Valorization of CBD-hemp through distillation to provide essential oil and improved cannabinoids profile.

Hemp (Cannabis sativa L.) synthesizes and accumulates a number of secondary metabolites such as terpenes and cannabinoids. They are mostly deposited as resin into the glandular trichomes occurring on the leaves and, to a major extent, on the flower bracts. In the last few years, hemp for production of high-value chemicals became a major commodity in the U.S. and across the world. The hypothesis was that hemp biomass valorization can be achieved through distillation and procurement of two high-value products: the essential oil (EO) and cannabinoids. Furthermore, the secondary hypothesis was that the distillation process will decarboxylate cannabinoids hence improving cannabinoid composition of extracted hemp biomass. Therefore, this study elucidated the effect of steam distillation on changes in the content and compositional profile of cannabinoids in the extracted biomass. Certified organic CBD-hemp strains (chemovars, varieties) Red Bordeaux, Cherry Wine and Umpqua (flowers and some upper leaves) and a T&H strain that included chopped whole-plant biomass, were subjected to steam distillation, and the EO and cannabinoids profile were analyzed by gas chromatography-mass spectrometry (GC-MS) and HPLC, respectively. The distillation of hemp resulted in apparent decarboxylation and conversion of cannabinoids in the distilled biomass. The study demonstrated a simple method for valorization of CBD-hemp through the production of two high-value chemicals, i.e. EO and cannabinoids with improved profile through the conversion of cannabidiolic acid (CBD-A) into cannabidiol (CBD), cannabichromenic acid (CBC-A) into cannabichromene (CBC), cannabidivarinic acid (CBDV-A) into cannabidivarin (CBDV), cannabigerolic acid (CBG-A) into cannabigerol (CBG), and δ-9-tetrahydrocannabinolic acid (THC-A) into δ-9-tetrahydrocannabinol (THC). In addition, the distilled biomass contained CBN while the non-distilled did not. Distillation improved the cannabinoids profile; e.g. the distilled hemp biomass had 3.4 times higher CBD in variety Red Bordeaux, 5.6 times in Cherry Wine, 9 times in variety Umpqua, and 6 times in T&H compared to the original non-distilled samples, respectively. Most of the cannabinoids remained in the distilled biomass and small amounts of CBD were transferred to the EO. The CBD concentration in the EO was as follows: 5.3% in the EO of Umpqua, 0.15% in the EO of Cherry Wine and Red Bordeaux and 0.06% in the EO of T&H. The main 3 EO constituents were similar but in different ratio; myrcene (23.2%), (E)-caryophyllene (16.7%) and selina-3,7(11)-diene (9.6%) in Cherry Wine; (E)-caryophyllene (~ 20%), myrcene (16.6%), selina-3,7(11)-diene (9.6%), α-humulene (8.0%) in Red Bordeaux; (E)-caryophyllene (18.2%) guaiol (7.0%), 10-epi-γ-eudesmol (6.9%) in Umpqua; and (E)-caryophyllene (30.5%), α-humulene (9.1%), and (E)-α-bisabolene (6.5%) in T&H. In addition, distillation reduced total THC in the distilled biomass. Scanning electron microscopy (SEM) analyses revealed that most of the glandular trichomes in the distilled biomass were not disturbed (remained intact); that suggest a possibility for terpenes evaporation through the epidermal membrane covering the glandular trichomes leaving the cannabinoids in the trichomes. This explained the fact that distillation resulted in terpene extraction while the cannabinoids remained in the distilled material.

Comparative Transcriptome Analysis Reveals the Potential Mechanism of Abortion in Tobacco sua-Cytoplasmic Male Sterility.

sua-CMS (cytoplasmic male sterility) is the only male sterile system in tobacco breeding, but the mechanism of abortion is unclear. Cytological characteristics show that abortion in the sua-CMS line msZY occurs before the differentiation of sporogenous cells. In this study, a comparative transcriptomic analysis was conducted on flower buds at the abortion stage of msZY and its male fertile control ZY. A total of 462 differentially expressed genes were identified in msZY and ZY, which were enriched via protein processing in the endoplasmic reticulum (ER), oxidative phosphorylation, photosynthesis, and circadian rhythm-plant by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Most genes were downregulated in the ER stress pathway, heat-shock protein family, F1F0-ATPase encoding by the mitochondrial genome, and differentiation of stamens. Genes in the programmed cell death (PCD) pathway were upregulated in msZY. The transcriptome results were consistent with those of qRT-PCR. Ultrastructural and physiological analyses indicted active vacuole PCD and low ATP content in msZY young flower buds. We speculated that PCD and a deficiency in ATP synthesis are essential for the abortion of sua-CMS. This study reveals the potential mechanism of abortion of tobacco sua-CMS.

