Mitochondrial Proteomics and Transcriptional Analysis of Cytoplasmic Male Sterility in Sugar Beet using iTRAQ and qRT–PCR

Sugar beet (Beta vulgaris L.) is an important raw material for the sugar industry, and its output is second only to sugar cane. Cytoplasmic male sterility (CMS) is a phenomenon of pollen abortion that has important implications in sugar beet hybrid breeding. Male plant sterility is usually considered to be associated with mitochondrial dysfunction. Although mitochondrial genes associated with male sterility have been well explored, the different mitochondrial proteomics of CMS in sugar beet are still poorly understood. In this study, differentially expressed mitochondrial proteomic analysis was performed on the flower buds of the male sterile line (DY5-CMS), its maintainer line (DY5-O) and a fertility restorer line (CL6), using an isobaric tag for relative and absolute quantitation (iTRAQ) technology. A total of 2260 proteins were identified by mass spectrometry, of which 538 were differentially expressed proteins. Most of them were involved in protein metabolism, carbohydrate and energy metabolism, and binding. More specifically, some cysteine and methionine metabolism proteins (A0A0J8BGE0, A0A0J8CZM6, A0A0J8D7W0 and A0A0J8BCR7) may play important roles during the formation of CMS. This study provided an in–depth understanding of the CMS molecular mechanism at the protein level in sugar beet.

TaEXPB5 is responsible for male fertility in thermo-sensitive male-sterility wheat with Aegilops kotschyi cytoplasm

Thermo-sensitive male sterility is of vital importance to heterosis, or hybrid vigor in crop production and hybrid breeding. Therefore, it is meaningful to study the function of the genes related to pollen development and male sterility, which is still not fully understand currently. Here, we conducted comparative analyses to screen fertility related genes using RNA-seq, iTRAQ, and PRM-based assay. A gene encoding expansin protein in wheat, TaEXPB5, was isolated in KTM3315A, which was in the cell wall and preferentially upregulated expression in the fertility anthers. The silencing of TaEXPB5 displayed pollen abortion, the declination or sterility of fertility. Further, cytological investigation indicated that the silencing of TaEXPB5 induced the early degradation of tapetum and abnormal development of pollen wall. These results revealed that the silencing of TaEXPB5 could eliminate the effects of temperature on male fertility, and resulting in functional loss of fertility conversion, which implied that TaEXPB5 may be essential for anther or pollen development and male fertility of KTM3315A. These findings provide a novel insight into molecular mechanism of fertility conversion for thermo-sensitive cytoplasmic male-sterility wheat, and contribute to the molecular breeding of hybrid wheat in the future.

Combined Transcriptome and Proteome Analysis of Anthers of AL-type Cytoplasmic Male Sterile Line and Its Maintainer Line Reveals New Insights into Mechanism of Male Sterility in Common Wheat

Cytoplasmic male sterility (CMS) plays an essential role in hybrid seeds production. In wheat, orf279 was reported as a CMS gene of AL-type male sterile line (AL18A), but its sterility mechanism is still unclear. Therefore, transcriptomic and proteomic analyses of the anthers of AL18A and its maintainer line (AL18B) were performed to interpret the sterility mechanism. Results showed that the electron transport chain and ROS scavenging enzyme expression levels changed in the early stages of the anther development. Biological processes, i.e., fatty acid synthesis, lipid transport, and polysaccharide metabolism, were abnormal, resulting in pollen abortion in AL18A. In addition, we identified several critical regulatory genes related to anther development through combined analysis of transcriptome and proteome. Most of the genes were enzymes or transcription factors, and 63 were partially homologous to the reported genic male sterile (GMS) genes. This study provides a new perspective of the sterility mechanism of AL18A and lays a foundation to study the functional genes of anther development.

High Temperature-induced Spindle Destabilization Results in Aborted Pollen Production in Populus

High temperature can induce the production of 2n gametes and aborted pollen during microsporogenesis in Populus canescens. However, the mechanism by which high temperature induces pollen abortion remains unknown. Here, pollen abortion was induced by exposing male flower buds of P. canescens to 38 and 41 °C; pollen morphology, meiotic abnormalities, defects of the meiotic microtubular cytoskeleton, and tapetum development were characterized, and expression analysis of the Actin gene was conducted. We found that the dominant meiotic stage, temperature, and duration of treatment significantly affected the percentage of high temperature-induced aborted pollen. Damaged spindle microtubules and depolymerized microtubular cytoskeletons were observed, which resulted in many lagging chromosomes at anaphase Ⅰ and Ⅱ, as well as aneuploid male gametes and micronuclei, generating aborted pollen grains. Tapetum disintegration was also delayed. However, the anther dehisced normally, and some viable pollen grains were released, suggesting that the delayed degradation of the tapetum was not responsible for pollen abortion. A significant reduction in PtActin gene expression was detected in treated cells, indicating that spindle actin was disrupted. The spindle actin appeared to protect cells against chromosome segregation errors during meiosis.

Long-term mild heat causes post-mitotic pollen abortion through a local effect on flowers

Crop reproductive success is significantly challenged by heatwaves, which are increasing in frequency and severity globally. A major reason is reduced male fertility due to deviations in pollen development, but the mechanism behind this is not well understood. Here, long-term mild heat (LTMH) treatment, mimicking a heatwave, was applied locally to flowers or to whole plants and followed up by cytological, transcriptomic and biochemical analyses. LTMH was shown to act directly on the flowers and not via a systemic effect on other plant tissue. The meiosis to early microspore stage was the most sensitive to LTMH and three days of exposure around this period was sufficient to significantly reduce pollen viability. Extensive cytological analysis showed that abnormalities in pollen development could first be observed after pollen mitosis I, while tapetum development appeared unaffected. Transcriptomic and biochemical analyses suggested that pollen development suffered from tapetal ER stress and that there was a limited role for oxidative stress. These characteristics differentiate the response of developing anthers and pollen to LTMH from the response to severe heat stress.

