Search

Tomato plants favor warmth, making them particularly susceptible to cold conditions, especially their reproductive development. Therefore, understanding how pollen reacts to cold stress is vital for selecting and improving cold-resistant tomato varieties. The programmed cell death (PCD) in the tapetum is particularly susceptible to cold temperatures which could hinder the degradation of the tapetal layer in the anthers, thus affecting pollen development. However, it is not clear yet how genes integral to tapetal degradation respond to cold stress. Here, we report that SlHB8, working upstream of the conserved genetic module DYT1-TDF1-AMS-MYB80, is crucial for regulating cold tolerance in tomato anthers. SlHB8 expression increases in the tapetum when exposed to low temperatures. CRISPR/Cas9-generated SlHB8-knockout mutants exhibit improved pollen cold tolerance due to the reduced temperature sensitivity of the tapetum. SlHB8 directly upregulates SlDYT1 and SlMYB80 by binding to their promoters. In normal anthers, cold treatment boosts SlHB8 levels, which then elevates the expression of genes like SlDYT1, SlTDF1, SlAMS, and SlMYB80; however, slhb8 mutants do not show this gene activation during cold stress, leading to a complete blockage of delayed tapetal programmed cell death (PCD). Furthermore, we found that SlHB8 can interact with both SlTDF1 and SlMYB80, suggesting the possibility that SlHB8 might regulate tapetal PCD at the protein level. This study sheds light on molecular mechanisms of anther adaptation to temperature fluctuations. [ABSTRACT FROM AUTHOR]

Key message: Two pollen-preferential thaumatin-like proteins show both common and distinctive expression profiles. Precocious expression of one of them drastically disturbs timely deposition and dissolution of callose during microsporogenesis, leading to microspore death. Thaumatin-like proteins (TLPs), members of the pathogenesis-related protein family 5 (PR-5), are involved in plant defenses against biotic and abiotic stresses through antifungal activity and enhanced tolerance. Accordingly, studies on TLPs have focused on their responses to various pathogens and stresses and on engineering agronomically valuable crops that can be cultivated in suboptimal environments. On the other hand, the role of TLP members in plant development and their genetic regulation remains largely unexplored. Recently, we reported that the generative cell internalization after pollen mitosis I, an essential pollen patterning step for the nonmotile sperm cell delivery through a pollen tube, depends on STICKY GENERATIVE CELL which suppresses callose deposition in the nascent generative cell and interacts with a germline cell preferential GCTLP1 in Arabidopsis. Here, we additionally identified GCTLP2 which is similarly expressed in the germline cells. We generated various transgenic lines and examined their expressions and phenotypes to elucidate GCTLP functions during pollen development. Expression profiles suggest two GCTLP proteins may have common but also distinctive roles during pollen development. Importantly, ectopic expression analyses show that precocious expression of GCTLP2 severely disturbs the timely deposition and degradation of callose during microsporogenesis which is essential to produce viable microspores. Therefore, our study broadens the knowledge of TLP function and callose regulation for successful pollen development in Arabidopsis. [ABSTRACT FROM AUTHOR]

Glycoside hydrolases (GHs), enzymes that break down glycosidic bonds in carbohydrates and between carbohydrates and non-carbohydrates, are prevalent in plants, animals, microorganisms, and other organisms. The tomato is a significant crop that contains the GH17 gene family. However, its role in tomatoes has yet to be fully investigated. In this study, we identified 43 GH17 genes from the tomato genome, distributed unevenly across 12 chromosomes. We further analyzed their gene structure, phylogenetic relationships, promoter elements, and expression patterns. The promoter element analysis indicated their potential roles in response to biotic and abiotic stresses as well as phytohormone effects on growth and development. The expression studies across different tomato tissues revealed that 10 genes were specifically expressed in floral organs, with SlA6 prominently expressed early during bud formation. By using CRISPR/Cas9 gene-editing technology, SlA6 knockout plants were generated. Phenotypic characterization showed that pollen viability, pollen tube germination, fruit weight, and seed number were significantly reduced in the Sla6 mutant, but the soluble solids content (TSS) was significantly higher in the Sla6 mutant, suggesting that SlA6 affects pollen development and fruit quality. [ABSTRACT FROM AUTHOR]

Cytoplasmic male sterility (CMS) is of major agronomical relevance in hybrid breeding. In gametophytic CMS, abortion of pollen is determined by the grain genotype, while in sporophytic CMS, it is determined by the mother plant genotype. While several CMS mechanisms have been dissected at the molecular level, gametophytic CMS has not been straightforwardly accessible. We used the gametophytic Sha-CMS in Arabidopsis to characterize the cause and process of pollen abortion by implementing in vivo biosensing in single pollen and mitoTALEN mutagenesis. We obtained conclusive evidence that orf117Sha is the CMS-causing gene, despite distinct characteristics from other CMS genes. We measured the in vivo cytosolic ATP content in single pollen, followed pollen development, and analyzed pollen mitochondrial volume in two genotypes that differed only by the presence of the orf117Sha locus. Our results showed that the Sha-CMS is not triggered by ATP deficiency. Instead, we observed desynchronization of a pollen developmental program. Pollen death occurred independently in pollen grains at diverse stages and was preceded by mitochondrial swelling. We conclude that pollen death is grain-autonomous in Sha-CMS and propose that mitochondrial permeability transition, which was previously described as a hallmark of developmental and environmental-triggered cell death programs, precedes pollen death in Sha-CMS. [ABSTRACT FROM AUTHOR]

Background: Understanding the mechanism of male sterility is crucial for producing hybrid seeds and developing sterile germplasm resources. However, only a few cytoplasmic male sterility (CMS) lines of cotton have been produced due to several challenges, like inadequate variation of agronomic traits, incomplete sterility, weak resilience of restorer lines, and difficulty in combining strong dominance. Therefore, the morphological and cytological identification of CMS in cotton will facilitate hybrid breeding. Results: Two F2 segregating populations of cotton were constructed from cytoplasmic male sterile lines (HaA and 01A, maternal) and restorer lines (HaR and 26R, paternal). Genetic analysis of these populations revealed a segregation ratio of 3:1 for fertile to sterile plants. Phenotypic analysis indicated no significant differences in traits of flower bud development between sterile and fertile plants. However, sterile plants exhibited smaller floral organs, shortened filament lengths, and anther atrophy on the flowering day in comparison with the fertile plants. When performed scanning electron microscopy (SEM), the two F2 populations revealed morphological variations in the anther epidermis. Cellular analysis showed no significant differences in pollen development before pollen maturation. Interestingly, between the pollen maturation and flowering stages, the tapetum layer of sterile plants degenerated prematurely, resulting in abnormal pollen grains and gradual pollen degradation. Conclusion: The results of this study suggest that fertility-restoring genes are controlled by a single dominant gene. Sterile plants exhibit distinctive floral morphology, which is characterized by stamen atrophy and abnormal anthers. Pollen abortion occurs between pollen maturity and flowering, indicating that premature tapetum degradation may be the primary cause of pollen abortion. Overall, our study provides a theoretical basis for utilizing CMS in hybrid breeding and in-depth investigation of the dominant configuration of cotton hybrid combinations, mechanisms of sterility, and the role of sterile and restorer genes. [ABSTRACT FROM AUTHOR]

Abstract Background Understanding the mechanism of male sterility is crucial for producing hybrid seeds and developing sterile germplasm resources. However, only a few cytoplasmic male sterility (CMS) lines of cotton have been produced due to several challenges, like inadequate variation of agronomic traits, incomplete sterility, weak resilience of restorer lines, and difficulty in combining strong dominance. Therefore, the morphological and cytological identification of CMS in cotton will facilitate hybrid breeding. Results Two F2 segregating populations of cotton were constructed from cytoplasmic male sterile lines (HaA and 01A, maternal) and restorer lines (HaR and 26R, paternal). Genetic analysis of these populations revealed a segregation ratio of 3:1 for fertile to sterile plants. Phenotypic analysis indicated no significant differences in traits of flower bud development between sterile and fertile plants. However, sterile plants exhibited smaller floral organs, shortened filament lengths, and anther atrophy on the flowering day in comparison with the fertile plants. When performed scanning electron microscopy (SEM), the two F2 populations revealed morphological variations in the anther epidermis. Cellular analysis showed no significant differences in pollen development before pollen maturation. Interestingly, between the pollen maturation and flowering stages, the tapetum layer of sterile plants degenerated prematurely, resulting in abnormal pollen grains and gradual pollen degradation. Conclusion The results of this study suggest that fertility-restoring genes are controlled by a single dominant gene. Sterile plants exhibit distinctive floral morphology, which is characterized by stamen atrophy and abnormal anthers. Pollen abortion occurs between pollen maturity and flowering, indicating that premature tapetum degradation may be the primary cause of pollen abortion. Overall, our study provides a theoretical basis for utilizing CMS in hybrid breeding and in-depth investigation of the dominant configuration of cotton hybrid combinations, mechanisms of sterility, and the role of sterile and restorer genes.

