Synchrotron micro-computed tomography unveils the three-dimensional structure and origin of staminodes in the Plains Prickly Pear Cactus Opuntia polyacantha Haw. (Cactaceae).

Floral appendages display an array of shapes and sizes. Among these organs, staminodes are morphologically diverse structures that have lost the ability to produce pollen, but in some instances, they produce fertile pollen grains. In the family Cactaceae staminodes are uncommon and range from simple linear to flat to spatulate structures, but studies describing their structural attributes are scanty. This study highlights the advantages of synchrotron radiation for sample preparation and as a research tool for plant biology. It describes the internal morphology of floral parts, particularly stamen, tepal, and staminode in the Plains Prickly Pear Cactus, Opuntia polyacantha, using synchrotron radiation micro-computed tomography (SR-µCT). It also shows the different anatomical features in reconstructed three-dimensional imaging of reproductive parts and discuss the advantages of the segmentation method to detect and characterize the configuration and intricate patterns of vascular networks and associated structures of tepal and androecial parts applying SR-µCT. This powerful technology led to substantial improvements in terms of resolution allowing a more comprehensive understanding of the anatomical organization underlying the vasculature of floral parts and inception of staminodes in O. polyacantha. Tepal and androecial parts have uniseriate epidermis enclosing loose mesophyll with mucilage secretory ducts, lumen, and scattered vascular bundles. Cryptic underlying structural attributes provide evidence of a vascularized pseudo-anther conjoint with tepals. The undefined contours of staminodial appendages (pseudo-anther) amalgamated to the tepals' blurred boundaries suggest that staminodes originate from tepals, a developmental pattern supporting the fading border model of floral organ identity for angiosperms.

Secretory Products in Petals of Centaurea cyanus L. Flowers: A Histochemistry, Ultrastructure, and Phytochemical Study of Volatile Compounds.

(1) Background: Centaurea cyanus L. is a medicinal plant whose flowers are widely used in herbal medicine. The aim of the study was to localise flower tissues that are responsible for the production of secretory products in petals and to analyse the volatile compounds. The volatile compounds of the flowers of this species have not been investigated to date. (2) Methods: Light, fluorescence, scanning and transmission electron microscopy techniques were used in the study. Lipophilic compounds were localised in the tissues using histochemical assays. Volatile compounds were determined with the use of solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS). (3) Results: The study showed production of secretion in the petal parenchyma, whose ultrastructure has features of a secretory tissue. The lipophilic secretion was localised in the cells and intercellular spaces of the parenchyma and in the walls and surface of epidermal cells, where it accumulated after release through cuticle microchannels. Sesquiterpenes were found to constitute the main group of volatile compounds, with the highest content of ß-caryophyllene (26.17%) and α-humulene (9.77%). (4) Conclusions: Given the presence of some volatile components that are often found in resins (caryophyllene, delta-cadinene) and the abundant secretion residues on the epidermal surface, we suppose that the C. cyanus secretion released by the flowers is a resinaceous mixture (oleoresin), which is frequently found in plants, as shown by literature data. This secretion may play an important role in the therapeutic effects of C. cyanus flowers.

Morpho-Physiological and Transcriptome Changes in Tomato Anthers of Different Developmental Stages under Drought Stress.

