the synthesis of enzymes and pectin; however, development of SER, diversiform leucoplasts with reticular sheaths, and chloroplasts with peripheral plastid reticulum also demonstrate the synthesis of lipid substances. The ultrastructural characteristics of the second type GTs indicate the primary synthesis of lipid components. Secretion is localized in a periplasmic space of the upper cell layers. The secretory products pass through the cell wall, accumulate in the subcuticular cavity, and rupture it.

Comparative anatomical properties of some Epidendroideae and Orchidoideae species distributed in NE Turkey.

In this research, anatomical, leaf micromorphological features of the samples belonging to 25 taxa (Anacamptis Rich., Cephalanthera Rich., Dactylorhiza Necker ex Nevski, Gymnadenia R.Br., Himantoglossum Spreng., Limodorum Boehm., Ophrys L., Orchis L., Platanthera Rich., Serapias L., Spiranthes Rich. and Steveniella Schltr.) spread in the Karadeniz Region have been evaluated comparatively. In anatomical studies, the transverse section from root, stem and leaf, and surface section from leaves of plants were examined. In addition, micromorphological properties of leaf were determined by electron microscopy. Morphometrical analyses were carried out using the anatomical and leaf micromophological characters of each taxa. The data matrices were obtained by examining the results of at least three samples collected from different localities. The data were evaluated using Statistical Package for the Social Sciences (SPSS) and PAleontological STatistics (PAST) statistical programs with PCA, linear discriminant analysis (LDA), and unweighted pair group method with arithmetic mean analysis. Anatomical characteristics of plants such as root epidermis cell length, cortex diameter and pith cell diameter, leaf upper epidermis length-width and bulliform cell length-width were determined to be important characteristics. It was concluded that these characters are especially important in grouping at the genus level.

Structural characterization of vegetative organs of the endangered Brazilian native species Hesperozygis ringens (Benth.) Epling.

The aim of this study was to describe the structural characterization of Hesperozygis ringens (Benth.) Epling vegetative organs. For this purpose, leaves, stems and roots of the endangered Lamiaceae were collected from a population located in Santo Antão, Santa Maria municipality, Rio Grande do Sul, Brazil. Results demonstrated that the H. ringens leaf blade presents glandular and non-glandular trichomes as well as two morphs of diallelocytic stomata, which are usually found above the epidermis level. The petiole is concave on ventral face and convex on its dorsal face, containing glandular and non-glandular trichomes as well as stomata in the epidermis. These types of trichomes were also detected in the stem. In addition, the presence of intercellular spaces within the organ is highlighted. Stomata above epidermis level also occurred in the stem. Phenolic idioblasts were found in the cortical region of plant root and deposit of lipophilic substance was observed in phloem cells. Great amount of apparently two different crystals were detected in all organs. Information obtained in this study provides knowledge about the characterizationof H. ringens, which may be used to distinguish characters of taxa and can help understand the species survival in its occurrence sites.

Proteome of olive non-glandular trichomes reveals protective protein network against (a)biotic challenge.

Olive is one of the most important fruit crop trees in the history of Mediterranean because of the high quality oil. Olive oil has a well-balanced fatty acid composition along with biophenols, which make it exceptional in human diet and provide an exceptional value to the olive oil. Leaf non-glandular peltate trichomes are specialized cell types representing a protective barrier against acute environmental conditions. To characterize the proteome of this highly differentiated cell type, we performed a comparative proteomic analysis among isolated trichomes and trichome-less leaves. Proteins were separated and identified using the 2-DE MALDI-TOF/MS method. A number of enzymes involved in abiotic and biotic stress responses are present and may be responsible for the adaptation to prolonged adverse environmental conditions. The results show that this highly differentiated cell type is physiologically active fulfilling the demands of the trichomes in furnishing the leaf with a highly protective mechanism.

Micromorphological and histochemical attributes of flowers and floral reward in Linaria vulgaris (Plantaginaceae).

The self-incompatible flowers of Linaria vulgaris have developed a range of mechanisms for attraction of insect visitors/pollinators and deterrence of ineffective pollinators and herbivores. These adaptive traits include the flower size and symmetry, the presence of a spur as a "secondary nectar presenter," olfactory (secondary metabolites) and sensual (scent, flower color, nectar guide-contrasting palate) signals, and floral rewards, i.e. pollen and nectar. Histochemical tests revealed that the floral glandular trichomes produced essential oils and flavonoids, and pollen grains contained flavonoids, terpenoids, and steroids, which play a role of olfactory attractants/repellents. The nectary gland is disc-shaped and located at the base of the ovary. Nectar is secreted through numerous modified stomata. Nectar secretion began in the bud stage and lasted to the end of anthesis. The amount of produced nectar depended on the flower age and ranged from 0.21 to 3.95 mg/flower (mean = 1.51 mg). The concentration of sugars in the nectar reached up to 57.0%. Both the nectar amount and sugar concentration demonstrated a significant year and population effect. Pollen production was variable between the years of the study. On average, a single flower of L. vulgaris produced 0.31 mg of pollen. The spectrum of insect visitors in the flowers of L. vulgaris differed significantly between populations. In the urban site, Bombus terrestris and Apis mellifera were the most common visitors, while a considerable number of visits of wasps and syrphid flies were noted in the rural site.

