Interaction of gibberellin and other hormones in almond anthers: phenotypic and physiological changes and transcriptomic reprogramming

Low temperature causes anther dysfunction, severe pollen sterility and, ultimately, major yield losses in crop plants. Previous studies have shown that the gibberellic acid (GA) metabolic pathway plays an important role in this process by regulating tapetum function and pollen development. However, the interaction mechanism of GA with other hormones mediating anther development is still unclear. Herein, we collected and analyzed almond (Amygdalus communis L.) anthers at the meiosis, tetrad, 1-nucleus, and mature 2-nucleus stages. The growth rate per 1000 anthers exhibited a significant positive correlation with the total bioactive GA compound content, and the levels of all bioactive GA compounds were highest in the 1-nucleus pollen stage. GA3 treatment experiments indicated that exogenous GA3 increased the levels of indole-3-acetic acid (IAA), trans-zeatin (tZ), and jasmonic acid (JA) and decreased the levels of salicylic acid (SA) and abscisic acid (ABA); moreover, GA3 improved pollen viability and quantities under cold conditions, whereas PP333 (paclobutrazol, an inhibitor of GA biosynthesis) was antagonistic with GA3 in controlling anther development. RNA-seq and qRT-PCR results showed that GA played an important role in anther development by regulating the expression of other phytohormone pathway genes, dehydration-responsive element-binding/C-repeat binding factor (DREB1/CBF)-mediated signaling genes, and anther development pathway genes. Our results reveal the novel finding that GA interacts with other hormones to balance anther development under normal- and low-temperature conditions in almond.

Integrated Proteo-Transcriptomic Analyses Reveal Insights into Regulation of Pollen Development Stages and Dynamics of Cellular Response to Apple Fruit Crinkle Viroid (AFCVd)-Infection in Nicotiana tabacum

Tobacco (Nicotiana tabacum) pollen is a well-suited model for studying many fundamental biological processes owing to its well-defined and distinct development stages. It is also one of the major agents involved in the transmission of infectious viroids, which is the primary mechanism of viroid pathogenicity in plants. However, some viroids are non-transmissible and may be possibly degraded or eliminated during the gradual process of pollen development maturation. The molecular details behind the response of developing pollen against the apple fruit crinkle viroid (AFCVd) infection and viroid eradication is largely unknown. In this study, we performed an integrative analysis of the transcriptome and proteome profiles to disentangle the molecular cascade of events governing the three pollen development stages: early bicellular pollen (stage 3, S3), late bicellular pollen (stage 5, S5), and 6 h-pollen tube (PT6). The integrated analysis delivered the molecular portraits of the developing pollen against AFCVd infection, including mechanistic insights into the viroid eradication during the last steps of pollen development. The isobaric tags for label-free relative quantification (iTRAQ) with digital gene expression (DGE) experiments led us to reliably identify subsets of 5321, 5286, and 6923 proteins and 64,033, 60,597, and 46,640 expressed genes in S3, S5, and PT6, respectively. In these subsets, 2234, 2108 proteins and 9207 and 14,065 mRNAs were differentially expressed in pairwise comparisons of three stages S5 vs. S3 and PT6 vs. S5 of control pollen in tobacco. Correlation analysis between the abundance of differentially expressed mRNAs (DEGs) and differentially expressed proteins (DEPs) in pairwise comparisons of three stages of pollen revealed numerous discordant changes in mRNA/protein pairs. Only a modest correlation was observed, indicative of divergent transcription, and its regulation and importance of post-transcriptional events in the determination of the fate of early and late pollen development in tobacco. The functional and enrichment analysis of correlated DEGs/DEPs revealed the activation in pathways involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, and cofactor as well as vitamin metabolism, which points to the importance of these metabolic pathways in pollen development. Furthermore, the detailed picture of AFCVd-infected correlated DEGs/DEPs was obtained in pairwise comparisons of three stages of infected pollen. The AFCVd infection caused the modulation of several genes involved in protein degradation, nuclear transport, phytohormone signaling, defense response, and phosphorylation. Intriguingly, we also identified several factors including, DNA-dependent RNA-polymerase, ribosomal protein, Argonaute (AGO) proteins, nucleotide binding proteins, and RNA exonucleases, which may plausibly involve in viroid stabilization and eradication during the last steps of pollen development. The present study provides essential insights into the transcriptional and translational dynamics of tobacco pollen, which further strengthens our understanding of plant-viroid interactions and support for future mechanistic studies directed at delineating the functional role of candidate factors involved in viroid elimination.

