Abnormal Tapetum Research Articles

NARROW AND ROLLED LEAF 2 regulates leaf shape, male fertility, and seed size in rice.

Grain yield in rice (Oryza sativa L.) is closely related to leaf and flower development. Coordinative regulation of leaf, pollen, and seed development in rice as a critical biological and agricultural question should be addressed. Here we identified two allelic rice mutants with narrow and semi-rolled leaves, named narrow and rolled leaf 2-1 (nrl2-1) and nrl2-2. Map-based molecular cloning revealed that NRL2 encodes a novel protein with unknown biochemical function. The mutation of NRL2 caused pleiotropic effects, including a reduction in the number of longitudinal veins, defective abaxial sclerenchymatous cell differentiation, abnormal tapetum degeneration and microspore development, and the formation of more slender seeds compared with the wild type (WT). The NRL2 protein interacted with Rolling-leaf (RL14), causing the leaves of the nrl2 mutants to have a higher cellulose content and lower lignin content than the WT, which may have been related to sclerenchymatous cell differentiation and tapetum degeneration. Thus, this gene is an essential developmental regulator controlling fundamental cellular and developmental processes, serving as a potential breeding target for high-yielding rice cultivars.

MID1 plays an important role in response to drought stress during reproductive development.

Drought during rice reproductive development results in yield loss. It is important to understand the functions of drought-responsive genes in reproductive tissues for improving rice yield under water-deficit conditions. We show here that MID1 (MYB Important for Drought Response1), encoding a putative R-R-type MYB-like transcription factor, can improve rice yield under drought. MID1 was primarily expressed in root and leaf vascular tissues, with low level in the tapetum, and was induced by drought and other abiotic stresses. Compared with wild type, MID1-overexpressing plants were more tolerant to drought at both vegetative and reproductive stages and produced more grains under water stress. MID1-overexpressing plants exhibited less severe anther defects such as deformed anther locules, abnormal tapetum, degenerated microspores and expanded middle layer, with improved pollen fertility and higher seed setting rate. MID1 was localized to the nucleus and could activate gene expression in yeast, and its homologs were identified in many other plants with high levels sequence similarity. In addition, candidate MID1-regulated genes were analyzed using RNA-seq and qRT-PCR, including genes crucial for stress responses and anther development, with altered expressions in the florets of MID1-overexpressing plants and RNAi lines. Furthermore, MID1 could bind to the promoters of two drought-related genes (Hsp17.0 and CYP707A5) and one anther developmental gene (KAR) according to ChIP-qPCR data. Our findings suggest that MID1 is a transcriptional regulator that promotes rice male development under drought by modulating the expressions of drought-related and anther developmental genes and provide valuable information for crop improvement.

Involvement of a universal amino acid synthesis impediment in cytoplasmic male sterility in pepper.

To explore the mechanisms of pepper (Capsicum annuum L.) cytoplasmic male sterility (CMS), we studied the different maturation processes of sterile and fertile pepper anthers. A paraffin section analysis of the sterile anthers indicated an abnormality of the tapetal layer and an over-vacuolization of the cells. The quantitative proteomics results showed that the expression of histidinol dehydrogenase (HDH), dihydroxy-acid dehydratase (DAD), aspartate aminotransferase (ATAAT), cysteine synthase (CS), delta-1-pyrroline-5-carboxylate synthase (P5CS), and glutamate synthetase (GS) in the amino acid synthesis pathway decreased by more than 1.5-fold. Furthermore, the mRNA and protein expression levels of DAD, ATAAT, CS and P5CS showed a 2- to 16-fold increase in the maintainer line anthers. We also found that most of the amino acid content levels decreased to varying degrees during the anther tapetum period of the sterile line, whereas these levels increased in the maintainer line. The results of our study indicate that during pepper anther development, changes in amino acid synthesis are significant and accompany abnormal tapetum maturity, which is most likely an important cause of male sterility in pepper.

Comparative analysis of microRNA profiles of rice anthers between cool-sensitive and cool-tolerant cultivars under cool-temperature stress.

Plants subjected to abiotic stress can regulate gene expression post-transcriptionally by means of small RNAs such as microRNAs. Cool-temperature stress causes abnormal tapetum hypertrophy in rice anthers, leading to pollen sterility. As a first step toward understanding the molecular mechanisms of cool tolerance in developing anthers of rice, we report here a comprehensive comparative analysis of microRNAs between cool-sensitive Sasanishiki and cool-tolerant Hitomebore cultivars. High-throughput Illumina sequencing revealed 241 known and 46 novel microRNAs. Interestingly, 15 of these microRNAs accumulated differentially in the two cultivars at the uninucleate microspore stage under cool conditions. Inverse correlations between expression patterns of microRNAs and their target genes were confirmed by quantitative RT-PCR analysis, and cleavage sites of some of the target genes were determined by 5' RNA ligase-mediated RACE experiments. Thus, our data are useful resources to elucidate microRNA-mediated mechanism(s) of cool tolerance in rice anthers at the booting stage.