Drought limits the growth and productivity of plants. Reproductive development is sensitive to drought but the underlying physiological and molecular mechanisms remain unclear in tomatoes. Here, we investigated the effect of drought on tomato floral development using morpho-physiological and transcriptome analyses. Drought-induced male sterility through abnormal anther development includes pollen abortion, inadequate pollen starch accumulation and anther indehiscence which caused floral bud and opened flower abortions and reduced fruit set/yield. Under drought stress (DS), pollen mother cell to meiotic (PMC-MEI) anthers survived whereas tetrad to vacuolated uninucleate microspore (TED-VUM) anthers aborted. PMC-MEI anthers had lower ABA increase, reduced IAA and elevated sugar contents under DS relative to well-watered tomato plants. However, TED-VUM anthers had higher ABA increase and IAA levels, and lower accumulation of soluble sugars, indicating abnormal carbohydrate and hormone metabolisms when exposed to drought-stress conditions. Moreover, RNA-Seq analysis identified altogether >15,000 differentially expressed genes that were assigned to multiple pathways, suggesting that tomato anthers utilize complicated mechanisms to cope with drought. In particular, we found that tapetum development and ABA homeostasis genes were drought-induced while sugar utilization and IAA metabolic genes were drought-repressed in PMC-MEI anthers. Our results suggest an important role of phytohormones metabolisms in anther development under DS and provide novel insight into the molecular mechanism underlying drought resistance in tomatoes.

Possible Role of Crystal-Bearing Cells in Tomato Fertility and Formation of Seedless Fruits.

Crystal-bearing cells or idioblasts, which deposit calcium oxalate, are located in various tissues and organs of many plant species. The functional significance of their formation is currently unclear. Idioblasts in the leaf parenchyma and the development of crystal-bearing cells in the anther tissues of transgenic tomato plants (Solanum lycopersicon L.), expressing the heterologous FeSOD gene and which showed a decrease in fertility, were studied by transmission and scanning electron microscopy. The amount of calcium oxalate crystals was found to increase significantly in the transgenic plants compared to the wild type (WT) ones in idioblasts and crystal-bearing cells of the upper part of the anther. At the same time, changes in the size and shape of the crystals and their location in anther organs were noted. It seems that the interruption in the break of the anther stomium in transgenic plants was associated with the formation and cell death regulation of a specialized group of crystal-bearing cells. This disturbance caused an increase in the pool of these cells and their localization in the upper part of the anther, where rupture is initiated. Perturbations were also noted in the lower part of the anther in transgenic plants, where the amount of calcium oxalate crystals in crystal-bearing cells was reduced that was accompanied by disturbances in the morphology of pollen grains. Thus, the induction of the formation of crystal-bearing cells and calcium oxalate crystals can have multidirectional effects, contributing to the regulation of oxalate metabolism in the generative and vegetative organs and preventing fertility when the ROS balance changes, in particular, during oxidative stresses accompanying most abiotic and biotic environmental factors.

Arabidopsis FAX1 mediated fatty acid export is required for the transcriptional regulation of anther development and pollen wall formation.

KEY MESSAGE: The mutation of FAX1 (Fatty Acid Export 1) disrupts ROS homeostasis and suppresses transcription activity of DYT1-TDF1-AMS-MS188 genetic network, leading to atypical tapetum PCD and defective pollen formation in Arabidopsis. Fatty acids (FAs) have multiple important biological functions and exert diverse cellular effects through modulating Reactive Oxygen Species (ROS) homeostasis. Arabidopsis FAX1 (Fatty Acid Export 1) mediates the export of de novo synthesized FA from chloroplast and loss of function of FAX1 impairs male fertility. However, mechanisms underlying the association of FAX1-mediated FA export with male sterility remain enigmatic. In this study, by using an integrated approach that included morphological, cytological, histological, and molecular analyses, we revealed that loss of function of FAX1 breaks cellular FA/lipid homeostasis, which disrupts ROS homeostasis and suppresses transcriptional activation of the DYT1-TDF1-AMS-MS188 genetic network of anther development, impairing tapetum development and pollen wall formation, and resulting in male sterility. This study provides new insights into the regulatory network for male reproduction in plants, highlighting an important role of FA export-mediated ROS homeostasis in the process.

CG Demethylation Leads to Sequence Mutations in an Anther Culture of Barley Due to the Presence of Cu, Ag Ions in the Medium and Culture Time.