Ogura cytoplasmic male sterility (Ogura CMS) is extensively applied in hybrid seed production in cruciferous crops. However, the post-transcriptional molecular basis of Ogura CMS in cruciferous crops remains elusive. Here, a data-independent acquisition-based proteomic approach coupled with a parallel reaction monitoring-based targeted proteomic assay was used to analyze the proteome dynamics of Ogura CMS cabbage line RM and its maintainer line RF during floral bud development to obtain insights into the mechanism underlying Ogura CMS in cruciferous crops. A total of 9 162 proteins corresponding to 61 464 peptides were identified in RM and RF floral buds. The proteomic fluctuation of RM was weaker than that of RF. Differences in protein expression between RM and RF gradually enlarged with floral bud development. Fifteen continually up-regulated and eight continually down-regulated proteins were found in RM relative to RF throughout floral bud development. Differentially expressed proteins between RM and RF during floral bud development were implicated in the endoplasmic reticulum (ER)-associated protein processing pathway, in which most of them exhibited down-regulated expression in RM. These data suggest that ER-associated protein processing may be involved in pollen abortion in Ogura CMS cabbage by inhibiting the expression of critical factors. Our findings not only deepen the understanding of the molecular mechanisms of Ogura CMS in cruciferous crops but also provide better guidance for applying Ogura CMS in the hybrid breeding of cruciferous crops.

Achieving seedlessness in citrus varieties is one of the important objectives of citrus breeding. Male sterility associated with abnormal pollen development is an important factor in seedlessness. However, our understanding of the regulatory mechanism underlying the seedlessness phenotype in citrus is still limited. Here, we determined that the miR159a‐DUO1 module played an important role in regulating pollen development in citrus, which further indirectly modulated seed development and fruit size. Both the overexpression of csi‐miR159a and the knocking out of DUO1 in Hong Kong kumquat (Fortunella hindsii) resulted in small and seedless fruit phenotypes. Moreover, pollen was severely aborted in both transgenic lines, with arrested pollen mitotic I and abnormal pollen starch metabolism. Through additional cross‐pollination experiments, DUO1 was proven to be the key target gene for miR159a to regulate male sterility in citrus. Based on DNA affinity purification sequencing (DAP‐seq), RNA‐seq, and verified interaction assays, YUC2/YUC6, SS4 and STP8 were identified as downstream target genes of DUO1, those were all positively regulated by DUO1. In transgenic F. hindsii lines, the miR159a‐DUO1 module down‐regulated the expression of YUC2/YUC6, which decreased indoleacetic acid (IAA) levels and modulated auxin signaling to repress pollen mitotic I. The miR159a‐DUO1 module reduced the expression of the starch synthesis gene SS4 and sugar transport gene STP8 to disrupt starch metabolism in pollen. Overall, this work reveals a new mechanism by which the miR159a‐DUO1 module regulates pollen development and elucidates the molecular regulatory network underlying male sterility in citrus. [ABSTRACT FROM AUTHOR]

In recent years, the frequency and intensity of exposure to heat stress have increased gradually, seriously hampering the production of maize. This paper presents a study designed to analyze how the development, physiology and dispersal of pollen from the heat‐resistant maize variety Zhengdan958 and the heat‐sensitive maize variety Xianyu335 are affected by exposure to heat stress during the tasselling stage. Our results showed That exposure to heat stress significantly increased the antioxidant enzyme activity in pollen. Upon visual inspection of the pollen, we found that the lower water content had given rise to wrinkles in the pollen surface, sunken germination pores, and morphological deformations. In addition, the anther dehiscence process was hindered, resulting in a reduced amount of pollen being dispersed. We also found elevated levels of abscisic acid (ABA), jasmonic acid (JA) and its derivatives, indole acetic acid (IAA) and gibberellin‐3 (GA3) in heat‐pollens, as well as elevated ratios of IAA to ABA and ABA to GA3. Ultimately impaired pollen fertility. Summarizing, our experiment revealed that reduced pollen quantity and quality are significant contributors to fertility losses in summer maize exposed to heat stress at anthesis, and we hope that our analysis of the physiological mechanisms involved can contribute to the development of crop management measures aimed at countering the increasingly detrimental effects of heat stress on the production of summer maize. [ABSTRACT FROM AUTHOR]

Main conclusion: Overexpression and loss of function of OsGEX3 reduce seed setting rates and affect pollen fertility in rice. OsGEX3 positively regulates osmotic stress response by regulating ROS scavenging. GEX3 proteins are conserved in plants. AtGEX3 encodes a plasma membrane protein that plays a crucial role in pollen tube guidance. However, the function of its homolog in rice, OsGEX3, has not been determined. Our results demonstrate that OsGEX3 is localized in the plasma membrane and the nucleus as shown by a transiently transformed assay using Nicotiana benthamiana leaves. The up-regulation of OsGEX3 was detected in response to treatments with polyethylene glycol (PEG) 4000, hydrogen peroxide, and abscisic acid (ABA) via RT-qPCR analysis. Interestingly, we observed a significant decline in the seed setting rates of OsGEX3-OE lines and mutants, compared to the wild type. Further investigations reveal that overexpression and loss of function of OsGEX3 affect pollen maturation. TEM observation revealed a significant decrease in the fertile pollen rates of OsGEX3-OE transgenic lines and Osgex3 mutants due to a delay in pollen development at the late vacuolated stage. Overexpression of OsGEX3 improved osmotic stress and oxidative stress tolerance by enhancing reactive oxygen species (ROS) scavenging in rice seedlings, whereas Osgex3 mutants exhibited an opposite phenotype in osmotic stress. These findings highlight the multifunctional roles of OsGEX3 in pollen development and the response to abiotic stress. The functional characterization of OsGEX3 provides a fundamental basis for rice molecular breeding and can facilitate efforts to cultivate drought resistance and yield-related varieties. [ABSTRACT FROM AUTHOR]

Summary: Receptor‐like kinases (RLKs) are evolved for plant cell–cell communications. The typical RLK protein contains an extracellular and hypervariable N‐terminus to perceive various signals, a transmembrane domain to anchor into plasma membrane, and a cytoplasmic, highly conserved kinase domain to phosphorylate target proteins. To date, RLKs have manifested their significance in a myriad of biological processes during plant reproductive growth, especially in male fertility. This review first summarizes a recent update on RLKs and their interacting protein partners controlling anther and pollen development, pollen release from dehisced anther, and pollen function during pollination and fertilization. Then, regulatory networks of RLK signaling pathways are proposed. In addition, we predict RLKs in maize and rice genome, obtain homologs of well‐studied RLKs from phylogeny of three subfamilies and then analyze their expression patterns in developing anthers of maize and rice to excavate potential RLKs regulating male fertility in crops. Finally, current challenges and future prospects regarding RLKs are discussed. This review will contribute to a better understanding of plant male fertility control by RLKs, creating potential male sterile lines, and inspiring innovative crop breeding methods. [ABSTRACT FROM AUTHOR]

Introduction: Wheat is a staple food crop for over one-third of the global population. However, the stability of wheat productivity is threatened by heat waves associated with climate change. Heat stress at the reproductive stage can result in pollen sterility and failure of grain development. Methods: This study used transcriptome data analysis to explore the specific expression of long non-coding RNAs (lncRNAs) in response to heat stress during pollen development in four wheat cultivars. Results and discussion: We identified 11,054 lncRNA-producing loci, of which 5,482 lncRNAs showed differential expression in response to heat stress. Heatresponsive lncRNAs could target protein-coding genes in cis and trans and in lncRNA-miRNA-mRNA regulatory networks. Gene ontology analysis predicted that target protein-coding genes of lncRNAs regulate various biological processes such as hormonal responses, protein modification and folding, response to stress, and biosynthetic and metabolic processes. We also noted some paired lncRNA/protein-coding gene modules and some lncRNA-miRNAmRNA regulatory modules shared in two or more wheat cultivars. These modules were related to regulating plant responses to heat stress, such as heat-shock proteins and transcription factors, and protein domains, such as MADS-box, Myc-type, and Alpha crystallin/Hsp20 domain. Conclusion: Our results provide the basic knowledge and molecular resources for future functional studies investigating wheat reproductive development under heat stress. [ABSTRACT FROM AUTHOR]

