Functional Pollen Research Articles

ALBA3 maintains male fertility under heat stress in plants.

Heat stress (HS) at the reproductive stage detrimentally affects crop yields and seed quality. However, the molecular mechanisms that protect reproductive processes in plants under HS remain largely unknown. Here, we report that Acetylation Lowers Binding Affinity 3 (ALBA3) is crucial for safeguarding male fertility against HS in Arabidopsis. ALBA3 is highly expressed in pollen, and ALBA3 is localized in the cytoplasm of both sperm and vegetative cells. Mutants lacking functional ALBA3 exhibit hypersensitivity to HS, with reduced silique length and fertility due to defects in pollen germination, pollination, pollen tube growth, and fertilization under HS. ALBA3 binds and stabilizes a subset of messenger RNAs (mRNAs) essential for pollen function, thereby protecting male fertility. Two residues in the Alba domain, K46 and L90, are critical for ALBA3's ability to bind and stabilize mRNAs and are necessary for its proper function. Interestingly, the loss of rice ALBA3 also leads to severe pollen abortion and male sterility under HS, highlighting the conserved role of ALBA3 in protecting male fertility across plant species. This study uncovers a conserved mechanism by which ALBA3 safeguards male fertility during HS by stabilizing specific mRNAs crucial for pollen function.

Heat stress during reproductive development inhibits fertilization in olives

In this study, we investigated the effect of heat exposure during olive inflorescence on reproductive development. We hypothesized that male development processes during this period are particularly sensitive to high temperatures. We characterized the development of flowers and inflorescences under natural conditions in the cultivars 'Arbequina' and 'Koroneiki', and defined eight phenological stages, beginning with the emergence of reproductive buds until anthesis (international standard BBCH scale of the phenological inflorescence development phases 53–60). In addition, we examined the response of olive inflorescence and flowering, to two heat scenarios: prolonged exposure to moderately high temperatures and a two-hour concentrated heat shock treatment. Analysis of our data revealed that both treatments had a detrimental effect on the development and functioning of the tapetum, the innermost layer of the anther, which is essential for the development and functioning of pollen. Additionally, we found that the 'Arbequina' cultivar displayed relative tolerance to heat compared to 'Koroneiki' in terms of pollen germinability after heat stress. In contrast to the response of the above cultivars, the 'Souri' exhibited an exceptionally high sensitivity to heat. An assessment of pollen viability after in vitro heat shock treatment of 54 cultivars revealed that pollen from seven varieties, 'Niedda de Oliana', 'Lechín de Sevilla', 'Tonda Oliana', 'Tamir', 'Sigoise', 'Taggiasca', and 'Carrasquenha', showed exceptional tolerance to heat. These cultivars show a greater degree of suitability for cultivation in warm climates, (or in areas susceptible to extreme hot-spells). This study demonstrated that heat has a negative impact on the male reproductive system in olives, during early flower development and in mature pollen grains, which are relatively resistant to heat. Heat stress impaired pollen tube elongation capacity, and germination rate. Additionally, heat stress affected the ultrastructure of the external wall of the pollen grain, the exine, potentially impairing its ability to adhere to the stigma and fertilize the ovule. The sensitivity of olive reproductive capacity to heat is a genotype-dependent trait, allowing for the identification of heat-resistant cultivars for use as pollen donors and for development of new heat-resistant cultivars.

Cytoplasmic male sterility in plants with special emphasis on sugar beet

The phenomenon of cytoplasmic male sterility (CMS) in plants is characterized by a disturbance in the development of functional pollen. The cause of this disorder is the incompatibility of the mitochondrial and nuclear genome. Determinants of CMS are found in the mitochondrial genome and are inherited maternally. Nuclear Rf genes are responsible for restoring male fertility in CMS plants most often by posttranscriptional and/or posttranslational activity on mitochondrial gene expression. Two fertility restoring genes have been identified in sugar beet plants, of which Rf1 is the best characterized. It is concluded that the translation product of this gene acts as a molecular chaperone leading to inactivation of a specific mitochondrial protein that is a marker for the CMS trait in sugar beet. The CMS phenomenon is applied in the commercial breeding of hybrid varieties of many crop species. Exploiting heterosis is easier with the knowledge of cytoplasmic (mitochondrial) determinants of sterility and corresponding restorer genes.

X-ray Microanalysis of Elemental Composition of Vitis sylvestris Pollen Grains.