During plant tissue cultures the changes affecting regenerants have a broad range of genetic and epigenetic implications. These changes can be seen at the DNA methylation and sequence variation levels. In light of the latest studies, DNA methylation change plays an essential role in determining doubled haploid (DH) regenerants. The present study focuses on exploring the relationship between DNA methylation in CG and CHG contexts, and sequence variation, mediated by microelements (CuSO4 and AgNO3) supplemented during barley anther incubation on induction medium. To estimate such a relationship, a mediation analysis was used based on the results previously obtained through metAFLP method. Here, an interaction was observed between DNA demethylation in the context of CG and the time of culture. It was also noted that the reduction in DNA methylation was associated with a total decrease in the amount of Cu and Ag ions in the induction medium. Moreover, the total increase in Cu and Ag ions increased sequence variation. The importance of the time of tissue culture in the light of the observed changes resulted from the grouping of regenerants obtained after incubation on the induction medium for 28 days. The present study demonstrated that under a relatively short time of tissue culture (28 days), the multiplication of the Cu2+ and Ag+ ion concentrations ('Cu*Ag') acts as a mediator of demethylation in CG context. Change (increase) in the demethylation in CG sequence results in the decrease of 'Cu*Ag', and that change induces sequence variation equal to the value of the indirect effect. Thus, Cu and Ag ions mediate sequence variation. It seems that the observed changes at the level of methylation and DNA sequence may accompany the transition from direct to indirect embryogenesis.

A Silent Exonic Mutation in a Rice Integrin-α FG-GAP Repeat-Containing Gene Causes Male-Sterility by Affecting mRNA Splicing.

Pollen development plays crucial roles in the life cycle of higher plants. Here we characterized a rice mutant with complete male-sterile phenotype, pollen-less 1 (pl1). pl1 exhibited smaller anthers with arrested pollen development, absent Ubisch bodies, necrosis-like tapetal hypertrophy, and smooth anther cuticular surface. Molecular mapping revealed a synonymous mutation in the fourth exon of PL1 co-segregated with the mutant phenotype. This mutation disrupts the exon-intron splice junction in PL1, generating aberrant mRNA species and truncated proteins. PL1 is highly expressed in the tapetal cells of developing anther, and its protein is co-localized with plasma membrane (PM) and endoplasmic reticulum (ER) signal. PL1 encodes an integrin-α FG-GAP repeat-containing protein, which has seven ß-sheets and putative Ca2+-binding motifs and is broadly conserved in terrestrial plants. Our findings therefore provide insights into both the role of integrin-α FG-GAP repeat-containing protein in rice male fertility and the influence of exonic mutation on intronic splice donor site selection.

Cytological observation of anther structure and genetic investigation of a thermo-sensitive genic male sterile line 373S in Brassica napus L.

BACKGROUND: Photoperiod and/or thermo-sensitive male sterility is an effective pollination control system in crop two-line hybrid breeding. We previously discovered the spontaneous mutation of a partially male sterile plant and developed a thermo-sensitive genic male sterile (TGMS) line 373S in Brassica napus L. The present study characterized this TGMS line through cytological observation, photoperiod/ temperature treatments, and genetic investigation. RESULTS: Microscopic observation revealed that the condensed cytoplasm and irregular exine of microspores and the abnormal degradation of tapetum are related to pollen abortion. Different temperature and photoperiod treatments in field and growth cabinet conditions indicated that the fertility alteration of 373S was mainly caused by temperature changes. The effects of photoperiod and interaction between temperature and photoperiod were insignificant. The critical temperature leading to fertility alteration ranged from 10 °C (15 °C/5 °C) to 12 °C (17 °C/7 °C), and the temperature-responding stage was coincident with anther development from pollen mother cell formation to meiosis stages. Genetic analysis indicated that the TGMS trait in 373S was controlled by one pair of genes, with male sterility as the recessive. Multiplex PCR analysis revealed that the cytoplasm of 373S is pol type. CONCLUSIONS: Our study suggested that the 373S line in B. napus has a novel thermo-sensitive gene Bnmst1 in Pol CMS cytoplasm background, and its fertility alteration is mainly caused by temperature changes. Our results will broaden the TGMS resources and lay the foundation for two-line hybrid breeding in B. napus.