Taxonomic traits in the microstructure of flowers of parasitic Orobanche picridis with particular emphasis on secretory structures.

Orobanche picridis is an obligate root parasite devoid of chlorophyll in aboveground organs, which infects various Picris species. Given the high level of phenotypic variability of the species, the considerable limitation of the number of taxonomically relevant traits (mainly in terms of generative elements), and the low morphological variation between species, Orobanche is regarded as one of the taxonomically most problematic genera. This study aimed to analyse the taxonomic traits of O. picridis flowers with the use of stereoscopic and bright-field microscopy as well as fluorescence, scanning, and transmission electron microscopy. The micromorphology of sepals, petals, stamens, and pistils was described. For the first time, the anatomy of parasitic Orobanche nectaries and the ultrastructure of nectaries and glandular trichomes were presented. Special attention was paid to the distribution and types of glandular and non-glandular trichomes as well as the types of metabolites contained in these structures. It was demonstrated that the nectary gland was located at the base of the gynoecium and nectar was secreted through modified nectarostomata. The secretory parenchyma cells contained nuclei, large amyloplasts with starch granules, mitochondria, and high content of endoplasmic reticulum profiles. Nectar was transported via symplastic and apoplastic routes. The results of histochemical assays and fluorescence tests revealed the presence of four groups of metabolites, i.e. polyphenols (tannins, flavonoids), lipids (acidic and neutral lipids, essential oil, sesquiterpenes, steroids), polysaccharides (acidic and neutral polysaccharides), and alkaloids, in the trichomes located on perianth elements and stamens.

Floral micromorphology of the bird-pollinated carnivorous plant species Utricularia menziesii R.Br. (Lentibulariaceae).

Background and Aims: Bird pollination is rare among species in the genus Utricularia, and has evolved independently in two lineages of this genus. In Western Australia, the Western Spinebill, Acanthorhynchus superciliosus, visits flowers of Utricularia menziesii (section Pleiochasia: subgenus Polypompholyx). This study aimed to examine the micromorphology of U. menziesii flowers to assess traits that might be linked to its pollination strategy. Methods: Light microscopy, histochemistry and scanning electron microscopy were used. Nectar sugar composition was analysed using high-performance liquid chromatography. Key Results: The flowers of U. menziesii fulfil many criteria that characterize bird-pollinated flowers: red colour, a large, tough nectary spur that can withstand contact with a hard beak, lack of visual nectar guides and fragrance. Trichomes at the palate and throat may act as tactile signals. Spur nectary trichomes did not form clearly visible patches, but were more frequently distributed along vascular bundles, and were small and sessile. Each trichome comprised a single basal cell, a unicellular short pedestal cell (barrier cell) and a multicelled head. These trichomes were much smaller than those of the U. vulgaris allies. Hexose-dominated nectar was detected in flower spurs. Fructose and glucose were present in equal quantities (43 ± 3.6 and 42 ± 3.6 g L-1). Sucrose was only detected in one sample, essentially at the limit of detection for the method used. This type of nectar is common in flowers pollinated by passerine perching birds. Conclusions: The architecture of nectary trichomes in U. menziesii was similar to that of capitate trichomes of insect-pollinated species in this genus; thus, the most important specializations to bird pollination were flower colour (red), and both spur shape and size modification. Bird pollination is probably a recent innovation in the genus Utricularia, subgenus Polypompholyx, and is likely to have evolved from bee-pollinated ancestors.

Exon junction complex (EJC) core genes play multiple developmental roles in Physalis floridana.