The flowering and fruit set of grapevines are determined by many morphological, physiological, and environmental factors. Although the elemental composition of pollen grains plays a crucial role in the fruit set, there is still a considerable gap in our knowledge. To date, no study has been conducted on the elemental composition of Vitis vinifera subsp. sylvestris (hereafter V. sylvestris) pollen grains. The aim of this work was to investigate the elemental composition of pollen grains of V. sylvestris using scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX). The pollen grains of ten V. sylvestris individuals (eight male and two female) and one hermaphrodite cultivar 'Plavac mali crni' were analyzed. SEM-EDX analysis revealed the presence of eight elements (carbon-C, oxygen-O, magnesium-Mg, phosphorus-P, potassium-K, calcium-Ca, molybdenum-Mo, and aluminum-Al) in the pollen grains. Interestingly, aluminum was detected exclusively in the pollen of the cultivated grape cultivar 'Plavac mali crni', while it was not present in the genotypes of V. sylvestris. No significant differences between genotypes were found for oxygen and phosphorus, while significant differences were found for other elements. Pollen dimorphism was not associated with differences in element composition, although principal component analysis separated the genotypes into two distinct groups, with two female individuals (Pak10 and Pak12) and one male (Im19) tending to form separate clusters. This study is the first report on the elemental composition of pollen grains of V. sylvestris genotypes and provides valuable insights for further studies on pollen functionality.

Mutations in nuclear genes encoding mitochondrial ribosome proteins restore pollen fertility in S male-sterile maize.

The interaction of plant mitochondrial and nuclear genetic systems is exemplified by mitochondria-encoded cytoplasmic male sterility (CMS) under the control of nuclear restorer-of-fertility genes. The S type of CMS in maize is characterized by a pollen collapse phenotype and a unique paradigm for fertility restoration in which numerous nuclear restorer-of-fertility lethal mutations rescue pollen function but condition homozygous-lethal seed phenotypes. Two nonallelic restorer mutations recovered from Mutator transposon-active lines were investigated to determine the mechanisms of pollen fertility restoration and seed lethality. Mu Illumina sequencing of transposon-flanking regions identified insertion alleles of nuclear genes encoding mitochondrial ribosomal proteins RPL6 and RPL14 as candidate restorer-of-fertility lethal mutations. Both candidates were associated with lowered abundance of mitochondria-encoded proteins in developing maize pollen, and the rpl14 mutant candidate was confirmed by independent insertion alleles. While the restored pollen functioned despite reduced accumulation of mitochondrial respiratory proteins, normal-cytoplasm plants heterozygous for the mutant alleles showed a significant pollen transmission bias in favor of the nonmutant Rpl6 and Rpl14 alleles. CMS-S fertility restoration affords a unique forward genetic approach to investigate the mitochondrial requirements for, and contributions to, pollen and seed development.

Battling arid adversity: unveiling the resilience of cotton in the face of drought and innovative mitigation approaches

Climate change has had significant impacts on agriculture, particularly on cotton production, where drought has emerged as a major threat worldwide. Long and intense dry periods in cotton-growing regions have become more frequent and severe. Drought stress severely affects various aspects of cotton plants, including chlorophyll pigments, carbohydrate metabolism, and enzyme activities related to fiber development, such as vacuolar invertase and sucrose synthase. Furthermore, drought stress disrupts the movement of nutrients toward the reproductive tissues in cotton, resulting in compromised pollen function, propagative failure, and fiber characteristics. To tackle these issues, scientists have made advancements in creating drought-resistant cotton varieties through transgenic methods or molecular breeding techniques, genome editing, CRISPR/Cas9, utilizing quantitative trait loci (QTL). Moreover, the application of plant growth regulators and mineral elements has displayed the potential to improve cotton’s ability to endure drought stress while also enhancing fiber yield and quality. These approaches activate stress-responsive signaling pathways, which could contribute to mitigating reproductive failure and improving fiber characteristics. While the impact of drought stress on cotton plants has been extensively studied, the variations in fiber quality resulting from drought stress are not yet completely understood. Current research has been focused on unraveling the mechanisms underlying these changes, including the physiological, biochemical, and molecular alterations during the multiplicative growth phase that contribute to poor fiber development. Understanding these mechanisms will facilitate the development of novel strategies to alleviate the adverse impact of worldwide weather changes on cotton growth and fiber quality. This research focuses on the drought stress in cotton cultivation and explores its different effects on cotton morphology, physiology, crop yield, and fiber characteristics as well as mechanisms by which cotton exhibits drought tolerance and highlights innovative strategies to mitigate drought stress.