Morphological characteristics and transcriptome analysis at different anther development stages of the male sterile mutant MS7–2 in Wucai (Brassica campestris L.)

Background The discovery of male sterile materials is of great significance for the development of plant fertility research. Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a variety of non-heading Chinese cabbage. There are few studies on the male sterility of wucai, and the mechanism of male sterility is not clear. In this study, the male sterile mutant MS7–2 and the wild-type fertile plant MF7–2 were studied. Results Phenotypic characteristics and cytological analysis showed that MS7–2 abortion occurred at the tetrad period. The content of related sugars in the flower buds of MS7–2 was significantly lower than that of MF7–2, and a large amount of reactive oxygen species (ROS) was accumulated. Through transcriptome sequencing of MS7–2 and MF7–2 flower buds at three different developmental stages (a–c), 2865, 3847, and 4981 differentially expressed genes were identified in MS7–2 at the flower bud development stage, stage c, and stage e, respectively, compared with MF7–2. Many of these genes were enriched in carbohydrate metabolism, phenylpropanoid metabolism, and oxidative phosphorylation, and most of them were down-regulated in MS7–2. The down-regulation of genes involved in carbohydrate and secondary metabolite synthesis as well as the accumulation of ROS in MS7–2 led to pollen abortion in MS7–2. Conclusions This study helps elucidate the mechanism of anther abortion in wucai, providing a basis for further research on the molecular regulatory mechanisms of male sterility and the screening and cloning of key genes in wucai.

A Novel Lateral Organ Boundary-domain Factor CmLBD2 Positively Regulates Pollen Development via Activating CmACOS5 in Chrysanthemum morifolium

ABSTRACT Male sterility, as a common reproductive characteristic in plants, plays an important role in breeding, in which pollen abortion is a key factor leading to male sterility. Here, based on a low expression level gene CmACOS5 in transcriptome of pollen abortive chrysanthemum, a new transcription factor CmLBD2 of the Lateral Organ Boundaries Domain family which could bind the promoter of CmACOS5 by yeast one-hybrid library was screened. This study revealed the origin and expression pattern of CmLBD2 in chrysanthemum, verified the functions of two genes in pollen development by transgenic means. Inhibiting the expression of CmACOS5 or CmLBD2 can lead to a large reduction in pollen and even abortion in chrysanthemum. Using yeast one/two-hybrid, electrophoretic mobility shift assays, and luciferase reporter assays, it was verified that CmLBD2 directly binds to the promoter of CmACOS5. These results suggest that LBD2 is a novel, key transcription factor regulating pollen development. This result will provide a new research background for enriching the function of LBD family proteins, and also lay a new foundation for the breeding of male sterile lines and the mechanism of pollen development.

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.

Comparative Transcriptome Identifies Gene Expression Networks Regulating Developmental Pollen Abortion in Ogura Cytoplasmic Male Sterility in Chinese Cabbage (Brassica rapa ssp. pekinensis)

Ogura cytoplasmic male sterility (Ogura CMS), originally identified in wild radish (Raphanus sativus), has enabled complete pollen sterility in Brassica plants, but the underlying mechanism in Ogura CMS Chinese cabbage (Brassica rapa ssp. pekinensis) remains unclear. In this study cytological analysis showed that during microsporogenesis the meiosis occurred normally, and the uninucleated pollens subsequently formed, but the development of both binucleated and trinucleated pollens was obviously disrupted due to defects of pollen mitosis in the Ogura CMS line (Tyms) compared with the corresponding maintainer line (231–330). In transcriptome profiling a total of 8052 differentially expressed genes (DEGs) were identified, among which 3890 were up-regulated and 4162 were down-regulated at the pollen abortion stages in an Ogura CMS line. KOG cluster analysis demonstrated that a large number of DEGs were related to the cytoskeleton’s dynamics, which may account for the failure of pollen mitosis during development in the Ogura CMS line. The pivotal genes related to the phenylpropane synthesis pathway (PAL, 4CL and CAD) were significantly down-regulated, which probably affected the formation and disposition of anther lignin and sporopollenin, and eventually led to abnormality in the pollen exine structure. In addition, several key up-regulated genes (GPX7, G6PD and PGD1) related to the glutathione oxidation-reduction (REDOX) reaction indicated that the accumulation of peroxides in Ogura CMS lines during this period affected the pollen development. Taken together, this cytological and molecular evidence is expected to advance our understanding of pollen abortion induced by Ogura cytoplasmic action in Chinese cabbage.

Air quality assessment using the Pollen Abortion assay in Tradescantia pallida in a Mid-sized City in Southern Brazil

Urbanization is an important source of air pollutants that can compromise human health. In developing countries, such as Brazil, most cities do not have air quality monitoring stations. Assessing air quality through plant species has gained recognized prominence, as they are sessile organisms and sensitive to environmental changes. Pollen abortion assay in Tradescantia pallida is a fast and low-cost bioassay that can be implemented in passive biomonitoring scenarios. The present study aimed to use the pollen abortion assay in T. pallida to assess air quality in the municipality of Rio Grande, RS, Brazil and the possible relationship with vehicular flow. A relation was found between the highest rate of pollen abortion and the sites where there was greater vehicular flow and ozone levels, while at the control point, the lowest rate of pollen abortion among the others was found, corroborating the hypothesis that air pollution together with high levels of ozone from vehicles, impair plant pollination.