KEY MESSAGE: Molecular and functional characterization of four gene families of the Physalis exon junction complex (EJC) core improved our understanding of the evolution and function of EJC core genes in plants. The exon junction complex (EJC) plays significant roles in posttranscriptional regulation of genes in eukaryotes. However, its developmental roles in plants are poorly known. We characterized four EJC core genes from Physalis floridana that were named PFMAGO, PFY14, PFeIF4AIII and PFBTZ. They shared a similar phylogenetic topology and were expressed in all examined organs. PFMAGO, PFY14 and PFeIF4AIII were localized in both the nucleus and cytoplasm while PFBTZ was mainly localized in the cytoplasm. No protein homodimerization was observed, but they could form heterodimers excluding the PFY14-PFBTZ heterodimerization. Virus-induced gene silencing (VIGS) of PFMAGO or PFY14 aborted pollen development and resulted in low plant survival due to a leaf-blight-like phenotype in the shoot apex. Carpel functionality was also impaired in the PFY14 knockdowns, whereas pollen maturation was uniquely affected in PFBTZ-VIGS plants. Once PFeIF4AIII was strongly downregulated, plant survival was reduced via a decomposing root collar after flowering and Chinese lantern morphology was distorted. The expression of Physalis orthologous genes in the DYT1-TDF1-AMS-bHLH91 regulatory cascade that is associated with pollen maturation was significantly downregulated in PFMAGO-, PFY14- and PFBTZ-VIGS flowers. Intron-retention in the transcripts of P. floridana dysfunctional tapetum1 (PFDYT1) occurred in these mutated flowers. Additionally, the expression level of WRKY genes in defense-related pathways in the shoot apex of PFMAGO- or PFY14-VIGS plants and in the root collar of PFeIF4AIII-VIGS plants was significantly downregulated. Taken together, the Physalis EJC core genes play multiple roles including a conserved role in male fertility and newly discovered roles in Chinese lantern development, carpel functionality and defense-related processes. These data increase our understanding of the evolution and functions of EJC core genes in plants.

Evolutionary diversification of CYC/TB1-like TCP homologs and their recruitment for the control of branching and floral morphology in Papaveraceae (basal eudicots).

Angiosperms possess enormous morphological variation in plant architectures and floral forms. Previous studies in Pentapetalae and monocots have demonstrated the involvement of TCP domain CYCLOIDEA/TEOSINTE BRANCHED1-like (CYC/TB1) genes in the control of floral symmetry and shoot branching. However, how TCP/CYC-like (CYL) genes originated, evolved and functionally diversified remain unclear. We conducted a comparative functional study in Ranunculales, the sister lineage to all other eudicots, between Eschscholzia californica and Cysticapnos vesicaria, two species of Papaveraceae with actinomorphic and zygomorphic flowers, respectively. Phylogenetic analysis indicates that CYL genes in Papaveraceae form two paralogous lineages, PapaCYL1 and PapaCYL2. Papaveraceae CYL genes show highly diversified expression patterns as well as functions. Enhanced branching by silencing of EscaCYL1 suggests that the role of CYC/TB1-like genes in branching control is conserved in Papaveraceae. In contrast to the arrest of stamen development in Pentapetalae, PapaCYL genes promote stamen initiation and growth. In addition, we demonstrate that CyveCYLs are involved in perianth development, specifying sepal and petal identity in Cysticapnos by regulating the B-class floral organ identity genes. Our data also suggest the involvement of CyveCYL genes in the regulation of flower symmetry in Cysticapnos. Our work provides evidence of the importance of TCP/CYC-like genes in the promotion of morphological diversity across angiosperms.

Does stigma curvature promote delayed selfing? An experimental investigation in Triodanis perfoliata (Campanulaceae).

Self-fertilisation that is delayed until after opportunities for outcrossing have ceased has been argued to provide both the reproductive assurance benefits of selfing and the genetic advantages of outcrossing. In the Campanulaceae, presentation of pollen on stylar hairs and progressive stigma curvature have been hypothesised to facilitate delayed selfing, but experimental tests are lacking. Stigma curvature is common in Campanula, a genus largely characterised by self-incompatibility, and therefore is unlikely to have initially evolved to promote self-fertilisation. In derived self-compatible species, however, stigma curvature might serve the secondary function of delayed selfing. We investigated delayed selfing in Triodanis perfoliata, a self-compatible relative of Campanula. Using floral manipulation experiments and pollen tube observations, we quantified the extent and timing of self-pollination. Further, we hypothesised that, if stigma curvature provides the benefit of delayed selfing in Triodanis, selection should have favoured retention of self-pollen through the loss of a stylar hair retraction mechanism. Results of a stigma removal experiment indicated that autonomous selfing produces partial seed set, but only some selfing was delayed. Pollen tube observations and a flower senescence assay also supported the finding of partial delayed selfing. Scanning electron microscopy revealed that pollen-collecting hairs retract during anthesis, which may limit the extent of delayed selfing. Delayed selfing appeared to be only partially effective in T. perfoliata. The stylar hair retraction in this species would seem to contradict selection for selfing. We suggest that caution and rigour are needed in interpreting floral traits as adaptive mechanisms for delayed selfing.