An S-locus F-box protein as pollen S determinant targets non-self S-RNase underlying self-incompatibility in Citrus.

Self-incompatibility (SI) is a crucial mechanism that prevents self-fertilization and inbreeding in flowering plants. Citrus exhibits SI regulated by a polymorphic S-locus containing an S-RNase gene and multiple S-locus F-box (SLF) genes. It has been documented that S-RNase functions as the pistil S determinant, but there is no direct evidence that the SLF genes closely linked with S-RNase function as pollen S determinants in Citrus. This study assembled the genomes of two pummelo (Citrus grandis) plants, obtained three novel complete and well-annotated S-haplotypes, and isolated 36 SLF or SLF-like alleles on the S-loci. Phylogenetic analysis of 138 SLFs revealed that the SLF genes were classified into 12 types, including six types with divergent or missing alleles. Furthermore, transformation experiments verified that the conserved S6-SLF7a protein can lead to the transition of SI to self-compatibility by recognizing non-self S8-RNase in 'Mini-Citrus' plants (S7S8 and S8S29, Fortunella hindsii), a model plant for citrus gene function studies. In vitro assays demonstrated interactions between SLFs of different S haplotypes and the Skp1-Cullin1-F-box subunit CgSSK1 protein. This study provides direct evidence that SLF controls the pollen function in Citrus, demonstrating its role in the 'non-self recognition' SI system.

Effect of Hybridization on the Nutrient Compositions of Two Cultivars of Solanum aethiopicum L. (Solanaceae) Found in Anambra State

The desire for modification of valuable crop species for human nutritional fulfilment and economic growth is essential. Two cultivars of Solanum aethiopicum L. found in Anambra State were hybridized to raise an F1 that might combine the attributes of the parental lines for enhancement in yield and biochemical contents for health nutritional requirements. The emasculation method was adopted, using Solanum aethiopicum var “Anara Adazi as the female parent and transference of functional pollen from Solanum aethiopicum var “Anara” to the stigmas of the emasculated plant at the correct time. Nutrient analysis of the fruits of F1 and the parental plants was carried out using the standard method. Analysis of variance was used for data analysis. Results revealed that parental plants and F1 contained investigated nutrients in varied amounts. Ash content, crude fiber and protein were higher in the fruit of F1 hybrid (10.90±0.11, 21.50±1.51, and 5.01±0.24) respectively when compared to the fruits of the parental plants. Data obtained indicated that when two Solanum aethiopicum plants of different but closely related cultivars are crossbred, a new hybrid that possesses enhanced characteristics of the two parent plants are produced. Results of the nutrients analysis demonstrated that hybridization could significantly increase food nutrients in eggplant (S. aethiopicum).

Environmental regulation of male fertility is mediated through Arabidopsis transcription factors bHLH89, 91, and 10.

Formation of functional pollen and successful fertilization rely on the spatial and temporal regulation of anther and pollen development. This process responds to environmental cues to maintain optimal fertility despite climatic changes. Arabidopsis transcription factors basic helix-loop-helix (bHLH) 10, 89, and 91 were previously thought to be functionally redundant in their control of male reproductive development, however here we show that they play distinct roles in the integration of light signals to maintain pollen development under different environmental conditions. Combinations of the double and triple bHLH10,89,91 mutants were analysed under normal (200 μmol m-2 s-1) and low (50 μmol m-2 s-1) light conditions to determine the impact on fertility. Transcriptomic analysis of a new conditionally sterile bhlh89,91 double mutant shows differential regulation of genes related to sexual reproduction, hormone signal transduction, and lipid storage and metabolism under low light. Here we have shown that bHLH89 and bHLH91 play a role in regulating fertility in response to light, suggesting that they function in mitigating environmental variation to ensure fertility is maintained under environmental stress.

How pollen tubes fight for food: the impact of sucrose carriers and invertases of Arabidopsis thaliana on pollen development and pollen tube growth.