Comparative transcriptome analysis indicates that a core transcriptional network mediates isonuclear alloplasmic male sterility in wheat (Triticum aestivum L.).

BACKGROUND: Cytoplasmic male sterility (CMS) plays a crucial role in the utilization of heterosis and various types of CMS often have different abortion mechanisms. Therefore, it is important to understand the molecular mechanisms related to anther abortion in wheat, which remain unclear at present. RESULTS: In this study, five isonuclear alloplasmic male sterile lines (IAMSLs) and their maintainer were investigated. Cytological analysis indicated that the abortion type was identical in IAMSLs, typical and stainable abortion, and the key abortive period was in the binucleate stage. Most of the 1,281 core shared differentially expressed genes identified by transcriptome sequencing compared with the maintainer in the vital abortive stage were involved in the metabolism of sugars, oxidative phosphorylation, phenylpropane biosynthesis, and phosphatidylinositol signaling, and they were downregulated in the IAMSLs. Key candidate genes encoding chalcone--flavonone isomerase, pectinesterase, and UDP-glucose pyrophosphorylase were screened and identified. Moreover, further verification elucidated that due to the impact of downregulated genes in these pathways, the male sterile anthers were deficient in sugar and energy, with excessive accumulations of ROS, blocked sporopollenin synthesis, and abnormal tapetum degradation. CONCLUSIONS: Through comparative transcriptome analysis, an intriguing core transcriptome-mediated male-sterility network was proposed and constructed for wheat and inferred that the downregulation of genes in important pathways may ultimately stunt the formation of the pollen outer wall in IAMSLs. These findings provide insights for predicting the functions of the candidate genes, and the comprehensive analysis of our results was helpful for studying the abortive interaction mechanism in CMS wheat.

Earlier Degraded Tapetum1 (EDT1) Encodes an ATP-Citrate Lyase Required for Tapetum Programmed Cell Death.

In flowering plants, the tapetum cells in anthers undergo programmed cell death (PCD) at the late meiotic stage, providing nutrients for further development of microspores, including the formation of the pollen wall. However, the molecular basis of tapetum PCD remains elusive. Here we report a tapetum PCD-related mutant in rice (Oryza sativa), earlier degraded tapetum 1 (edt1), that shows complete pollen abortion associated with earlier-than-programmed tapetum cell death. EDT1 encodes a subunit of ATP-citrate lyase (ACL), and is specifically expressed in the tapetum of anthers. EDT1 localized in both the nucleus and the cytoplasm as observed in rice protoplast transient assays. We demonstrated that the A and B subunits of ACL interacted with each other and might function as a heteromultimer in the cytoplasm. EDT1 catalyzes the critical steps in cytosolic acetyl-CoA synthesis. Our data indicated a decrease in ATP level, energy charge, and fatty acid content in mutant edt1 anthers. In addition, the genes encoding secretory proteases or lipid transporters, and the transcription factors known to regulate PCD, were downregulated. Our results demonstrate that the timing of tapetum PCD must be tightly regulated for successful pollen development, and that EDT1 is involved in the tapetum PCD process. This study furthers our understanding of the molecular basis of pollen fertility and fecundity in rice and may also be relevant to other flowering plants.

Report: Pharmacognostic and physicochemical screening of Euphorbia nivulia Buch.-Ham.

Euphorbia nivulia Buch.-Ham. (Euphorbiaceae) is commonly known as Indian Spurge Tree in English, and "Saj Thor" or "Jhanami booti" in local language. The plant is used traditionally in the treatment of various diseases like inflammation, fever, worm infection, asthma, cough, wounds and diabetes. In current study fresh as well as dried aerial parts of the plant and cut sections were examined, both macroscopically and microscopically. The study also deals with fluorescence analysis and phytochemical characteristics and other WHO recommended methods for standardization. WHO guidelines on quality control for medicinal plants materials were used for pharmacognostical evaluation of E. nivulia, phytochemical screening helps in determining the predominant classes of active constituents responsible for the activity. The present work will be helpful in identification of the fresh and dried samples of aerial parts pharmacognostically and anatomically. These studies will serve as a reference for correct identification and may be helpful in checking any type of adulteration. These observations will also help in differentiating this species from closely related species of the same genus and family.