Glycoside hydrolases (GHs), enzymes that break down glycosidic bonds in carbohydrates and between carbohydrates and non-carbohydrates, are prevalent in plants, animals, microorganisms, and other organisms. The tomato is a significant crop that contains the GH17 gene family. However, its role in tomatoes has yet to be fully investigated. In this study, we identified 43 GH17 genes from the tomato genome, distributed unevenly across 12 chromosomes. We further analyzed their gene structure, phylogenetic relationships, promoter elements, and expression patterns. The promoter element analysis indicated their potential roles in response to biotic and abiotic stresses as well as phytohormone effects on growth and development. The expression studies across different tomato tissues revealed that 10 genes were specifically expressed in floral organs, with SlA6 prominently expressed early during bud formation. By using CRISPR/Cas9 gene-editing technology, SlA6 knockout plants were generated. Phenotypic characterization showed that pollen viability, pollen tube germination, fruit weight, and seed number were significantly reduced in the Sla6 mutant, but the soluble solids content (TSS) was significantly higher in the Sla6 mutant, suggesting that SlA6 affects pollen development and fruit quality.

Tomato plants favor warmth, making them particularly susceptible to cold conditions, especially their reproductive development. Therefore, understanding how pollen reacts to cold stress is vital for selecting and improving cold-resistant tomato varieties. The programmed cell death (PCD) in the tapetum is particularly susceptible to cold temperatures which could hinder the degradation of the tapetal layer in the anthers, thus affecting pollen development. However, it is not clear yet how genes integral to tapetal degradation respond to cold stress. Here, we report that SlHB8, working upstream of the conserved genetic module DYT1-TDF1-AMS-MYB80, is crucial for regulating cold tolerance in tomato anthers. SlHB8 expression increases in the tapetum when exposed to low temperatures. CRISPR/Cas9-generated SlHB8-knockout mutants exhibit improved pollen cold tolerance due to the reduced temperature sensitivity of the tapetum. SlHB8 directly upregulates SlDYT1 and SlMYB80 by binding to their promoters. In normal anthers, cold treatment boosts SlHB8 levels, which then elevates the expression of genes like SlDYT1, SlTDF1, SlAMS, and SlMYB80; however, slhb8 mutants do not show this gene activation during cold stress, leading to a complete blockage of delayed tapetal programmed cell death (PCD). Furthermore, we found that SlHB8 can interact with both SlTDF1 and SlMYB80, suggesting the possibility that SlHB8 might regulate tapetal PCD at the protein level. This study sheds light on molecular mechanisms of anther adaptation to temperature fluctuations.

Pollen cells require large amounts of sugars from the anther to support their development, which is critical for plant sexual reproduction and crop yield. Sugars Will Eventually be Exported Transporters (SWEETs) have been shown to play an important role in the apoplasmic unloading of sugars from anther tissues into symplasmically isolated developing pollen cells and thereby affect the sugar supply for pollen development. However, among the 17 CsSWEET genes identified in the cucumber (Cucumis sativus L.) genome, the CsSWEET gene involved in this process has not been identified. Here, a member of the SWEET gene family, CsSWEET5a, was identified and characterized. The quantitative real-time PCR and β-glucuronidase expression analysis revealed that CsSWEET5a is highly expressed in the anthers and pollen cells of male cucumber flowers from the microsporocyte stage (stage 9) to the mature pollen stage (stage 12). Its subcellular localization indicated that the CsSWEET5a protein is localized to the plasma membrane. The heterologous expression assays in yeast demonstrated that CsSWEET5a encodes a hexose transporter that can complement both glucose and fructose transport deficiencies. CsSWEET5a can significantly rescue the pollen viability and fertility of atsweet8 mutant Arabidopsis plants. The possible role of CsSWEET5a in supplying hexose to developing pollen cells via the apoplast is also discussed. [ABSTRACT FROM AUTHOR]

The increment in global temperature reduces crop productivity, which in turn threatens food security. Currently, most of our food supply is produced by plants and the human population is estimated to reach 9 billion by 2050. Gaining insights into how plants navigate heat stress in their reproductive phase is essential for effectively overseeing the future of agricultural productivity. The reproductive success of numerous plant species can be jeopardized by just one exceptionally hot day. While the effects of heat stress on seedlings germination and root development have been extensively investigated, studies on reproduction are limited. The intricate processes of gamete development and fertilization unfold within a brief timeframe, largely concealed within the flower. Nonetheless, heat stress is known to have important effects on reproduction. Considering that heat stress typically affects both male and female reproductive structures concurrently, it remains crucial to identify cultivars with thermotolerance. In such cultivars, ovules and pollen can successfully undergo development despite the challenges posed by heat stress, enabling the completion of the fertilization process and resulting in a robust seed yield. Hereby, we review the current understanding of the molecular mechanisms underlying plant resistance to abiotic heat stress, focusing on the reproductive process in the model systems of Arabidopsis and Oryza sativa. [ABSTRACT FROM AUTHOR]

The membrane-less organelles in cytoplasm that are presented as cytoplasmic foci were successively identified. Although multiple CCCH zinc-finger proteins have been found to be localized in cytoplasmic foci, the relationship between their specific localization and functions still needs further clarification. Here, we report that the heterologous expression of two Brassica campestris CCCH zinc-finger protein genes (BcMF30a and BcMF30c) in Arabidopsis thaliana can affect microgametogenesis by involving the formation of cytoplasmic foci. By monitoring the distribution of proteins and observing pollen phenotypes, we found that, when these two proteins were moderately expressed in pollen, they were mainly dispersed in the cytoplasm, and the pollen developed normally. However, high expression induced the assembly of cytoplasmic foci, leading to pollen abortion. These findings suggested that the continuous formation of BcMF30a/BcMF30c-associated cytoplasmic foci due to high expression was the inducement of male sterility. A co-localization analysis further showed that these two proteins can be recruited into two well-studied cytoplasmic foci, processing bodies (PBs), and stress granules (SGs), which were confirmed to function in mRNA metabolism. Together, our data suggested that BcMF30a and BcMF30c play component roles in the assembly of pollen cytoplasmic foci. Combined with our previous study on the homologous gene of BcMF30a/c in Arabidopsis, we concluded that the function of these homologous genes is conserved and that cytoplasmic foci containing BcMF30a/c may participate in the regulation of gene expression in pollen by regulating mRNA metabolism. [ABSTRACT FROM AUTHOR]

Transcription factor IIB (TFIIB) is a general transcription factor for RNA polymerase II, exerting its influence across various biological contexts. In the majority of eukaryotes, TFIIB typically has two homologs, serving as general transcription factors for RNA polymerase I and III. In plants, however, the TFIIB-related protein family has expanded greatly, with 14 and 9 members in Arabidopsis and rice, respectively. BRP5/pollen-expressed transcription factor 2 (PTF2) proteins belong to a subfamily of TFIIB-related proteins found only in plants and algae. The prior analysis of an Arabidopsis atbrp5 mutant, characterized by a T-DNA insertion at the 5′ untranslated region, demonstrated the essential role of BRP5/PTF2 during the process of pollen germination and embryogenesis in Arabidopsis. Using a rice transformation system based on CRISPR/Cas9 technology, we have generated transgenic rice plants containing loss-of-function frameshift mutations in the BRP5/PTF2 gene. Unlike in the Arabidopsis atbrp5 mutant, the brp5/ptf2 frameshift mutations were not transmitted to progeny in rice, indicating an essential role of BRP5/PTF2 in both male and female gamete development or viability. The silencing of rice BRP5/PTF2 expression through RNA interference (RNAi) had little effect on vegetative growth and panicle formation but strongly affected pollen development and grain formation. Genetic analysis revealed that strong RNAi silencing of rice BRP5/PTF2 was still transmissible to progeny almost exclusively through female gametes, as found in the Arabidopsis atbrp5 knockdown mutant. Thus, reduced rice BRP5/PTF2 expression impacted pollen preferentially by interfering with male gamete development or viability. Drawing upon these findings, we posit that BRP5/PTF2 assumes a distinct and imperative function in the realm of plant sexual reproduction. [ABSTRACT FROM AUTHOR]