Analyzing trichomes and spatio-temporal expression of a cysteine protease gene Mucunain in Mucuna pruriens L. (DC).

Mucuna pruriens is a well-known legume for the itching attributes of the trichome and a valuable medicinal herb that is used for the treatment of Parkinson's disease, sexual debilities, etc. Its cultivation was deprived due to its itching behavior. The wild genotype of M. pruriens have the largest trichome length (2015 ± 29 µm) compared to other genotype and mutants. The white-seeded variety of M. pruriens was found to be the most suitable for large-scale cultivation due to the small trichome size and less trichome density on the pod. The external surface trichomes have protuberance with unknown function. The unicellular trichomes of Mucuna show the flowing fluid or cytoplasm inside the trichome. The unigenes regulating the differentiation and development of the trichome such as GLABRA-1, GLABRA-2, and cpr-5 have been identified in M. pruriens transcriptome of the leaf. The Mucunain shows a higher transcript abundance in the flower and pod cover compared to the seeds. The Mucunain was found in every stage of plant growth, but it was highly expressed during maturity (about 170 days) with a high fragment per kilobase per million value.

A part of the upstream promoter region of SHN2 gene directs trichome specific expression in Arabidopsis thaliana and heterologous plants.

A promoter trap mutant line of Arabidopsis carrying a promoterless ß-glucuronidase (uidA) gene exhibited GUS expression predominantly in all the trichomes. In this mutant, the T-DNA insertion was localized at 147bp upstream of the putative start codon, ATG, of the At5g11190 (SHN2) gene. Transcript profiling of the SHN2 suggested a constitutive expression of the gene in all the tissues. Deletion analysis of the upstream sequences established that a 565bp (-594/-30) region confers trichome-specific gene expression. The trichomes isolated from young, mature and senesced leaf tissues also showed the presence of SHN2 transcript. The occurrence of multiple TSSs on the SHN2 gene sequence, presence of the SHN2 transcript in the homozygous trip mutant, despite an insertional mutation event, and diverse reporter gene expression pattern driven by 5' and 3' promoter deletion fragments, suggest a complex transcriptional regulation of SHN2 gene in Arabidopsis. The promoter sequence -594/-30 showed a conserved functional role in conferring non-glandular trichome-specific expression in other heterologous systems like Brassica juncea and Solanum lycopersicon. Thus, in the present study T-DNA tagging has led to the identification of a trichome-specific regulatory sequence in the upstream region of a constitutively expressed SHN2 gene. The study also suggests a complex regulation of SHN2 gene. Isolated trichome specific region retains its functions in other systems like Brassica and tomato, hence could be effectively exploited in engineering trichome cells in heterologous crop plants to manipulate traits like biopharming and insect herbivory.

Complex patterns of multiple biomineralization in single-celled plant trichomes of the Loasaceae.

PREMISE OF THE STUDY: Plants of the family Loasaceae are characterized by a usually dense indument of various trichome types, including two basically different types of mineralized, unicellular trichomes (stinging hairs or setae and scabrid-glochidiate trichomes). Mineralized trichomes have long been known to have silicified or calcified walls, but recent studies demonstrated that trichomes of Loasaceae may also contain calcium phosphate. The current study investigates the distribution of different biominerals in the mineralized trichomes across several different taxa. METHODS: Plants from cultivation were studied with scanning electron microscopy including energy dispersive x-ray analyses and element mapping. KEY RESULTS: The vast majority of the 31 species investigated had at least two different biominerals in their trichomes, and 22 had three different biominerals in their trichomes. Thirty of the species had calcium phosphate in their trichomes. Loasa was mostly free of silica, but contained calcium phosphate in trichome tips and barbs, whereas calcium phosphate and silica were found in representatives of other genera of the family (Blumenbachia, Caiophora, Nasa). CONCLUSIONS: Biomineralization is remarkably diversified between species, different trichome types and parts of the same trichome. Individual genera largely had different patterns of biomineralization. The presence of three biominerals in the trichomes of the basally branching Eucnide urens indicates either an early evolution and subsequent loss or several independent origins of multiple biomineralization. Differential biomineralization of the parts of individual, unicellular trichomes clearly indicates an extraordinary degree of physiological control over this process.