Spatio-temporal orientation of microtubules controls conical cell shape in Arabidopsis thaliana petals.

The physiological functions of epidermal cells are largely determined by their diverse morphologies. Most flowering plants have special conical-shaped petal epidermal cells that are thought to influence light capture and reflectance, and provide pollinator grips, but the molecular mechanisms controlling conical cell shape remain largely unknown. Here, we developed a live-confocal imaging approach to quantify geometric parameters of conical cells in Arabidopsis thaliana (A. thaliana). Through genetic screens, we identified katanin (KTN1) mutants showing a phenotype of decreased tip sharpening of conical cells. Furthermore, we demonstrated that SPIKE1 and Rho of Plants (ROP) GTPases were required for the final shape formation of conical cells, as KTN1 does. Live-cell imaging showed that wild-type cells exhibited random orientation of cortical microtubule arrays at early developmental stages but displayed a well-ordered circumferential orientation of microtubule arrays at later stages. By contrast, loss of KTN1 prevented random microtubule networks from shifting into well-ordered arrays. We further showed that the filamentous actin cap, which is a typical feature of several plant epidermal cell types including root hairs and leaf trichomes, was not observed in the growth apexes of conical cells during cell development. Moreover, our genetic and pharmacological data suggested that microtubules but not actin are required for conical cell shaping. Together, our results provide a novel imaging approach for studying petal conical cell morphogenesis and suggest that the spatio-temporal organization of microtubule arrays plays crucial roles in controlling conical cell shape.

The VviMYB80 Gene is Abnormally Expressed in Vitis vinifera L. cv. 'Zhong Shan Hong' and its Expression in Tobacco Driven by the 35S Promoter Causes Male Sterility.

Anther development is a very precise and complicated process. In Arabidopsis, the AtMYB80 transcription factor regulates genes involved in pollen development and controls the timing of tapetal programmed cell death (PCD). In this study, we isolated and characterized cDNA for VviMYB80 expressed in flower buds of male-sterile Vitis vinifera L. cv. 'Zhong Shan Hong', a late-maturing cultivar derived from self-progeny of cv. 'Wink'. VviMYB80 belongs to the MYB80 subfamily and clusters with AtMYB35/TDF1 in a distinct clade. We found that in flower buds, expression of the VviMYB80 gene in cv. 'Zhong Shan Hong' sharply increased at the tetrad stage, resulting in a higher and earlier transcript level than that found in cv. 'Wink'. Expression of the VviMYB80 gene, driven by the 35S promoter, caused pleiotropic effects on the stamens, including smaller and shriveled anthers, delayed dehiscence, fewer seeds, shorter anther filaments, distorted pollen shape and a lack of cytoplasm, with the tapetum exhibiting hypertrophy in transformed tobacco. These results suggest that VviMYB80 may play an important role in stamen development and that expression of VviMYB80 driven by the 35S promoter in tobacco induces male sterility.

Histone H2B Monoubiquitination Mediated by HISTONE MONOUBIQUITINATION1 and HISTONE MONOUBIQUITINATION2 Is Involved in Anther Development by Regulating Tapetum Degradation-Related Genes in Rice.