Climate change is one of the biggest threats that human society currently needs to face. Heat waves associated with global warming negatively affect plant growth and development and will increase in intensity and frequency in the coming years. Tomato is one of the most produced and consumed fruit in the world but remarkable yield losses occur every year due to the sensitivity of many cultivars to heat stress (HS). New insights into how tomato plants are responding to HS will contribute to the development of cultivars with high yields under harsh temperature conditions. In this study, the analysis of microsporogenesis and pollen germination rate of eleven tomato cultivars after exposure to a chronic HS revealed differences between genotypes. Pollen development was either delayed and/or desynchronized by HS depending on the cultivar considered. In addition, except for two, pollen germination was abolished by HS in all cultivars. The transcriptome of floral buds at two developmental stages (tetrad and pollen floral buds) of five cultivars revealed common and specific molecular responses implemented by tomato cultivars to cope with chronic HS. These data provide valuable insights into the diversity of the genetic response of floral buds from different cultivars to HS and may contribute to the development of future climate resilient tomato varieties. [ABSTRACT FROM AUTHOR]

Purpose: Crop sensitivity to root zone salinity can vary over time, which can lead to severe damage when high sensitivity coincides with high soil salinity. The variation in salinity sensitivity of sunflower during its growth cycle is unknown. Methods: Two pot experiments were conducted in sand culture with a complete nutrient solution. Solutions were flushed through pots in excess to maintain specified salt concentrations in the soil solution. In Experiment 1, salt-sensitive stages were determined by applying solutions with an electrical conductivity of < 0.7, 2, 4 or 8 dS m− 1 through the vegetative, flowering or grain filling stages. In Experiment 2, the most sensitive stage to root-zone salinity was determined by exposing plants to 10-day periods of salinity (< 0.7, 8, 16 and 24 dS m− 1) overlapping by 4 days starting from 13-leaf to grain filling. Results: In both experiments, decreases in seed yield were associated with exposure of plants to elevated EC during the period before opening of disk flower to ~ 95% anthesis, while grain filling was the least sensitive. The decline in yield was correlated with a decrease in mature seed number. Increasing salinity from 4 to 8 dS m− 1 during flowering increased the area without seeds at the centre of the disk. In experiment 2, measurements of leaf ion concentrations and photosynthesis suggested that Na+ toxicity decreased yield due to decreases in availability of photosynthate to flowers. Conclusion: To maximise sunflower yield in saline soils, it is important to minimise salinity stress from before flower opening to anthesis. [ABSTRACT FROM AUTHOR]

The biosynthesis of C27–29 sterols from their C30 precursor squalene involves C24-alkylation and the removal of three methyl groups, including two at the C4 position. The two C4 demethylation reactions require a bifunctional enzyme known as 3β-hydroxysteroid dehydrogenase/C4-decarboxylase (3βHSD/D), which removes an oxidized methyl (carboxylic) group at C4 while simultaneously catalyzing the 3β-hydroxyl→3-keto oxidation. Its loss-of-function mutations cause ergosterol-dependent growth in yeast and congenital hemidysplasia with ichthyosiform erythroderma and limb defect (CHILD) syndrome in humans. Although plant 3βHSD/D enzymes were well studied enzymatically, their developmental functions remain unknown. Here we employed a CRISPR/Cas9-based genome-editing approach to generate knockout mutants for two Arabidopsis 3βHSD/D genes, HSD1 and HSD2, and discovered the male gametophytic lethality for the hsd1 hsd2 double mutation. Pollen-specific expression of HSD2 in the heterozygous hsd1 hsd2/+ mutant not only rescued the pollen lethality but also revealed the critical roles of the two HSD genes in embryogenesis. Our study thus demonstrated the essential functions of the two Arabidopsis 3βHSD/D genes in male gametogenesis and embryogenesis. [ABSTRACT FROM AUTHOR]

Commercially, leguminous crops (Fabaceae) are the second most important group of cultivated plants, just after grasses (Poaceae). This study focuses on the analysis of pollen development and stainability in two species belonging to the Fabaceae family: Vicia faba L. and Lupinus angustifolius L. Morphological analysis of the anthers at various stages of flower development allowed us to trace the processes of microsporogenesis and microgametogenesis. Nine different cell staining protocols with diverse mechanisms of action, including acetocarmine, Alexander's dye, aniline blue in lactophenol, Calcein AM, FDA, MTT, TTC, Lugol's iodine, and aceto-orcein, were tested for their suitability in assessing the viability of microspores as well as pollen grains in both species. Among the applied dyes, four allowed for the discrimination between viable and nonviable microspores in V. faba, and six dyes allowed for this in L. angustifolius. For mature pollen grains, all dyes enabled differentiation between viable and nonviable cells in both species. The highest viability indications for V. faba microspores were obtained with acetocarmine (94.6%), while for mature pollen, aniline blue in lactophenol, MTT, and aceto-orcein yielded the highest viability indications (90.8–96.3%). In L. angustifolius, the highest percentages of viable microspores (64.9–66.5%) were obtained with the acetocarmine, aniline blue in lactophenol, and TTC dyes. For mature pollen, the highest viability indications (83.4%–92.9%) were obtained with acetocarmine, aniline blue in lactophenol, Lugol's iodine, and aceto-orcein. The viability of V. faba pollen grains in an in vitro germination test showed that the highest pollen germination (61.3%) was observed on the BK medium (rich in minerals with 10% sucrose). In L. angustifolius, the highest pollen germination was observed on the media containing boric acid and 5% sucrose (70.5%) and on the medium containing 10% sucrose only (74.2%). [ABSTRACT FROM AUTHOR]

IntroductionWheat is a staple food crop for over one-third of the global population. However, the stability of wheat productivity is threatened by heat waves associated with climate change. Heat stress at the reproductive stage can result in pollen sterility and failure of grain development.MethodsThis study used transcriptome data analysis to explore the specific expression of long non-coding RNAs (lncRNAs) in response to heat stress during pollen development in four wheat cultivars. Results and discussionWe identified 11,054 lncRNA-producing loci, of which 5,482 lncRNAs showed differential expression in response to heat stress. Heat-responsive lncRNAs could target protein-coding genes in cis and trans and in lncRNA-miRNA-mRNA regulatory networks. Gene ontology analysis predicted that target protein-coding genes of lncRNAs regulate various biological processes such as hormonal responses, protein modification and folding, response to stress, and biosynthetic and metabolic processes. We also noted some paired lncRNA/protein-coding gene modules and some lncRNA-miRNA-mRNA regulatory modules shared in two or more wheat cultivars. These modules were related to regulating plant responses to heat stress, such as heat-shock proteins and transcription factors, and protein domains, such as MADS-box, Myc-type, and Alpha crystallin/Hsp20 domain. ConclusionOur results provide the basic knowledge and molecular resources for future functional studies investigating wheat reproductive development under heat stress.

The increment in global temperature reduces crop productivity, which in turn threatens food security. Currently, most of our food supply is produced by plants and the human population is estimated to reach 9 billion by 2050. Gaining insights into how plants navigate heat stress in their reproductive phase is essential for effectively overseeing the future of agricultural productivity. The reproductive success of numerous plant species can be jeopardized by just one exceptionally hot day. While the effects of heat stress on seedlings germination and root development have been extensively investigated, studies on reproduction are limited. The intricate processes of gamete development and fertilization unfold within a brief timeframe, largely concealed within the flower. Nonetheless, heat stress is known to have important effects on reproduction. Considering that heat stress typically affects both male and female reproductive structures concurrently, it remains crucial to identify cultivars with thermotolerance. In such cultivars, ovules and pollen can successfully undergo development despite the challenges posed by heat stress, enabling the completion of the fertilization process and resulting in a robust seed yield. Hereby, we review the current understanding of the molecular mechanisms underlying plant resistance to abiotic heat stress, focusing on the reproductive process in the model systems of Arabidopsis and Oryza sativa.

Arabinogalactan-proteins (AGPs) are a family of hyperglycosylated hydroxyproline-rich cell wall proteins found throughout the plant kingdom. To date, eight Hydroxyproline-galactosyltransferases (Hyp-GALTs), named GALT2-GALT9, are known to catalyze the addition of the first galactose sugar to Hyp residues in AGP protein cores. The generation and characterization of galt23456789 octuple mutants using CRISPR-Cas9 gene editing technology, provided strong reverse genetic evidence that AG glycans are essential for normal vegetative and reproductive growth, as these mutants demonstrated stunted growth, greatly delayed flowering and significant defects in floral organ development and morphogenesis. Compared to the lower seed set of galt25789 quintuple mutants being more so contributed by female gametophytic defects, dramatically low seed-set of octuple mutants was largely due to impaired male reproductive function, specifically due to shorter filaments, delayed anther dehiscence, and large decreases in pollen quantity and viability. Octuple mutant pollen had severely distorted reticulate exine, tectum patterning and intine thickness. Reduced amounts of galactose and arabinose in overall lower amounts of β-Yariv precipitated AGPs illustrated how biological functions of AGPs are affected by abnormal glycosylation.