Combined elevated temperature and soil waterlogging stresses inhibit cell elongation by altering osmolyte composition of the developing cotton (Gossypium hirsutum L.) fiber.

Soil waterlogging events and high temperature conditions occur frequently in the Yangtze River Valley, yet the effects of these co-occurring stresses on fiber elongation have received little attention. In the current study, the combined effect of elevated temperature (ET) and soil waterlogging (SW) more negatively affected final fiber length (reduced by 5.4%-11.3%) than either stress alone by altering the composition of osmotically active solutes (sucrose, malate, and K+), where SW had the most pronounced effect. High temperature accelerated early fiber development, but limited the duration of elongation, thereby limiting final fiber length. Treatment of ET alone altered fiber sucrose content mainly through decreased source strength and the expression of the sucrose transporter gene GhSUT-1, making sucrose availability the primary determinant of final fiber length under ET. Waterlogging stress alone decreased source strength, down-regulated GhSUT-1 expression and enhanced SuSy catalytic activity for sucrose reduction. Waterlogging treatment alone also limited fiber malate production by down-regulating GhPEPC-1 & -2. However, combined elevated temperature and waterlogging limited primary cell wall synthesis by affecting GhCESAs genes and showed a negative impact on all three major osmotic solutes through the regulation of GhSUT-1, GhPEPC-1 & -2 and GhKT-1 expression and altered SuSy activity, which functioned together to produce a shorter fiber length.

Comparative micromorphology and anatomy of flowers and floral secretory structures in two Viburnum species.

In entomogamous plants, the presence and function of floral secretory structures, whose main role is to attract pollinators, is strictly associated with the pollination ecology and hence the reproductive success of the plant. The aims of the present paper were to analyse the micromorphology and anatomy of flower nectaries and stigmas in Viburnum opulus and V. lantana and to determine the function and microstructure of inflorescence trichomes in both taxa using light and scanning electron microscopy as well as histochemical assays. It was found that stigmas were formed by papillae, which contained lipids, polysaccharides, tannins, and pigments. Stigmatic secretion proceeded via cuticular pores. Floral nectaries formed a thick layer around the styles, and nectar was secreted through numerous nectarostomata. There were no traces of vascular bundles penetrating the nectary tissue. In turn, numerous tannin deposits were observed in the cells of the glandular parenchyma. Pedicels, hypanthia, and bracts had mainly peltate and capitate glandular trichomes as well as stellate non-glandular trichomes (in V. lantana). The trichomes were shown to contain lipids, mucilage, and tannins. Many similarities in the flower and nectaries microstructure and considerable heterogeneity were observed in the examined Viburnum species. Knowledge of the microstructural characteristics of flowers, nectaries, and trichomes may be important for the phylogenesis and taxonomy of the genus Viburnum and the family Adoxaceae. Additionally, floral and nectaries features are helpful in assessment of the relatedness between taxa and provide better understanding of the floral biology and pollination ecology.

The phylogenetic significance of leaf anatomical traits of southern African Oxalis.

BACKGROUND: The southern African Oxalis radiation is extremely morphologically variable. Despite recent progress in the phylogenetics of the genus, there are few morphological synapomorphies supporting DNA-based clades. Leaflet anatomy can provide an understudied and potentially valuable source of information on the evolutionary history and systematics of this lineage. Fifty-nine leaflet anatomical traits of 109 southern African Oxalis species were assessed in search of phylogenetically significant characters that delineate clades. RESULTS: A combination of 6 leaflet anatomical traits (stomatal position, adaxial epidermal cells, abaxial epidermal cells, mesophyll, sheath around vascular tissue, degree of leaflet conduplication) clearly support various clades defined by previous DNA-based phylogenetic work. Other, mostly continuous leaflet anatomical traits were highly variable and showed less phylogenetic pattern. CONCLUSIONS: Major and unexpected findings include the transition from ancestral hypostomatic leaflets to adaxially-located stomata in the vast majority of southern African Oxalis, the loss of semi-swollen AB epidermal cells and the gain of swollen adaxial and abaxial epidermal cells in selected clades, and multiple changes from ancestral bifacial mesophyll to isobilateral or homogenous mesophyll types. The information gathered in this study will aid in the taxonomic revision of this speciose member of the Greater Cape Floristic Region and provide a basis for future hypotheses regarding its radiation.