The DYT1-interacting proteins bHLH010, bHLH089 and bHLH091 are redundantly required for Arabidopsis anther development and transcriptome.

The anther is the male reproductive organ of flowering plants, and the Arabidopsis bHLH transcription factors encoded by DYSFUNCTIONAL TAPETUM1 (DYT1) and ABORTED MICROSPORE (AMS) are required for control of the complex transcriptional networks regulating anther development. Knowledge of the mechanisms by which the bHLH proteins affect this diverse gene expression is quite limited. We examine here three recently duplicated Arabidopsis bHLH genes, bHLH010, bHLH089 and bHLH091, using evolutionary, genetic, morphological and transcriptomic approaches, and uncover their redundant functions in anther development. These three genes are relatively highly expressed in the tapetum of the Arabidopsis anther; single mutants at each of the bHLH010, bHLH089 and bHLH091 loci are developmentally normal, but the various double and triple combinations progressively exhibit increasingly defective anther phenotypes (abnormal tapetum morphology, delayed callose degeneration, and aborted pollen development), indicating their redundant functions in male fertility. Further transcriptomic and molecular analyses suggest that these three proteins act slightly later than DYT1, and also form protein complexes with DYT1, subsequently affecting the correct expression of many DYT1 target genes in the anther development transcriptional network. This study demonstrated that bHLH010, bHLH089 and bHLH091 together are important for the normal transcriptome of the developing Arabidopsis anther, possibly by forming a feed-forward loop with DYT1.

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.

Tomato Male sterile 1035 is essential for pollen development and meiosis in anthers.

Male fertility in flowering plants depends on proper cellular differentiation in anthers. Meiosis and tapetum development are particularly important processes in pollen production. In this study, we showed that the tomato male sterile (ms10(35)) mutant of cultivated tomato (Solanum lycopersicum) exhibited dysfunctional meiosis and an abnormal tapetum during anther development, resulting in no pollen production. We demonstrated that Ms10(35) encodes a basic helix-loop-helix transcription factor that is specifically expressed in meiocyte and tapetal tissue from pre-meiotic to tetrad stages. Transgenic expression of the Ms10(35) gene from its native promoter complemented the male sterility of the ms10(35) mutant. In addition, RNA-sequencing-based transcriptome analysis revealed that Ms10(35) regulates 246 genes involved in anther development processes such as meiosis, tapetum development, cell-wall degradation, pollen wall formation, transport, and lipid metabolism. Our results indicate that Ms10(35) plays key roles in regulating both meiosis and programmed cell death of the tapetum during microsporogenesis.

Deficiency in a very-long-chain fatty acid β-ketoacyl-coenzyme a synthase of tomato impairs microgametogenesis and causes floral organ fusion.

Previously, it was shown that β-ketoacyl-coenzyme A synthase ECERIFERUM6 (CER6) is necessary for the biosynthesis of very-long-chain fatty acids with chain lengths beyond C₂₈ in tomato (Solanum lycopersicum) fruits and C₂₆ in Arabidopsis (Arabidopsis thaliana) leaves and the pollen coat. CER6 loss of function in Arabidopsis resulted in conditional male sterility, since pollen coat lipids are responsible for contact-mediated pollen hydration. In tomato, on the contrary, pollen hydration does not rely on pollen coat lipids. Nevertheless, mutation in SlCER6 impairs fertility and floral morphology. Here, the contribution of SlCER6 to the sexual reproduction and flower development of tomato was addressed. Cytological analysis and cross-pollination experiments revealed that the slcer6 mutant has male sterility caused by (1) hampered pollen dispersal and (2) abnormal tapetum development. SlCER6 loss of function provokes a decrease of n- and iso-alkanes with chain lengths of C₂₇ or greater and of anteiso-alkanes with chain lengths of C₂₈ or greater in flower cuticular waxes, but it has no impact on flower cuticle ultrastructure and cutin content. Expression analysis confirmed high transcription levels of SlCER6 in the anther and the petal, preferentially in sites subject to epidermal fusion. Hence, wax deficiency was proposed to be the primary reason for the flower fusion phenomenon in tomato. The SlCER6 substrate specificity was revisited. It might be involved in elongation of not only linear but also branched very-long-chain fatty acids, leading to production of the corresponding alkanes. SlCER6 implements a function in the sexual reproduction of tomato that is different from the one in Arabidopsis: SlCER6 is essential for the regulation of timely tapetum degradation and, consequently, microgametogenesis.