Histological and Cytological Characterization of Anther and Appendage Development in Asian Lotus (Nelumbo nucifera Gaertn.).

The lotus (Nelumbo Adans.) is a perennial aquatic plant with important value in horticulture, medicine, food, religion, and culture. It is rich in germplasm and more than 2000 cultivars have been cultivated through hybridization and natural selection. Microsporogenesis and male gametogenesis in the anther are important for hybridization in flowering plants. However, little is known about the cytological events, especially related to the stamen, during the reproduction of the lotus. To better understand the mechanism controlling the male reproductive development of the lotus, we investigated the flower structure of the Asian lotus (N. nucifera). The cytological analysis of anther morphogenesis showed both the common and specialized cytological events as well as the formation of mature pollen grains via meiosis and mitosis during lotus anther development. Intriguingly, an anatomical difference in anther appendage structures was observed between the Asian lotus and the American lotus (N. lutea). To facilitate future study on lotus male reproduction, we categorized pollen development into 11 stages according to the characterized cytological events. This discovery expands our knowledge on the pollen and appendage development of the lotus as well as improving the understanding of the species differentiation of N. nucifera and N. lutea.

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress.

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H2 DCFDA-staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into 'low-ROS' and 'high-ROS' subpopulations. Pollen germination assays following fluorescence-activated cell sorting revealed that the high-ROS pollen germinated with a frequency that was 35-fold higher than the low-ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose-dependent alterations in DCF-fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry-based approaches to investigate metabolic changes during stress responses in pollen.

OsERF101, an ERF family transcription factor, regulates drought stress response in reproductive tissues.

KEY MESSAGE: An ERF transcription factor OsERF101 is predominantly expressed in rice reproductive tissues and plays an important role in improving rice seed setting rate under drought stress. Drought reduces grain yield due to the cumulative damage effects to plant vegetative and reproductive developmental processes. However, the genes involved in these processes are still not completely understood. In this study, we identified a gene named OsERF101 as an important positive regulator in the adaptive responses to dehydration stress during the reproductive and vegetative stages. This gene encodes a member of APETALA2/Ethylene-Responsive Element Binding Protein (AP2/EREBP) family. OsERF101 was predominantly expressed in flowers, particularly in the tapetum and microspores under normal growth conditions. It was induced by drought, PEG6000 and abscisic acid (ABA) in leaves. During the vegetative stage, OsERF101-overexpression plants were more resistant to osmotic stress caused by PEG6000 compared to the control plants. They also had higher survival and seed setting rates than wild type when subjected to reproductive-stage drought stress. Further physiological analysis revealed that the pollen fertility was improved in the overexpression lines, while the knockout mutant and RNAi lines showed reduced pollen fertility and compromised drought tolerance during the reproductive stage. The increased proline content and peroxidase activity in OsERF101-overexpression plants might contribute to the improved drought-tolerance of plants. In addition, OsERF101-overexpression plants displayed ABA susceptible phenotype, in which the expression levels of ABA-responsive genes RD22, LEA3, and PODs were up-regulated. Taken together, our results indicate that OsERF101 is a gene that regulates dehydration responses during the vegetative and reproductive stages.

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.

Variations in Volatile Oil Yield and Composition of "Xin-yi" (Magnolia biondii Pamp. Flower Buds) at Different Growth Stages.