Histone H2B monoubiquitination (H2Bub1) is an important regulatory mechanism in eukaryotic gene transcription and is essential for normal plant development. However, the function of H2Bub1 in reproductive development remains elusive. Here, we report rice (Oryza sativa) HISTONE MONOUBIQUITINATION1 (OsHUB1) and OsHUB2, the homologs of Arabidopsis (Arabidopsis thaliana) HUB1 and HUB2 proteins, which function as E3 ligases in H2Bub1, are involved in late anther development in rice. oshub mutants exhibit abnormal tapetum development and aborted pollen in postmeiotic anthers. Knockout of OsHUB1 or OsHUB2 results in the loss of H2Bub1 and a reduction in the levels of dimethylated lysine-4 on histone 3 (H3K4me2). Anther transcriptome analysis revealed that several key tapetum degradation-related genes including OsC4, rice Cysteine Protease1 (OsCP1), and Undeveloped Tapetum1 (UDT1) were down-regulated in the mutants. Further, chromatin immunoprecipitation assays demonstrate that H2Bub1 directly targets OsC4, OsCP1, and UDT1 genes, and enrichment of H2Bub1 and H3K4me2 in the targets is consistent to some degree. Our studies suggest that histone H2B monoubiquitination, mediated by OsHUB1 and OsHUB2, is an important epigenetic modification that in concert with H3K4me2, modulates transcriptional regulation of anther development in rice.

Effect of the ultrasound-assisted preliminary maceration on the efficiency of the essential oil distillation from selected herbal raw materials.

The objective of the study was the estimation of the ultrasound-assisted preliminary maceration effect on the efficiency of essential oil distillation and on its qualitative and quantitative composition. The experiment included analyses on three herbal materials, i.e. peppermint leaves (Mentha piperita L.), marjoram herb (Origanum majorana L.), and chamomile flowers (Chamomilla recutita (L.) Rauschert). The study showed that the application of preliminary water maceration of raw material, augmented with ultrasounds, had a statistically significant increasing effect on the amount of oil distilled, in the case of mint leaf from 1.32% to 1.46% v/w, and in the case of marjoram herb from 1.13% to 1.27% v/w. In the case of chamomile flowers no significant effect of ultrasound on the amount of oil obtained was observed. Generally, comparing the composition of essential oils obtained in the experiments with the requirements of the relevant standards no unfavourable effect of the distillation augmenting techniques applied was noted. Therefore, it should be expected that the studied distillates of volatile fractions will have the desired biological activity.

SCI1 is a component of the auxin-dependent control of cell proliferation in Arabidopsis upper pistil.

To characterize the recently described SCI1 (stigma/style cell cycle inhibitor 1) gene relationship with the auxin pathway, we have taken the advantage of the Arabidopsis model system and its available tools. At first, we have analyzed the At1g79200 T-DNA insertion mutants and constructed various transgenic plants. The loss- and gain-of-function plants displayed cell number alterations in upper pistils that were controlled by the amino-terminal domain of the protein. These data also confirmed that this locus holds the functional homolog (AtSCI1) of the Nicotiana tabacum SCI1 gene. Then, we have provided some evidences the auxin synthesis/signaling pathways are required for downstream proper AtSCI1 control of cell number: (a) its expression is downregulated in yuc2yuc6 and npy1 auxin-deficient mutants, (b) triple (yuc2yuc6sci1) and double (npy1sci1) mutants mimicked the auxin-deficient phenotypes, with no synergistic interactions, and (c) the increased upper pistil phenotype in these last mutants, which is a consequence of an increased cell number, was able to be complemented by AtSCI1 overexpression. Taken together, our data strongly suggests SCI1 as a component of the auxin signaling transduction pathway to control cell proliferation/differentiation in stigma/style, representing a molecular effector of this hormone on pistil development.

Annular floral nectary with oil-producing trichomes in Salvia farinacea (Lamiaceae): Anatomy, histochemistry, ultrastructure, and significance.

PREMISE OF THE STUDY: Many angiosperms produce nectar that entices pollinator visits. Each floral nectary tends to embody a singular form, such as the receptacular ring arising beneath the ovary in mint flowers (Lamiaceae). Exceptionally, the annular floral nectary in Salvia farinacea possesses modified stomata plus secretory trichomes. This first study of nectary ultrastructure within the largest genus of Lamiaceae examined this unusual condition. METHODS: Nectary anatomy, histochemistry, and ultrastructure were investigated from fresh and fixed material using light microscopy and scanning electron and transmission electron microscopy. KEY RESULTS: The annular nectary encircled the ovary plus extended ventrally as a projection. Modified stomata occurred only in the projection's abaxial epidermis. Conversely, peltate trichomes with a basal cell, a stalk cell, and 4-7 head cells were interspersed among the ovary lobes and covered the projection's adaxial surface. Phloem and xylem supplied the nectary interior, where parenchyma cells had numerous mitochondria and plastids with little starch, but few dictyosomes and little endoplasmic reticulum. Nectar accumulated as a drop opposite the projection's abaxial surface, escaping through stomatal pores and probably the cuticle. However, the annular nectary's glistening trichomes secreted a Sudan-positive product largely retained below the distended cuticle, but not nectar. CONCLUSIONS: This first ultrastructural study of co-occurring secretory trichomes and modified stomata on a mint nectary suggests multiple interactive functions for this atypical structure. These trichomes-possibly generating a substance informative to pollinators or as an ovarian defense against phytophagy-produced oil in an aqueous milieu, rather than contributing fluid to nectar.