The study of reproductive mechanisms is of particular interest for a real understanding of seed plant evolution. Spermatophytes comprise angiosperms and four orders of gymnosperms (Cycadales, Ginkgoales, Coniferales, and Gnetales) whose main characteristic is the reproduction via seeds. Ginkgo and cycads form a sister clade to the other gymnosperms and occupy a key phylogenetic position in-between the extinct Paleozoic seed ferns and the other extant gymnosperms. This review focuses on the similarities and differences between the reproductive mechanisms of Ginkgo and Cycas, from the morphogenesis of the male and female organs to the pollination and fertilization events. Together with the morphological and cytological description, the latest available molecular data on the reproductive organ development of the two plant genera are discussed. This will, hopefully, pave the path for new studies aiming at filling the gaps in our understanding of the hormonal and genetic regulation of their reproductive mechanisms. The whole reproductive process is presented in detail, providing a comprehensive and organic picture together with complete illustrations and photographic material. Each phase of the reproductive process is dissected, pointing out the main similarities and differences found among the two genera. The comparison comprises the male and female reproductive organs development, with a focus on pollen ontogeny, shape, ultrastructure, and germination as well as ovule development and patterning, female gametophyte formation, and ovule integument differentiation. Particular attention is given to the pollination and fertilization events focusing on the role of reproductive fluids as well as zoogamy. [ABSTRACT FROM AUTHOR]

Climate change is one of the biggest threats that human society currently needs to face. Heat waves associated with global warming negatively affect plant growth and development and will increase in intensity and frequency in the coming years. Tomato is one of the most produced and consumed fruit in the world but remarkable yield losses occur every year due to the sensitivity of many cultivars to heat stress (HS). New insights into how tomato plants are responding to HS will contribute to the development of cultivars with high yields under harsh temperature conditions. In this study, the analysis of microsporogenesis and pollen germination rate of eleven tomato cultivars after exposure to a chronic HS revealed differences between genotypes. Pollen development was either delayed and/or desynchronized by HS depending on the cultivar considered. In addition, except for two, pollen germination was abolished by HS in all cultivars. The transcriptome of floral buds at two developmental stages (tetrad and pollen floral buds) of five cultivars revealed common and specific molecular responses implemented by tomato cultivars to cope with chronic HS. These data provide valuable insights into the diversity of the genetic response of floral buds from different cultivars to HS and may contribute to the development of future climate resilient tomato varieties.

The microspore can follow two different developmental pathways. In vivo microspores follow the gametophytic program to produce pollen grains. In vitro, isolated microspores can be reprogrammed by stress treatments and follow the embryogenic program, producing doubled-haploid embryos. In the present study, we analyzed the dynamics and role of endogenous auxin in microspore development during these two different scenarios, in Brassica napus. We analyzed auxin concentration, cellular accumulation, the expression of the TAA1 auxin biosynthesis gene, and the PIN1-like efflux carrier gene, as well as the effects of inhibiting auxin biosynthesis by kynurenine on microspore embryogenesis. During the gametophytic pathway, auxin levels and TAA1 and PIN1-like expression were high at early stages, in tetrads and tapetum, while they progressively decreased during gametogenesis in both pollen and tapetum cells. In contrast, in microspore embryogenesis, TAA1 and PIN1-like genes were upregulated, and auxin concentration increased from the first embryogenic divisions. Kynurenine treatment decreased both embryogenesis induction and embryo production, indicating that auxin biosynthesis is required for microspore embryogenesis initiation and progression. The findings indicate that auxin exhibits two opposite profiles during these two microspore developmental pathways, which determine the different cell fates of the microspore. [ABSTRACT FROM AUTHOR]

SUMMARY: Pollen development is critical to plant reproduction, but the underlying regulatory molecular mechanisms have not been fully elucidated. The Arabidopsis (Arabidopsis thaliana) EFR3 OF PLANT 3 (EFOP3) and EFR3 OF PLANT 4 (EFOP4) genes encode members of the Armadillo (ARM) repeat superfamily that play key roles in pollen development. Herein, we demonstrate that EFOP3 and EFOP4 are co‐expressed in pollen at anther stages 10–12, but loss‐of‐function of both EFOP3 and EFOP4 leads to male gametophyte sterility, irregular intine, and shriveled pollen grains at anther stage 12. We further established that full‐length EFOP3 and EFOP4 specifically localize to the plasma membrane, and the integrity of these proteins is essential for pollen development. We observed uneven intine, less organized cellulose and reduced pectin content in mutant pollen compared with the wild‐type. These, together with the misexpression of several genes related to cell wall metabolism in efop3−/− efop4+/− mutants, suggest that EFOP3 and EFOP4 may indirectly regulate the expression of these genes to affect intine formation, thus controlling Arabidopsis pollen fertility in a functionally redundant manner. Moreover, transcriptome analysis showed that the absence of EFOP3 and EFOP4 function affects multiple pollen development pathways. These results enhance our understanding of EFOPs proteins and their role in pollen development. Significance Statement: We show that the integrity of EFOP3 and EFOP4 proteins is essential for their specific association with the plasma membrane of pollen, and is required for pollen intine development in Arabidopsis (Arabidopsis thaliana). EFOP3 and EFOP4 may indirectly regulate the expression of cell wall metabolism‐related genes to affect intine formation, thus controlling Arabidopsis pollen fertility in a functionally redundant manner. [ABSTRACT FROM AUTHOR]

Industrial chicory (Cichorium intybus var. sativum) is a biannual crop mostly cultivated for extraction of inulin, a fructose polymer used as a dietary fiber. F1 hybrid breeding is a promising breeding strategy in chicory but relies on stable male sterile lines to prevent self-pollination. Here, we report the assembly and annotation of a new industrial chicory reference genome. Additionally, we performed RNA-Seq on subsequent stages of flower bud development of a fertile line and two cytoplasmic male sterile (CMS) clones. Comparison of fertile and CMS flower bud transcriptomes combined with morphological microscopic analysis of anthers, provided a molecular understanding of anther development and identified key genes in a range of underlying processes, including tapetum development, sink establishment, pollen wall development and anther dehiscence. We also described the role of phytohormones in the regulation of these processes under normal fertile flower bud development. In parallel, we evaluated which processes are disturbed in CMS clones and could contribute to the male sterile phenotype. Taken together, this study provides a state-of-the-art industrial chicory reference genome, an annotated and curated candidate gene set related to anther development and male sterility as well as a detailed molecular timetable of flower bud development in fertile and CMS lines. [ABSTRACT FROM AUTHOR]

Drought stress inducing pollen sterility can reduce crop yield worldwide. The regulatory crosstalk associated with the effects of drought on pollen formation at the cellular level has not been explored in detail so far. In this study, we performed morphological and cytoembryological analysis of anther perturbations and examined pollen development in two spring barley genotypes that differ in earliness and drought tolerance. The Syrian breeding line CamB (drought-tolerant) and the European cultivar Lubuski (drought-sensitive) were used as experimental materials to analyze the drought-induced changes in yield performance, chlorophyll fluorescence kinetics, the pollen grain micromorphology and ultrastructure during critical stages of plant development. In addition, fluctuations in HvGAMYB expression were studied, as this transcription factor is closely associated with the development of the anther. In the experiments, the studied plants were affected by drought, as was confirmed by the analyses of yield performance and chlorophyll fluorescence kinetics. However, contrary to our expectations, the pollen development of plants grown under specific conditions was not severely affected. The results also suggest that growth modification, as well as the perturbation in light distribution, can affect the HvGAMYB expression. This study demonstrated that the duration of the vegetation period can influence plant drought responses and, as a consequence, the processes associated with pollen development as every growth modification changes the dynamics of drought effects as well as the duration of plant exposition to drought. [ABSTRACT FROM AUTHOR]