Pollen tubes of higher plants grow very rapidly until they reach the ovules to fertilize the female gametes. This growth process is energy demanding, however, the nutrition strategies of pollen are largely unexplored. Here, we studied the function of sucrose transporters and invertases during pollen germination and pollen tube growth. RT-PCR analyses, reporter lines and knockout mutants were used to study gene expression and protein function in pollen. The genome of Arabidopsis thaliana contains eight genes that encode functional sucrose/H+ symporters. Apart from AtSUC2, which is companion cell specific, all other AtSUC genes are expressed in pollen tubes. AtSUC1 is present in developing pollen and seems to be the most important sucrose transporter during the fertilization process. Pollen of an Atsuc1 knockout plant contain less sucrose and have defects in pollen germination and pollen tube growth. The loss of other sucrose carriers affects neither pollen germination nor pollen tube growth. A multiple knockout line Atsuc1Atsuc3Atsuc8Atsuc9 shows a phenotype that is comparable to the Atsuc1 mutant line. Loss of AtSUC1 can`t be complemented by AtSUC9, suggesting a special function of AtSUC1. Besides sucrose carriers, pollen tubes also synthesize monosaccharide carriers of the AtSTP family as well as invertases. We could show that AtcwINV2 and AtcwINV4 are expressed in pollen, AtcwINV1 in the transmitting tissue and AtcwINV5 in the funiculi of the ovary. The vacuolar invertase AtVI2 is also expressed in pollen, and a knockout of AtVI2 leads to a severe reduction in pollen germination. Our data indicate that AtSUC1 mediated sucrose accumulation during late stages of pollen development and cleavage of vacuolar sucrose into monosaccharides is important for the process of pollen germination.

Simulated microgravity affects directional growth of pollen tubes in candidate space crops

BackgroundLong-term space missions will necessarily require producing viable seeds to be used for plant cultivation over time under altered gravity conditions. Pollen is known to play a key role in determining seed and fruit production over seed-to-seed cycles, but very few studies have evaluated pollen functionality under altered gravity.MethodsWe performed ground-based experiments to test how simulated microgravity can affect the directional growth of pollen tubes as a potential bottleneck in seed and fruit sets. The effect of clinorotation was assessed in the pollen of Solanum lycopersicum L. cv. ‘Micro-Tom’ and Brassica rapa L. var. silvestris, both eligible for cultivation in space. Pollen tube length and tortuosity were compared under 1g and simulated microgravity with a uniaxial clinostat.ResultsThe main results highlighted that simulated microgravity significantly increased pollen tube length and tortuosity compared to 1g conditions. Further, clinorotation prompted a differential effect on pollen germination between S. lycopersicum and B. rapa. A more in-depth analysis evaluating the effect of gravity on the directional growth of pollen tubes excluded gravitropic responses as responsible for the tube tip position reached after germination.DiscussionThis research provides new insights into how altered gravity can interfere with plant reproduction and, in particular, microgametophyte functionality. Our findings represent a basis for further studies aimed at understanding the effect of real microgravity on plant reproduction and developing countermeasures to ensure seed-to-seed cultivation in long-term space missions and achieve self-sufficiency in food supplies from Earth.

Polyphenol oxidases regulate pollen development through modulating flavonoids homeostasis in tobacco

Pollen development is critical in plant reproduction. Polyphenol oxidases (PPOs) genes encode defense-related enzymes, but the role of PPOs in pollen development remains largely unexplored. Here, we characterized NtPPO genes, and then investigated their function in pollen via creating NtPPO9/10 double knockout mutant (cas-1), overexpression 35S::NtPPO10 (cosp) line and RNAi lines against all NtPPOs in Nicotiana tabacum. NtPPOs were abundantly expressed in the anther and pollen (especially NtPPO9/10). The pollen germination, polarity ratio and fruit weights were significantly reduced in the NtPPO-RNAi and cosp lines, while they were normal in cas-1 likely due to compensation by other NtPPO isoforms. Comparisons of metabolites and transcripts between the pollen of WT and NtPPO-RNAi, or cosp showed that decreased enzymatic activity of NtPPOs led to hyper-accumulation of flavonoids. This accumulation might reduce the content of ROS. Ca2+ and actin levels also decreased in pollen of the transgenic lines.Thus, the NtPPOs regulate pollen germination through the flavonoid homeostasis and ROS signal pathway. This finding provides novel insights into the native physiological functions of PPOs in pollen during reproduction.