Dried flower buds of Magnolia biondii Pamp. are the main ingredient in "Xin-yi" in China, and the volatile oils of M. biondii flower buds are the principal medicinal component. Gas chromatographymass spectrometry (GC-MS) and microscopic techniques were employed to detect the volatile yields of M. biondii flowers at various growth stages. The volatile oil yields of M. biondii flowers differed significantly at different growth stages and were closely related to flower dry weight, oil cell density and degree of oil accumulation. In February 2016, flower buds had the highest dry weight, the maximum percentage of oil cells at the oil saturation stage and the highest density of oil cells, which coincided with the highest oil yield. In March 2016, flower buds had a lower dry weight, a higher percentage of oil cells at the oil-degrading stage and the lowest oil cell density, resulting in decreased oil yields. The total amounts of the major medicinal components in the M. biondii flower also showed regular changes at different growth stages. In January and February of 2016, M. biondii flowers had a higher dry weight, volatile oil yield and total content of medicinal ingredients, which was the best time for harvesting high-quality medicinal components. Our study reveals that volatile oil content and chemical composition are closely related to the growth stage of M. biondii flower buds. The results provide a scientific morphology and composition index for evaluating the medicinal value and harvesting of high-quality M. biondii medicinal herbs.

Functional anatomy reveals secretory activity in papillose anthers of a buzz-pollinated Solanum species (Cyphomandra clade - Solanaceae).

Pollination in Solanum (Solanaceae) species is commonly performed by female bees, which vibrate anthers to extract pollen. Another pollen removal type is by male euglossine bees, milking the anthers when searching for floral scents produced by secretory tissues (osmophorous) at the swollen connective of the anthers of species in the Cyphomandra clade. Some species of this clade, however, are buzz-pollinated and present papillate anthers that should also have secretory activity, a hypothesis here tested. The anthers of Solanum luridifuscescens were fixed at different stages of development and analysed under light microscopy, SEM and TEM. Histochemical tests for the detection of starch and lipids were done. Epidermal cells of the abaxial surface of the anthers were visibly papillose, had large nuclei and dense cytoplasm rich in organelles such as mitochondria and plastids, typical features of secretory tissues. In this site, lipid droplets were detected, concomitantly with starch consumption, compatible with the secretory process in osmophores. No exudate or accumulation of substances was seen on the surface; in agreement with a previous pollination study performed in field conditions, where no pollinators were observed collecting floral scents, only pollen. The histochemical and structural analyses have evidenced the lipidic composition of the secretion, strongly pointing to terpenes as the secreted compounds. Ours findings show that papillae of the anthers have secretory activities that produce lipophilic compounds. This does not result in resources for bees, but could be an evolutionary step to the development of more specialised anthers in the Cyphomandra clade.

Abnormal development of floral meristem triggers defective morphogenesis of generative system in transgenic tomatoes.

Parthenocarpy and fruit malformations are common among independent transgenic tomato lines, expressing genes encoding different pathogenesis-related (PR) protein and antimicrobal peptides. Abnormal phenotype developed independently of the expression and type of target genes, but distinctive features during flower and fruit development were detected in each transgenic line. We analyzed the morphology, anatomy, and cytoembryology of abnormal flowers and fruits from these transgenic tomato lines and compared them with flowers and fruits of wild tomatoes, line YaLF used for transformation, and transgenic plants with normal phenotype. We confirmed that the main cause of abnormal flower and fruit development was the alterations of determinate growth of generative meristem. These alterations triggered different types of anomalous growth, affecting the number of growing ectopic shoots and formation of new flowers. Investigation of the ovule ontogenesis did not show anomalies in embryo sac development, but fertilization did not occur and embryo sac degenerated. Nevertheless, the ovule continued to differentiate due to proliferation of endothelium cells. The latter substituted embryo sac and formed pseudoembryonic tissue. This process imitated embryogenesis and stimulated ovary growth, leading to the development of parthenocarpic fruit. We demonstrated that failed fertilization occurred due to defective male gametophyte formation, which was manifested in blocked division of the nucleus in the microspore and arrest of vegetative and generative cell formation. Maturing pollen grains were overgrown microspores, not competent for fertilization but capable to induce proliferation of endothelium and development of parthenocarpic ovary. Thus, our study provided new data on the structural transformations of reproductive organs during development of parthenocarpic fruits in transgenic tomato.