Over-expression of Arginine Decarboxylase Gene in Tapetal Tissue Results in Male Sterility in Tomato Plants

Homeostasis of cellular Polyamines (PAs) is very important for proper growth and development of living organisms. Development of male sex organ and gametophyte in plants is a complex process involving many known and unknown pathways. PAs are actively involved in the development of anther and pollen. In the present study, oat arginine decarboxylase (ADC) gene has been over-expressed in tapetum of tomato using tapetum-specific TA29 promoter to perturb the PA homeostasis in order to see its implication on male fertility. Tapetum-specific ADC overexpression resulted in PA accumulation, mainly in free and bound fractions in the anther of transgenics. Transgenic lines displayed abnormal pollen and tapetum development. Pollen grains were aborted and distorted in morphology and tapetum showed premature degeneration in ADC transgenics. These transgenics failed to set seeds, but cross pollination with normal pollen from wild type plants restored the seed setting indicating that s ADC over-expression did not affect female fertility. ADC over-expression also did not affect the growth and morphology of transgenics. The present study suggests that the excess of PAs in anther is lethal for pollen and tapetum development and overexpression of ADC gene can be an effective approach for the engineering of male sterility in plants.

Brassinosteroids control male fertility by regulating the expression of key genes involved in Arabidopsis anther and pollen development

The development of anther and pollen is important for male reproduction, and this process is coordinately regulated by many external and internal cues. In this study, we systematically examined the male reproductive phenotypes of a series of brassinosteroid biosynthetic and signaling mutants and found that, besides the expected cell-expansion defects, these mutants also showed reduced pollen number, viability, and release efficiency. These defects were related with abnormal tapetum and microspore development. Using both real-time quantitative RT-PCR and microarray experiments, we found that the expression of many key genes required for anther and pollen development was suppressed in these mutants. ChIP analysis demonstrated that BES1, an important transcription factor for brassinosteroid signaling, could directly bind to the promoter regions of genes encoding transcription factors essential for anther and pollen development, SPL/NZZ, TDF1, AMS, MS1, and MS2. Taken together, these data lead us to propose that brassinosteroids control male fertility at least in part via directly regulating key genes for anther and pollen development in Arabidopsis. Our work provides a unique mechanism to explain how a phytohormone regulates an essential genetic program for plant development.

Suppression and restoration of male fertility using a transcription factor

The Arabidopsis AtMYB103 gene codes for an R2R3 MYB domain protein whose expression is restricted to the tapetum of developing anthers and to trichomes. Down-regulation of expression using anti-sense leads to abnormal tapetum and pollen development, although seed setting still occurs (Higginson, T., Li, S.F. and Parish, R.W. (2003) AtMYB103 regulates tapetum and trichome development in Arabidopsis thaliana. Plant J. 35, 177-192). In this study, we show that blocking the function of the AtMYB103 gene, employing either an insertion mutant or an AtMYB103EAR chimeric repressor construct under the control of the AtMYB103 promoter, results in complete male sterility and failure to set seed. These plants exhibit similar abnormalities in tapetum and pollen development, with the tapetum becoming highly vacuolated at early stages and degenerating prematurely. No exine is deposited on to the pollen wall. The degeneration of pollen grains commences prior to pollen mitosis, the pollen collapsing and largely lacking cytoplasmic content. A restorer containing the AtMYB103 gene under the control of a stronger anther-specific promoter was introduced into pollen donor plants and crossed into the male sterile plants transgenic for the repressor. The male fertility of F1 plants was restored. The chimeric repressor and the restorer constitute a reversible male sterility system which could be adapted for hybrid seed production. This is the first reversible male sterility system targeting a transcription factor essential for pollen development. Strategies for generating inducible male sterility and maintainable male sterility for the production of hybrid crops are discussed.

The implications of aberrant anatomical and histochemical features in the anthers of male sterile dsf-15 sunflower - Helianthus petiolaris Nutt.

Present investigation aims at determining the developmental stage at which the male sterile anthers of DSF-15 Helianthus petiolaris Nutt. deviate from their isogenic fertile counterparts and the underlying histochemical factors responsible for that deviation. The flower buds of fertile and male sterile H.petiolaris were fixed in the Carnoy’s fluid, dehydrated in ethyl alcohol series, embedded in paraffin and microtomed. The sections were stained for the localization of insoluble polysaccharides, RNA and total proteins using periodic acid Schiff’s toluidine blue and mercuric bromophenol blue reagents respectively. The results of the present investigation reveal that, during premeiotic stages, anthers of fertile and sterile lines are indistinguishable except in the occurrence of starch storage in the sterile anthers during the onset of meiosis. In the sterile anthers, precocious degeneration of callose failure of microscope wall information, persistent abnormal tapetum (hypertrophied or premature plasmodial formation), failure of formation of endothecial thickenings accompany the abortion process of microspores. The malfunctioning of the tapetum seems to be responsible for male sterility in DSF-15 H.petiolaris.