The presence of a pollinium is a distinct character in Apocynaceae which is important for phylogenetic analysis. The pollinium of Hoya has an outer sporopollenin wall and a pellucid margin which are adaptive features. However, their ontogeny and related evolutionary implications are not entirely understood. Therefore, a representative species Hoya carnosa was selected to investigate the pollinium development using light and electron microscopy and cytochemical tests. In contrast to the microsporogenesis in most angiosperms, which is associated with callose, the non-callosic intersporal walls in Hoya carnosa, together with the successive cytokinesis and linear form of the tetrad, represent an alternative pattern of microsporogenesis. This pattern has specific implication for the early stages of pollen morphogenesis. The absence of exine and apertures in the pollen grains in the pollinium could result from a combination of factors including the absence of callose in the early stages and the modifications in later developmental pathways, e.g., the sporopollenin accumulation pathway. The pollinium wall is an exine without stratification, its surface lacks sculptures, and it provides structural support and protection. The pollen tubes germinate through the pellucid margin and germinating ridge which are specialized features. The pellucid margin originates from aborted microspores. The germinating ridge that lies on the outer side of the pellucid margin develops in the same way as a classic pollen exine. The pollen grains are aggregated by intine fusion which is favorable for tube germination and growth. Comparing Asclepiadoideae with the other two subfamilies of Apocynaceae that develop a pollinium, the pollinium of Asclepiadoideae has reduced deposition of sporopollenin in the inner walls but an increase in the outer pollinium wall, thus making the inner walls more reduced and simplified, and the outer walls more solid. The adaptive characters of the pollen wall structure and the cohesion mechanism suggest that the pollinium of Hoya carnosa is a derived form of pollen aggregation. [ABSTRACT FROM AUTHOR]

Cold stress usually causes the abscission of floral organs and a decline in fruit setting rate, seriously reducing tomato yield. Auxin is one of the key hormones that affects the abscission of plant floral organs; the YUCCA (YUC) family is a key gene in the auxin biosynthesis pathway, but there are few research reports on the abscission of tomato flower organs. This experiment found that, under low temperature stress, the expression of auxin synthesis genes increased in stamens but decreased in pistils. Low temperature treatment decreased pollen vigor and pollen germination rate. Low night temperature reduced the tomato fruit setting rate and led to parthenocarpy, and the treatment effect was most obvious in the early stage of tomato pollen development. The abscission rate of tomato pTRV-Slfzy3 and pTRV-Slfzy5 silenced plants was higher than that of the control, which is the key auxin synthesis gene affecting the abscission rate. The expression of Solyc07g043580 was down-regulated after low night temperature treatment. Solyc07g043580 encodes the bHLH-type transcription factor SlPIF4. It has been reported that PIF4 regulates the expression of auxin synthesis and synthesis genes, and is a key protein in the interaction between low temperature stress and light in regulating plant development. [ABSTRACT FROM AUTHOR]

Background: Long-intergenic non-coding RNAs (lincRNAs) originate from intergenic regions and have no coding potential. LincRNAs have emerged as key players in the regulation of various biological processes in plant development. Cytoplasmic male-sterility (CMS) in association with restorer-of-fertility (Rf) systems makes it a highly reliable tool for exploring heterosis for producing commercial hybrid seeds. To date, there have been no reports of lincRNAs during pollen development in CMS and fertility restorer lines in pigeon pea. Objective: Identification of lincRNAs in the floral buds of cytoplasmic male-sterile (AKCMS11) and fertility restorer (AKPR303) pigeon pea lines. Methods: We employed a computational approach to identify lincRNAs in the floral buds of cytoplasmic male-sterile (AKCMS11) and fertility restorer (AKPR303) pigeon pea lines using RNA-Seq data. Results: We predicted a total of 2145 potential lincRNAs of which 966 were observed to be differentially expressed between the sterile and fertile pollen. We identified, 927 cis-regulated and 383 trans-regulated target genes of the lincRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the target genes revealed that these genes were specifically enriched in pathways like pollen and pollen tube development, oxidative phosphorylation, etc. We detected 23 lincRNAs that were co-expressed with 17 pollen-related genes with known functions. Fifty-nine lincRNAs were predicted to be endogenous target mimics (eTMs) for 25 miRNAs, and found to be associated with pollen development. The, lincRNA regulatory networks revealed that different lincRNA-miRNA-mRNA networks might be associated with CMS and fertility restoration. Conclusion: Thus, this study provides valuable information by highlighting the functions of lincRNAs as regulators during pollen development in pigeon pea and utilization in hybrid seed production. [ABSTRACT FROM AUTHOR]

Abstract Background Cytoplasmic male sterility (CMS) is a maternally inherited failure to produce functional pollen that most commonly results from expression of novel, chimeric mitochondrial genes. In Zea mays, cytoplasmic male sterility type S (CMS-S) is characterized by the collapse of immature, bi-cellular pollen. Molecular and cellular features of developing CMS-S and normal (N) cytoplasm pollen were compared to determine the role of mitochondria in these differing developmental fates. Results Terminal deoxynucleotidyl transferase dUTP nick end labeling revealed both chromatin and nuclear fragmentation in the collapsed CMS-S pollen, demonstrating a programmed cell death (PCD) event sharing morphological features with mitochondria-signaled apoptosis in animals. Maize plants expressing mitochondria-targeted green fluorescent protein (GFP) demonstrated dynamic changes in mitochondrial morphology and association with actin filaments through the course of N-cytoplasm pollen development, whereas mitochondrial targeting of GFP was lost and actin filaments were disorganized in developing CMS-S pollen. Immunoblotting revealed significant developmental regulation of mitochondrial biogenesis in both CMS-S and N mito-types. Nuclear and mitochondrial genome encoded components of the cytochrome respiratory pathway and ATP synthase were of low abundance at the microspore stage, but microspores accumulated abundant nuclear-encoded alternative oxidase (AOX). Cytochrome pathway and ATP synthase components accumulated whereas AOX levels declined during the maturation of N bi-cellular pollen. Increased abundance of cytochrome pathway components and declining AOX also characterized collapsed CMS-S pollen. The accumulation and robust RNA editing of mitochondrial transcripts implicated translational or post-translational control for the developmentally regulated accumulation of mitochondria-encoded proteins in both mito-types. Conclusions CMS-S pollen collapse is a PCD event coincident with developmentally programmed mitochondrial events including the accumulation of mitochondrial respiratory proteins and declining protection against mitochondrial generation of reactive oxygen species.

Ogura cytoplasmic male sterility (CMS) lines are widely used breeding materials in cruciferous crops and play important roles in heterosis utilization; however, the sterility mechanism remains unclear. To investigate the microspore development process and gene expression changes after the introduction of orf138 and Rfo, cytological observation and transcriptome analysis were performed using a maintainer line, an Ogura CMS line, and a restorer line. Semithin sections of microspores at different developmental stages showed that the degradation of tapetal cells began at the tetrad stage in the Ogura CMS line, while it occurred at the bicellular microspore stage to the tricellular microspore stage in the maintainer and restorer lines. Therefore, early degradation of tapetal cells may be the cause of pollen abortion. Transcriptome analysis results showed that a total of 1287 DEGs had consistent expression trends in the maintainer line and restorer line, but were significantly up- or down-regulated in the Ogura CMS line, indicating that they may be closely related to pollen abortion. Functional annotation showed that the 1287 core DEGs included a large number of genes related to pollen development, oxidative phosphorylation, carbohydrate, lipid, and protein metabolism. In addition, further verification elucidated that down-regulated expression of genes related to energy metabolism led to decreased ATP content and excessive ROS accumulation in the anthers of Ogura CMS. Based on these results, we propose a transcriptome-mediated induction and regulatory network for cabbage Ogura CMS. Our research provides new insights into the mechanism of pollen abortion and fertility restoration in Ogura CMS. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are endogenously expressed, non-coding RNAs that play essential roles in numerous developmental and physiological processes in plants. However, there is a lack of data available on regulating fertility in cabbage (Brassica oleracea var. capitata L.). This study examined the expression of miRNAs during cabbage pollen development using high-throughput sequencing. Two small RNA libraries were constructed from flower buds from the cytoplasmic male-sterile (CMS) line and its maintainer fertile (MF) linee of cabbage, resulting in over 10 million sequence reads. A total of 211 new miRNAs and 7 conserved miRNAs were discovered. Eight randomly chosen miRNAs were found to have differential expression between CMS and MF, as determined by stem-loop quantitative real-time PCR (qRT-PCR). The expression patterns were similar to those detected with Illumina sequencing. Moreover, the possible targets of these putative miRNAs were inferred using a BlastX search and Gene Ontology (GO) analysis. The majority of targets have been indicated to be essential for the development of pollen or other plant parts. The discovery and characterization of miRNAs involved in pollen development in CMS and MF cabbage lines might drive research on the regulatory mechanisms of male sterility, leading to a better understanding of the crucial roles performed by miRNAs in this process. [ABSTRACT FROM AUTHOR]