Ultrastructural characterization of microlipophagy induced by the interaction of vacuoles and lipid bodies around generative and sperm cells in Arabidopsis pollen

During pollen maturation, various organelles change their distribution and function during development as male gametophytes. We analyzed the behavior of lipid bodies and vacuoles involved in lipophagy in Arabidopsis pollen using serial section SEM and conventional TEM. At the bicellular pollen stage, lipid bodies in the vegetative cells lined up at the surface of the generative cell. Vacuoles then tightly attached, drew in, and degraded the lipid bodies and eventually occupied the space of the lipid bodies. Degradation of lipid began before transfer of the entire contents of the lipid body. At the tricellular stage, vacuoles instead of lipid bodies surrounded the sperm cells. The degradation of lipid bodies is morphologically considered microautophagy. The atg2-1 Arabidopsis mutant is deficient in one autophagy-related gene (ATG). In this mutant, the assembly of vacuoles around sperm cells was sparser than that in wild-type pollen. The deficiency of ATG2 likely prevents or slows lipid degradation, although it does not prevent contact between organelles. These results demonstrate the involvement of microlipophagy in the pollen development of Arabidopsis.

OsmiR528 regulates rice-pollen intine formation by targeting an uclacyanin to influence flavonoid metabolism

The intine, the inner layer of the pollen wall, is essential for the normal development and germination of pollen. However, the composition and developmental regulation of the intine in rice (Oryza sativa) remain largely unknown. Here, we identify a microRNA, OsmiR528, which regulates the formation of the pollen intine and thus male fertility in rice. Themir528knockout mutant aborted pollen development at the late binucleate pollen stage, significantly decreasing the seed-setting rate. We further demonstrated that OsmiR528 affects pollen development by directly targeting the uclacyanin geneOsUCL23(encoding a member of the plant-specific blue copper protein family of phytocyanins) and regulating intine deposition.OsUCL23overexpression phenocopied themir528mutant. The OsUCL23 protein localized in the prevacuolar compartments (PVCs) and multivesicular bodies (MVBs). We further revealed that OsUCL23 interacts with a member of the proton-dependent oligopeptide transport (POT) family of transporters to regulate various metabolic components, especially flavonoids. We propose a model in which OsmiR528 regulates pollen intine formation by directly targetingOsUCL23and in which OsUCL23 interacts with the POT protein on the PVCs and MVBs to regulate the production of metabolites during pollen development. The study thus reveals the functions of OsmiR528 and an uclacyanin during pollen development.

Cytological and transcriptome analyses reveal OsPUB73 defect affects the gene expression associated with tapetum or pollen exine abnormality in rice

Background As one of the main crops in the world, sterility of rice (Oryza sativa L.) significantly affects the production and leads to yield decrease. Our previous research showed that OsPUB73, which encodes U-box domain-containing protein 73, may be associated with male sterility. However, little information is available on this gene that is required for anther development. In the present study, we knocked out OsPUB73 by using the CRISPR/Cas9 system and studied the cytological and transcriptome of the gene-defect associated with pollen development and sterility in the rice variety (Taichung 65). Results The sequence analysis indicated that OsPUB73 was comprised of 3 exons and 2 introns, of which CDS encoded 586 amino acids including a U-box domain. The expression pattern of OsPUB73 showed that it was highly expressed in the anther during meiosis stage. The ospub73 displayed low pollen fertility (19.45%), which was significantly lower than wild type (WT) (85.37%). Cytological observation showed tapetum vacuolated at the meiosis stage and pollen exine was abnormal at the bi-cellular pollen stage of ospub73. RNA-seq analysis detected 2240 down and 571 up-regulated genes in anther of ospub73 compared with WT during meiosis stage. Among of 2240 down-regulated genes, seven known genes were associated with tapetal cell death or pollen exine development, including CYP703A3 (Cytochrome P450 Hydroxylase703A3), CYP704B2 (Cytochrome P450 Hydroxylase704B2), DPW (Defective Pollen Wall), PTC1 (Persistant Tapetal Cell1), UDT1 (Undeveloped Tapetum1), OsAP37 (Aspartic protease37) and OsABCG15 (ATP binding cassette G15), which were validated by quantitative real-time polymerase chain reaction (qRT-PCR). These results suggested OsPUB73 may play an important role in tapetal or pollen exine development and resulted in pollen partial sterility. Conclusion Our results revealed that OsPUB73 plays an important role in rice male reproductive development, which provides valuable information about the molecular mechanisms of the U-box in rice male reproductive development.

AtNOT1 Is a Novel Regulator of Gene Expression during Pollen Development

Development of pollen, the male gametophyte of flowering plants, is tightly controlled by dynamic changes in gene expression. Recent research to clarify the molecular aspects of pollen development has revealed the involvement of several transcription factors in the induction of gene expression. However, limited information is available about the factors involved in the negative regulation of gene expression to eliminate unnecessary transcripts during pollen development. In this study, we revealed that AtNOT1 is an essential protein for proper pollen development and germination capacity. AtNOT1 is a scaffold protein of the AtCCR4–NOT complex, which includes multiple components related to mRNA turnover control in Arabidopsis. Phenotypic analysis using atnot1 heterozygote mutant pollen showed that the mature mutant pollen failed to germinate and also revealed abnormal localization of nuclei and a specific protein at the tricellular pollen stage. Furthermore, transcriptome analysis of atnot1 heterozygote mutant pollen showed that the downregulation of a large number of transcripts, along with the upregulation of specific transcripts required for pollen tube germination by AtNOT1 during late microgametogenesis, is important for proper pollen development and germination. Overall, our findings provide new insights into the negative regulation of gene expression during pollen development, by showing the severely defective phonotype of atnot1 heterozygote mutant pollen.