EAT1 transcription factor, a non-cell-autonomous regulator of pollen production, activates meiotic small RNA biogenesis in rice anther tapetum.

The 24-nucleotides (nt) phased secondary small interfering RNA (phasiRNA) is a unique class of plant small RNAs abundantly expressed in monocot anthers at early meiosis. Previously, 44 intergenic regions were identified as the loci for longer precursor RNAs of 24-nt phasiRNAs (24-PHASs) in the rice genome. However, the regulatory mechanism that determines spatiotemporal expression of these RNAs has remained elusive. ETERNAL TAPETUM1 (EAT1) is a basic-helix-loop-helix (bHLH) transcription factor indispensable for induction of programmed cell death (PCD) in postmeiotic anther tapetum, the somatic nursery for pollen production. In this study, EAT1-dependent non-cell-autonomous regulation of male meiosis was evidenced from microscopic observation of the eat1 mutant, in which meiosis with aberrantly decondensed chromosomes was retarded but accomplished somehow, eventually resulting in abortive microspores due to an aberrant tapetal PCD. EAT1 protein accumulated in tapetal-cell nuclei at early meiosis and postmeiotic microspore stages. Meiotic EAT1 promoted transcription of 24-PHAS RNAs at 101 loci, and importantly, also activated DICER-LIKE5 (DCL5, previous DCL3b in rice) mRNA transcription that is required for processing of double-stranded 24-PHASs into 24-nt lengths. From the results of the chromatin-immunoprecipitation and transient expression analyses, another tapetum-expressing bHLH protein, TDR INTERACTING PROTEIN2 (TIP2), was suggested to be involved in meiotic small-RNA biogenesis. The transient assay also demonstrated that UNDEVELOPED TAPETUM1 (UDT1)/bHLH164 is a potential interacting partner of both EAT1 and TIP2 during early meiosis. This study indicates that EAT1 is one of key regulators triggering meiotic phasiRNA biogenesis in anther tapetum, and that other bHLH proteins, TIP2 and UDT1, also play some important roles in this process. Spatiotemporal expression control of these bHLH proteins is a clue to orchestrate precise meiosis progression and subsequent pollen production non-cell-autonomously.

[Study on effect elements of anthers opening-closing movement and its cell morphology regulatory mechanism of Paris polyphylla var. yunnanensis].

In order to study the effect elements of anthers opening-closing movement of Paris polyphylla var. yunnanensis, and its cell morphology regulatory mechanism. Anthers daily opening in the morning and closing in the evening and its corresponding ecological elements changes were recorded. Different light, temperature, humidity experiment and artificial rainfall experiment were designed to observe the effect on anthers opening-closing movement, paraffin sections were made to observe the cell morphology change when the anthers daily opening and closing. The result showed that the movement of anthers daily opening and closing was regulated by ecological elements. The overall trend was high temperature and strong light, low humidity was favorable for anther opening, and low temperature, weak light, high humidity was favorable for anther closing. In this experiment, the effect of these ecological elements on the movement of anthers opening from strong to weak was humidity, temperature, light. The effect of these ecological elements on the movement of anthers closing from strong to weak was light, humidity, temperature. The direct contact of the raindrops causes the rapid closing of the anthers in the rain. Observing the cell morphology change when anthers opening and closing, it was a pollen sac dehydration and water-absorption process, different light, temperature and humidity conditions induce different physiological activities in the cell, which caused the cell osmotic pressure change, eventually resulting in anther opening and closing movement. But anthers closing caused by the rain was a simple physical adjustment process, the raindrops fell on the anthers directly, which caused anthers soaked water and change of the cell osmotic pressure, then resulting in anther closing.