Key message: The genomic location and stage-specific expression pattern of many long non-coding RNAs reveal their critical role in regulating protein-coding genes crucial in pollen developmental progression and male germ line specification. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 bp with no apparent protein-coding potential. Multiple investigations have revealed high expression of lncRNAs in plant reproductive organs in a cell and tissue-specific manner. However, their potential role as essential regulators of molecular processes involved in sexual reproduction remains largely unexplored. We have used developing field mustard (Brassica rapa) pollen as a model system for investigating the potential role of lncRNAs in reproductive development. Reference-based transcriptome assembly performed to update the existing genome annotation identified novel expressed protein-coding genes and long non-coding RNAs (lncRNAs), including 4347 long intergenic non-coding RNAs (lincRNAs, 1058 expressed) and 2,045 lncRNAs overlapping protein-coding genes on the opposite strand (lncNATs, 780 expressed). The analysis of expression profiles reveals that lncRNAs are significant and stage-specific contributors to the gene expression profile of developing pollen. Gene co-expression networks accompanied by genome location analysis identified 38 cis-acting lincRNA, 31 cis-acting lncNAT, 7 trans-acting lincRNA and 14 trans-acting lncNAT to be substantially co-expressed with target protein-coding genes involved in biological processes regulating pollen development and male lineage specification. These findings provide a foundation for future research aiming at developing strategies to employ lncRNAs as regulatory tools for gene expression control during reproductive development. [ABSTRACT FROM AUTHOR]

In mature pollen grains, lipids are primarily stored in the form of lipid droplets that provide energy and act as a carbon source for normal pollen development and germination. Triacylglycerol (TAG) is the major form of stored plant lipids. Diacylglycerol transferase, which is encoded by DGAT1 in Arabidopsis thaliana , is an important enzyme regulating triacylglycerol synthesis. Within the seeds of the DGAT1 mutant as11 , the content of TAG is significantly decreased and the fatty acid composition also differs from the wild type. Transcriptome data of mature anthers showed that the genes involved in the TAG synthesis pathway were downregulated in as11. Analysis of gene expression patterns via transcriptome data also revealed that the expression of PDAT1 , which functions in a manner complementary to the DGAT1 gene, was significantly decreased in as11 , whereas the amylopectin synthase genes SS1 and SS2 were upregulated in mutant as11. We also detected lower total lipid, TAG and fatty acid contents in mature as11 pollen, with palmitic acid (C16:0) and linolenic acid (C18:3) being the major fatty acids in mature pollen. The cytological results showed that the lipid droplet content was reduced in mature as11 pollen. In the binuclear pollen grain II stage, WT pollen contained lipid droplets that were primarily accumulated around the generative nucleus, whereas the pollen in the mutant as11 was rich in starch grains that were primarily distributed around the vegetative nucleus. Ultrastructural analysis indicated that during pollen development in as11 , the amount of endoplasmic reticulum in tapetal cells and pollen grains decreased, whereas the Golgi body content increased, which directly or indirectly led to a decrease in the levels of lipidosomes and an increase in the starch content in as11. Changes in the lipid content and fatty acid composition of the mutant as11 differ from those in the wild type during pollen development. [ABSTRACT FROM AUTHOR]

Fragaria × ananassa 'Maehyang' strawberries are the main exported Korean-bred cultivar due to their excellent quality and storability. However, an intrinsic issue associated with this cultivar is a high incidence of malformed fruits during greenhouse cultivation. Despite the importance for controlling the cultivation conditions and achieving high, stable yields, knowledge of flower development in F. × ananassa is lacking. Morphological and physiological changes in the anthers, pollens, and pistils of 'Maehyang' strawberry were reported at the ultrastructural level during development. 'Maehyang' strawberry plants were transplanted in a greenhouse. Before anthesis, the flower buds were sampled at different stages according to bud diameter and observed by using transmission electron microscopy, scanning electron microscopy, and confocal laser scanning microscopy. Flower development was classified into the following seven stages based on cytological events and significant structures: the sporogenous cell, microspore mother cell, tetrad, free microspore, vacuolated microspore, bicellular pollen, and mature pollen stages. In male gametophytes, the tapetum changed rapidly and substantially during anther development in coordination with different stages of pollen development. The pollen grains exhibited thickened exine wall at the free microspore and vacuolated microspore stages, with thickened sexine and sculptural elements having sharp apices. The endothecium thickening pattern was of the annular rib type, which shows unconnected rings of fibrous bands laid down diagonally. In female gametophytes, the stigmatic papillae were complex and diverse. These morphological characteristics could affect pollen development, pollination, fertilization, and flower susceptibility to unfavorable weather. These results expand the current understanding of the floral biology of F. × ananassa 'Maehyang' and will aid future comparative studies with other species or cultivars to improve yield and reduce damage caused by unfavorable weather, thereby increasing yield. [ABSTRACT FROM AUTHOR]

Industrial chicory (Cichorium intybus var. sativum) is a biannual crop mostly cultivated for extraction of inulin, a fructose polymer used as a dietary fiber. F1 hybrid breeding is a promising breeding strategy in chicory but relies on stable male sterile lines to prevent self-pollination. Here, we report the assembly and annotation of a new industrial chicory reference genome. Additionally, we performed RNA-Seq on subsequent stages of flower bud development of a fertile line and two cytoplasmic male sterile (CMS) clones. Comparison of fertile and CMS flower bud transcriptomes combined with morphological microscopic analysis of anthers, provided a molecular understanding of anther development and identified key genes in a range of underlying processes, including tapetum development, sink establishment, pollen wall development and anther dehiscence. We also described the role of phytohormones in the regulation of these processes under normal fertile flower bud development. In parallel, we evaluated which processes are disturbed in CMS clones and could contribute to the male sterile phenotype. Taken together, this study provides a state-of-the-art industrial chicory reference genome, an annotated and curated candidate gene set related to anther development and male sterility as well as a detailed molecular timetable of flower bud development in fertile and CMS lines.

Pollen cells require large amounts of sugars from the anther to support their development, which is critical for plant sexual reproduction and crop yield. Sugars Will Eventually be Exported Transporters (SWEETs) have been shown to play an important role in the apoplasmic unloading of sugars from anther tissues into symplasmically isolated developing pollen cells and thereby affect the sugar supply for pollen development. However, among the 17 CsSWEET genes identified in the cucumber (Cucumis sativus L.) genome, the CsSWEET gene involved in this process has not been identified. Here, a member of the SWEET gene family, CsSWEET5a, was identified and characterized. The quantitative real-time PCR and β-glucuronidase expression analysis revealed that CsSWEET5a is highly expressed in the anthers and pollen cells of male cucumber flowers from the microsporocyte stage (stage 9) to the mature pollen stage (stage 12). Its subcellular localization indicated that the CsSWEET5a protein is localized to the plasma membrane. The heterologous expression assays in yeast demonstrated that CsSWEET5a encodes a hexose transporter that can complement both glucose and fructose transport deficiencies. CsSWEET5a can significantly rescue the pollen viability and fertility of atsweet8 mutant Arabidopsis plants. The possible role of CsSWEET5a in supplying hexose to developing pollen cells via the apoplast is also discussed.

The membrane-less organelles in cytoplasm that are presented as cytoplasmic foci were successively identified. Although multiple CCCH zinc-finger proteins have been found to be localized in cytoplasmic foci, the relationship between their specific localization and functions still needs further clarification. Here, we report that the heterologous expression of two Brassica campestris CCCH zinc-finger protein genes (BcMF30a and BcMF30c) in Arabidopsis thaliana can affect microgametogenesis by involving the formation of cytoplasmic foci. By monitoring the distribution of proteins and observing pollen phenotypes, we found that, when these two proteins were moderately expressed in pollen, they were mainly dispersed in the cytoplasm, and the pollen developed normally. However, high expression induced the assembly of cytoplasmic foci, leading to pollen abortion. These findings suggested that the continuous formation of BcMF30a/BcMF30c-associated cytoplasmic foci due to high expression was the inducement of male sterility. A co-localization analysis further showed that these two proteins can be recruited into two well-studied cytoplasmic foci, processing bodies (PBs), and stress granules (SGs), which were confirmed to function in mRNA metabolism. Together, our data suggested that BcMF30a and BcMF30c play component roles in the assembly of pollen cytoplasmic foci. Combined with our previous study on the homologous gene of BcMF30a/c in Arabidopsis, we concluded that the function of these homologous genes is conserved and that cytoplasmic foci containing BcMF30a/c may participate in the regulation of gene expression in pollen by regulating mRNA metabolism.