Metabolic Alterations in Male-Sterile Potato as Compared to Male-Fertile

The common potato, Solanum tuberosum L., is the fourth most important agricultural crop worldwide. Until recently, vegetative propagation by tubers has been the main method of potato cultivation. A shift of interest to sexual potato reproduction by true botanical seeds is due to the appearance of a new hybrid seed breeding strategy whose successful application for many crop species has been supported by male sterility. This investigation was focused on the study of differences in the metabolite profiles of anthers at the mature pollen stage from male-fertile and male-sterile genotypes of S. tuberosum. Application of gas chromatography coupled with a mass spectrometry method allowed detection of metabolic profiles for 192 compounds. Further data analysis with several libraries fully identified 75 metabolites; a similar amount was defined up to the classes. Metabolic profiles in the anthers of fertile genotypes were significantly distinguished from male-sterile ones by the accumulation of carbohydrates, while the anthers of sterile genotypes contained a higher amount of amino acids. In comparison with male-fertile plants, male-sterile genotypes had undeveloped pollen grain characters; i.e., smaller grain size, a thicker exine, “permanent tetrads” that failed to disintegrate into microspores, and the absence of pollen apertures that might be due to a disorder in the metabolism of carbohydrates and fatty acids.

Meiotic Defects and Premature Tapetal Degeneration Are Involved in the Low Fertility of Oncidesa Gower Ramsey, an Important Cut-flower Orchid

The dancing-lady orchid, Oncidesa Gower Ramsey, is an important cultivar for cut-flower production, but it has low pollen fertility in breeding programs. In this study, we compared the pollen germination in vitro, sporad type, and pollinia development of Oncsa. Gower Ramsey and a diploid species, Oncidium sphacelatum Lindl (one of its grandparents). In Oncsa. Gower Ramsey, the pollen germination in vitro was lower as compared with those in Onc. sphacelatum. In addition, the frequency of abnormal sporads in Oncsa. Gower Ramsey was higher than those in Onc. sphacelatum. In Oncsa. Gower Ramsey, the middle layer and the tapetum were disorganized before meiosis, and subsequently they degenerated at the early tetrad stage. In contrast, the middle layer and the tapetum of Onc. sphacelatum began to degenerate at the early tetrad stage and fully disappeared at the bicellular pollen stage. These results suggested that the abnormal meiosis caused by unbalanced genomes and the premature degeneration of the middle layer and the tapetum could probably result in the abnormal pollen development and the low fertility of Oncsa. Gower Ramsey.

Ultrastructural study of maturing pollen in Arabidopsis thaliana (L.) Heynh. (Brassicaceae)

Ultrastructural changes in <em>Arabidopsis thaliana</em> pollen, between late microspore stage and mature pollen stage were described. When the generative cell was peeled off from the intine, it was of spherical shape and had all usual organelles with the exception of plastids. The cytoplasm transformation of the vegetative cell included an increase in the number of mitochondria and changes in the accumulation of starch and lipid bodies. The starch plastids were observed at the bicellular and early tricellular pollen stages and next starch was utilized during the maturation procces. The lipid bodies of the vegetative cell form a very regular sheath around the generative cell and then, around the sperm cells. Before anthesis the lipid bodies were dispersed within the whole vegetative cell cytoplasm.

Immunolocalization of Lipoxygenase in the Anther Wall Cells of Lathyrus undulatus Boiss. during Programmed Cell Death

Lipoxygenase catalyzes oxygenation of long chain fatty acids to hydroperoxides and is involved in the degradation of membranes occuring in some types of programmed cell death (PCD). The localization of lipoxygenase in the anther wall layers of L. undulatus during cellular degradation was analyzed by immunogold labeling technique at young and vacuolated pollen stage, due to the close relation between lipoxygenase activity and membrane degradation in programmed cell death. Immunoreaction to lipoxygenase was monitored slightly at young pollen stage in the anther wall cells. As programmed cell death signals progress, lipoxygenase revealed in anther wall cells intensely. At vacuolated pollen stage tapetal cells came forward with ultrastructural changes such as cell, organelle and membrane disintegration. At the indicated stage immunogold particles indicating sites of LOX PAb-binding epitopes were located in the nucleus (chromatin was condensed and lined at the periphery), cytoplasm and close to long dilated rough endoplasmic reticulum (RER) cisterna. In conclusion lipoxygenase increase which has a role in the membrane degeneration, possibly induced the collapse of tonoplast, nuclear and plasma membrane and triggered programmed cell death in the tapetal cells of L. undulatus as well as the other wall cells.