Estructura, ontogenia y vascularización de las flores e inflorescencias de Drimys granadensis (Winteraceae) / Structure, ontogeny and vascularization of the flowers and inflorescences of Drimys granadensis (Winteraceae)

Drimys granadensis is a widespread species in montane forests of South and Central America. In this research, the structure, ontogeny, phyllotaxis and vascularization of the flowers and inflorescences of this species was studied in a population from the Eastern hills of Sabana de Bogota, Colombia. The methods used applied both optical microscopy, with astra blue-fuchsin staining, and scanning electron microscopy, using critical point dryed and gold-paladium metallized samples. Besides, results were compared with those of Drimys winteri, a widely studied species distributed in Chile and Argentina. Additionally, we studied the detail of the floral anatomy to determine the bracteal or calicine identity of the caliptra. I confirmed the proliferative status of the monothelic inflorescence, discarding alternative explanations of the terminal flower identity. I found that uniflorescences have an acropetal development until the terminal meristem becomes the terminal flower, then this flower develops rapidly resulting in a determined uniflorescence. I found pseudosyphonosthelic vascularization in peduncles and pedicels. Besides, I discovered some evidence in the vascular and anatomical structures, to consider the caliptra as the fusion product of various structures and therefore of calicine origin. The caliptra showed a whorled phyllotaxis, but the petals, stamens and carpels presented a spiral condition; phyllotaxis change was explained by the long time lapse between the initiation of the calyx and the corolla. I found great similarities among the inflorescences of D. granadensis and D. winteri; they were different in the proliferation start time, and in the frequent presence of nomophylls in D. granadensis, in contrast to the presence of reduced bracts and bracteoles in D. winteri inflorescences.

Drimys granadensis es una especie de amplia distribución en los bosques montanos de Sur y Centro América. En esta investigación se estudiaron, mediante microscopía óptica y electrónica de barrido, la estructura, ontogenia, filotaxis y vascularización de sus flores e inflorescencias, y fueron comparadas con las de Drimys winteri, especie distribuida en Chile y Argentina. Adicionalmente, se buscó evidencia para determinar la identidad bracteal o calicina de la caliptra de sus flores. Se confirmó la condición monotélica proliferante de la inflorescencia, descartando explicaciones alternativas de identidad de la flor terminal. Las uniflorescencias presentan un desarrollo acrópeto, hasta que el meristemo terminal se transforma en flor terminal, entonces esta flor se desarrolla rápidamente dando lugar a una uniflorescencia determinada. La vascularización es pseudosifonostélica para pedúnculos y pedicelos. Se encontró evidencia en la vascularización y estructura anatómica para considerar la caliptra como el producto de fusión de varias estructuras y, por tanto, de origen calicino. La caliptra presentó una filotaxis verticilada, pero los pétalos, estambres y carpelos presentaron una filotaxis espiralada; el cambio se explicó por el tiempo prolongado entre la iniciación de cáliz y corola. Las inflorescencias de D. granadensis y D. winteri son muy similares; siendo diferente el tiempo de inicio de la proliferación y la frecuente presencia de nomófilos en las inflorescencias de D. granadensis, en contraste con la presencia de brácteas y bracteolas reducidas en D. winteri.

The cysteine protease CEP1, a key executor involved in tapetal programmed cell death, regulates pollen development in Arabidopsis.

Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant PCD. We identified a papain-like cysteine protease, CEP1, which is involved in tapetal PCD and pollen development in Arabidopsis thaliana. CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development. The CEP1 protein first appears as a proenzyme in precursor protease vesicles and is then transported to the vacuole and transformed into the mature enzyme before rupture of the vacuole. cep1 mutants exhibited aborted tapetal PCD and decreased pollen fertility with abnormal pollen exine. A transcriptomic analysis revealed that 872 genes showed significantly altered expression in the cep1 mutants, and most of them are important for tapetal cell wall organization, tapetal secretory structure formation, and pollen development. CEP1 overexpression caused premature tapetal PCD and pollen infertility. ELISA and quantitative RT-PCR analyses confirmed that the CEP1 expression level showed a strong relationship to the degree of tapetal PCD and pollen fertility. Our results reveal that CEP1 is a crucial executor during tapetal PCD and that proper CEP1 expression is necessary for timely degeneration of tapetal cells and functional pollen formation.

Roles of hydroxyproline-rich glycoproteins in the pollen tube and style cell growth of tobacco (Nicotiana tabacum L.).

Hydroxyproline-rich glycoproteins (HRGPs) are plant cell wall proteins related to plant growth and development, and extensins (EXTs) are a subfamily of HRGPs. In this study, the function of HRGPs, especially EXTs, was investigated in the pollen tube and style cell growth of Nicotiana tabacum L. By using the techniques of protein blot and immunohistochemistry, the JIM20-recognized epitopes of EXTs were abundantly expressed in vivo for pollen tubes and transmitting tissue. A hydroxyproline synthesis inhibitor, 3,4-dehydro-l-proline (3,4-DHP), was used to investigate the functions of HRGPs. The addition of 3,4-DHP decreased the speed of pollen tube growth and shortened the length of style. Moreover, the hydroxyproline assay and JIM20 immunolocalization confirmed that 3,4-DHP treatment reduced the level of hydroxyproline and EXTs in the treated styles, respectively. These results indicate that HRGPs, most likely EXTs, may play important roles in the pollen tube and style cell growth.

Glandular trichomes and essential oil of Thymus quinquecostatus.

The distribution and types of glandular trichomes and essential oil chemistry of Thymus quinquecostatus were studied. The glandular trichomes are distributed on the surface of stem, leaf, rachis, calyx and corolla, except petiole, pistil and stamen. Three morphologically distinct types of glandular trichomes are described. Peltate trichomes, consisting of a basal cell, a stalk cell and a 12-celled head, are distributed on the stem, leaf, corolla and outer side of calyx. Capitate trichomes, consisting of a unicellular base, a 1-2-celled stalk and a unicellular head, are distributed more diffusely than peltate ones, existing on stem, leaf, rachis and calyx. Digitiform trichomes are just distributed on the outer side of corolla, consisting of 1 basal cell, 3 stalk cells and 1 head cell. All three types of glandular trichomes can secrete essential oil, and in small capitate trichomes of rachis, all peltate trichomes and digitiform trichomes, essential oil is stored in a large subcuticular space, released by cuticle rupture, whereas, in other capitate trichomes, essential oil crosses the thin cuticle. The essential oil of T. quinquecostatus is yellow, and its content is highest in the growth period. 68 constituents were identified in the essential oils. The main constituent is linalool.

Morphoanatomy of the flower of Syagrus inajai (SPRUCE) Becc. (Arecaceae- Arecoideae- Attaleinae), Amazon / Morfoanatomia floral de Syagrus inajai (Spruce) Becc. (Arecaceae- Arecoideae- Attaleinae), Amazônia

The occurrence of Syagrus inajai (Spruce) Becc., popularly known as pupunha palm, among other names, has been registered in the Guianas and in the North of Brazil in areas of terra firme (non-flooding) and gallery forests. In order to characterize the inflorescence and further knowledge of this family, a morphoanatomical study was carried out of the palm S. inajai in a green area of the Campus of the Federal University of Amazonas - UFAM, Manaus, Amazonas. The inflorescences are branched to one order, pedunculate, and interfoliar, measuring 62-82 cm in length, with woody bracts with longitudinal grooves on the external surface, and flowers in triads. The number of flowers to each inflorescence varies from 5,904 to 17,316 for staminate flowers, and from 180 to 3,528 for pistillate flowers. Staminate flowers with six anthers and one vascular bundle each; three-lobed pistillodium, vascularized pistillodium. Its pistillate flowers have six staminodia joined to form a circle, syncarpic, tricarpellary, trilocular gynoecium, one ovule to each locule, synascidiate in the ovary, and plicated above. Tripartite stigma, apical and sessile, with epidermis composed of elongated papillary cells, pattern of epidermis that is maintained throughout the stylar canal. Bitegmented, anatrope, pachychalazal ovule.