Transcription factor IIB (TFIIB) is a general transcription factor for RNA polymerase II, exerting its influence across various biological contexts. In the majority of eukaryotes, TFIIB typically has two homologs, serving as general transcription factors for RNA polymerase I and III. In plants, however, the TFIIB-related protein family has expanded greatly, with 14 and 9 members in Arabidopsis and rice, respectively. BRP5/pollen-expressed transcription factor 2 (PTF2) proteins belong to a subfamily of TFIIB-related proteins found only in plants and algae. The prior analysis of an Arabidopsis atbrp5 mutant, characterized by a T-DNA insertion at the 5′ untranslated region, demonstrated the essential role of BRP5/PTF2 during the process of pollen germination and embryogenesis in Arabidopsis. Using a rice transformation system based on CRISPR/Cas9 technology, we have generated transgenic rice plants containing loss-of-function frameshift mutations in the BRP5/PTF2 gene. Unlike in the Arabidopsis atbrp5 mutant, the brp5/ptf2 frameshift mutations were not transmitted to progeny in rice, indicating an essential role of BRP5/PTF2 in both male and female gamete development or viability. The silencing of rice BRP5/PTF2 expression through RNA interference (RNAi) had little effect on vegetative growth and panicle formation but strongly affected pollen development and grain formation. Genetic analysis revealed that strong RNAi silencing of rice BRP5/PTF2 was still transmissible to progeny almost exclusively through female gametes, as found in the Arabidopsis atbrp5 knockdown mutant. Thus, reduced rice BRP5/PTF2 expression impacted pollen preferentially by interfering with male gamete development or viability. Drawing upon these findings, we posit that BRP5/PTF2 assumes a distinct and imperative function in the realm of plant sexual reproduction.

In flowering plants, pollen development is a key process that is essential for sexual reproduction and seed set. Molecular and genetic studies indicate that pollen development is coordinatedly regulated by both gametophytic and sporophytic factors. Tapetum, the somatic cell layer adjacent to the developing male meiocytes, plays an essential role during pollen development. In the early anther development stage, the tapetal cells secrete nutrients, proteins, lipids, and enzymes for microsporocytes and microspore development, while initiating programmed cell death to provide critical materials for pollen wall formation in the late stage. Therefore, disrupting tapetum specification, development, or function usually leads to serious defects in pollen development. In this review, we aim to summarize the current understanding of tapetum-mediated pollen development and illuminate the underlying molecular mechanism in Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

Nitrate Transporter 1/Peptide Transporter Family (NPF) genes encode membrane transporters involved in the transport of diverse substrates. However, little is known about the diversity and functions of NPFs in Brassica rapa. In this study, 85 NPFs were identified in B. rapa (BrNPFs) which comprised eight subfamilies. Gene structure and conserved motif analysis suggested that BrNFPs were conserved throughout the genus. Stress and hormone-responsive cis-acting elements and transcription factor binding sites were identified in BrNPF promoters. Syntenic analysis suggested that tandem duplication contributed to the expansion of BrNPFs in B. rapa. Transcriptomic profiling analysis indicated that BrNPF2.6, BrNPF2.15, BrNPF7.6, and BrNPF8.9 were expressed in fertile floral buds, suggesting important roles in pollen development. Thirty-nine BrNPFs were responsive to low nitrate availability in shoots or roots. BrNPF2.10, BrNPF2.19, BrNPF2.3, BrNPF5.12, BrNPF5.16, BrNPF5.8, and BrNPF6.3 were only up-regulated in roots under low nitrate conditions, indicating that they play positive roles in nitrate absorption. Furthermore, many genes were identified in contrasting genotypes that responded to vernalization and clubroot disease. Our results increase understanding of BrNPFs as candidate genes for genetic improvement studies of B. rapa to promote low nitrate availability tolerance and for generating sterile male lines based on gene editing methods. [ABSTRACT FROM AUTHOR]

Main conclusion: The loss of TaGAMYB function in Arabidopsis results in abnormal pollen development and leads to decreased fertility. This process may be regulated by microRNAs, which suppress the expression of fatty acid pathway genes. Development of the anthers and pollen is significantly influenced by the transcription factor GAMYB. However, our knowledge of GAMYB in wheat is limited. Here, under fertility and sterility conditions, we identified the distinct transcripts TaGAMYB-d and TaGAMYB-g in thermosensitive genic sterile wheat YanZhan 4110S and confirmed their functions. TaGAMYB-g overexpression decreased the pollen vigor and germination rates, thereby reducing fertility. TaGAMYB-d overexpression lines exhibited early flowering. Due to aberrant pollen germination, the mutant homologous TaGAMYB genes in Arabidopsis thaliana also resulted in lower fertility. Our findings indicate that TaGAMYB controls the fertility and flowering time in transgenic Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

Polyamines, including putrescine, spermidine, and spermine, play critical roles in cell physiology by different forms. As a rate-limiting enzyme that converts ornithine to putrescine, ornithine decarboxylase (ODC, EC 1.1.1.37) has been studied in detail in animals and microorganisms, but its specific functions are poorly understood in plants. In this study, the metabolic and developmental roles of the ODC gene were studied through RNAi-mediated suppression of the ODC gene (AbODC) in A. belladonna. Suppression of AbODC reduced the production of precursors of medicinal tropane alkaloids, including putrescine and N -methylputrescine, as well as hyoscyamine and scopolamine. In AbODC -RNAi roots, the production of putrescine and spermidine in free form was reduced, but in the AbODC -RNAi leaves, the content of free polyamines was not altered. In the roots/leaves of AbODC -RNAi plants, the production of conjugated and bound polyamines was reduced. In addition, suppression of the ODC gene resulted in reduction of polyamines and pollen sterility in AbODC- RNAi flowers. In floral organs, GUS-staining results indicated that AbODC was domainantly expressed in pollen. In summary, ornithine decarboxylase not only plays a key role in regulating the biosynthesis of diverse forms of polyamines and medicinal tropane alkaloids, but also participates in pollen development. [Display omitted] • Suppression of AbODC reduced the yield of tropane alkaloids and its precursors. • Inhibition of AbODC expression suppressed the biosynthesis of various polyamines. • In flower, AbODC was dominantly expressed in pollen. • Suppression of the ODC gene resulted in pollen sterility in AbODC- RNAi plants. [ABSTRACT FROM AUTHOR]

Background: Cytoplasmic male sterility (CMS) is a maternally inherited failure to produce functional pollen that most commonly results from expression of novel, chimeric mitochondrial genes. In Zea mays, cytoplasmic male sterility type S (CMS-S) is characterized by the collapse of immature, bi-cellular pollen. Molecular and cellular features of developing CMS-S and normal (N) cytoplasm pollen were compared to determine the role of mitochondria in these differing developmental fates. Results: Terminal deoxynucleotidyl transferase dUTP nick end labeling revealed both chromatin and nuclear fragmentation in the collapsed CMS-S pollen, demonstrating a programmed cell death (PCD) event sharing morphological features with mitochondria-signaled apoptosis in animals. Maize plants expressing mitochondria-targeted green fluorescent protein (GFP) demonstrated dynamic changes in mitochondrial morphology and association with actin filaments through the course of N-cytoplasm pollen development, whereas mitochondrial targeting of GFP was lost and actin filaments were disorganized in developing CMS-S pollen. Immunoblotting revealed significant developmental regulation of mitochondrial biogenesis in both CMS-S and N mito-types. Nuclear and mitochondrial genome encoded components of the cytochrome respiratory pathway and ATP synthase were of low abundance at the microspore stage, but microspores accumulated abundant nuclear-encoded alternative oxidase (AOX). Cytochrome pathway and ATP synthase components accumulated whereas AOX levels declined during the maturation of N bi-cellular pollen. Increased abundance of cytochrome pathway components and declining AOX also characterized collapsed CMS-S pollen. The accumulation and robust RNA editing of mitochondrial transcripts implicated translational or post-translational control for the developmentally regulated accumulation of mitochondria-encoded proteins in both mito-types. Conclusions: CMS-S pollen collapse is a PCD event coincident with developmentally programmed mitochondrial events including the accumulation of mitochondrial respiratory proteins and declining protection against mitochondrial generation of reactive oxygen species. [ABSTRACT FROM AUTHOR]