Syagrus inajai (Spruce) Becc., popularmente conhecida como pupunha-brava entre outras denominações, teve sua ocorrência registrada para Guianas e no norte do Brasil em áreas de floresta de terra firme e floresta de galeria. Com o intuito de caracterizar a inflorescência ampliando o conhecimento a respeito da família foi realizado o estudo morfoanatômico da palmeira S. inajai, em área verde do Campus da Universidade Federal do Amazonas - UFAM, Manaus, Amazonas. A inflorescência é ramificada em primeira ordem, pedunculada, interfoliar com 62-82 cm de comprimento, bráctea lenhosa com fissuras longitudinais na superfície externa e flores em tríades. O número de flores por inflorescência varia entre 5.904 - 17.316 para flores estaminadas e 180 - 3.528 para as flores pistiladas. Flores estaminadas com seis anteras e um feixe vascular cada; pistilódio trífido e vascularizado. Flores pistiladas com seis estaminódios unidos formando um círculo. Gineceu sincárpico, tricarpelar, trilocular, um óvulo por lóculo, sinascidiado no ovário e plicado acima. Estigma tripartido, apical e séssil, com epiderme composta por células papilosas alongadas, padrão de epiderme que se mantem por todo canal estilar. Óvulo é anátropo, paquicalazal e bitegumentado.

Pollination induces autophagy in petunia petals via ethylene.

Autophagy is one of the main mechanisms of degradation and remobilization of macromolecules, and it appears to play an important role in petal senescence. However, little is known about the regulatory mechanisms of autophagy in petal senescence. Autophagic processes were observed by electron microscopy and monodansylcadaverine staining of senescing petals of petunia (Petunia hybrida); autophagy-related gene 8 (ATG8) homologues were isolated from petunia and the regulation of expression was analysed. Nutrient remobilization was also examined during pollination-induced petal senescence. Active autophagic processes were observed in the mesophyll cells of senescing petunia petals. Pollination induced the expression of PhATG8 homologues and was accompanied by an increase in ethylene production. Ethylene inhibitor treatment in pollinated flowers delayed the induction of PhATG8 homologues, and ethylene treatment rapidly upregulated PhATG8 homologues in petunia petals. Dry weight and nitrogen content were decreased in the petals and increased in the ovaries after pollination in detached flowers. These results indicated that pollination induces autophagy and that ethylene is a key regulator of autophagy in petal senescence of petunia. The data also demonstrated the translocation of nutrients from the petals to the ovaries during pollination-induced petal senescence.

A cystathionine-ß-synthase domain-containing protein, CBSX2, regulates endothecial secondary cell wall thickening in anther development.

Anther formation and dehiscence are complex pivotal processes in reproductive development. The secondary wall thickening in endothecial cells of the anther is a known prerequisite for successful anther dehiscence. However, many gaps remain in our understanding of the regulatory mechanisms underlying anther dehiscence in planta, including a possible role for jasmonic acid (JA) and H(2)O(2) in secondary wall thickening of endothecial cells. Here, we report that the cystathionine ß-synthase domain-containing protein CBSX2 located in the chloroplast plays a critical role in thickening of the secondary cell walls of the endothecium during anther dehiscence in Arabidopsis. A T-DNA insertion mutant of CBSX2 (cbsx2) showed increased secondary wall thickening of endothecial cells and early anther dehiscence. Consistently, overexpression of CBSX2 resulted in anther indehiscence. Exogenous JA application induced secondary wall thickening and caused flower infertility in the cbsx2 mutant, whereas it partially restored fertility in the CBSX2-overexpressing lines lacking the wall thickening. CBSX2 directly modulated thioredoxin (Trx) in chloroplasts, which affected the level of H(2)O(2) and, consequently, expression of the genes involved in secondary cell wall thickening. Our findings have revealed that CBSX2 modulates the H(2)O(2) status, which is linked to the JA response and in turn controls secondary wall thickening of the endothecial cells in anthers for dehiscence to occur.