Palynological studies and their implication in the compatibility of tuberose cultivars

Determination of palynological characters of commercial cultivars is significant in determining thecompatibility and intraspecific relationship, an important criterion in tuberose breeding. The presence ofself-incompatibility and seed sterility in tuberose has greatly hindered the conventional breeding methods asconfirmed through various studies. However, regulation of the incompatibility system and the principal factorbehind it has not been thoroughly investigated. The understanding of both male and female gametophytes andtheir functioning is a prerequisite which is still a less explored area in tuberose. Seventeen tuberose genotypesstudied showed the same inclination towards viability and germination determining a positive correlation.Analysis of pollen morphology and ultra-structure using a scanning electron microscope (SEM) revealed markedvariations among the genotypes. The distinct pollen ultra-structures revealed would serve as important featuresfor classification and character identification in taxonomy. It also ascertains the pollen function as evidenced bythe shrunken pollen of genotypes previously reported as sterile. These findings are vital to fully comprehendingthe breeding systems and reproduction biology of tuberose cultivars.

Developmentally regulated mitochondrial biogenesis and cell death competence in maize pollen

BackgroundCytoplasmic 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.ResultsTerminal 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.ConclusionsCMS-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.

Histological characterization of anther structure in Tetep-cytoplasmic male sterility and fine mapping of restorer-of-fertility gene in rice.

Cytoplasmic male sterility (CMS) is a maternally inherited trait that inhibits plants from producing or releasing viable pollen. CMS is caused by mitochondrial-nuclear interaction, and can be rescued by introducing functional nuclear restorer-of-fertility (Rf) gene. The Tetep-CMS/Rf lines were developed through successive inter-subspecific backcrosses between indica and japonica rice accessions. Phenotypic characterization of Tetep-CMS lines revealed abnormal anther dehiscence and the inability to release, while possessing functional pollen. Transverse sections of developing anthers collected from CMS plants showed connective tissue deformities and aberrant dehydration of endothecium and epidermis. Fine mapping of Rf-Tetep using a series of segregating populations, delimited the candidate region to an approximately 109 kb genomic interval between M2099 and FM07 flanking markers. Nanopore long-read sequencing and genome assembly, proceeded by gene prediction and annotation revealed 11 open reading frames (ORFs) within the candidate region, and suggest ORF6 annotated as pentatricopeptide repeat motif containing gene 1 (PPR1), as a possible candidate gene responsible for fertility restoration. This study suggests that tissue-specific abnormalities in anthers are responsible for indehiscence-based sterility, and propose that the functional Rf gene is derived from allelic variation between inter-subspecies in rice.

Mutation of glucose-methanol-choline oxidoreductase leads to thermosensitive genic male sterility in rice and Arabidopsis.

SummaryThermosensitive genic male sterility (TGMS) lines serve as the major genetic resource for two‐line hybrid breeding in rice. However, their unstable sterility under occasional low temperatures in summer highly limits their application. In this study, we identified a novel rice TGMS line, ostms18, of cultivar ZH11 (Oryza sativa ssp. japonica). ostms18 sterility is more stable in summer than the TGMS line carrying the widely used locus tms5 in the ZH11 genetic background, suggesting its potential application for rice breeding. The ostms18 TGMS trait is caused by the point mutation from Gly to Ser in a glucose‐methanol‐choline (GMC) oxidoreductase; knockout of the oxidoreductase was previously reported to cause complete male sterility. Cellular analysis revealed the pollen wall of ostms18 to be defective, leading to aborted pollen under high temperature. Further analysis showed that the tapetal transcription factor OsMS188 directly regulates OsTMS18 for pollen wall formation. Under low temperature, the flawed pollen wall in ostms18 is sufficient to protect its microspore, allowing for development of functional pollen and restoring fertility. We identified the orthologous gene in Arabidopsis. Although mutants for the gene were fertile under normal conditions (24°C), fertility was significantly reduced under high temperature (28°C), exhibiting a TGMS trait. A cellular mechanism integrated with genetic mutations and different plant species for fertility restoration of TGMS lines is proposed.

Flowering and pollen resilience to high temperature of apricot cultivars

Prunus armeniaca L. is widely cultivated in the Mediterranean area including Southern Italy where local cultivars are recognized for their excellent quality. Gradual warming and abrupt variations of seasonal temperatures are expected to significantly impact the Mediterranean area with potential implications on many crops including apricot. In this scenario, the identification of physiological processes involved in heat-stress responses and the selection of genotypes resilient/tolerant to high temperatures and heat waves are necessary. The aim of this study was to investigate possible differences in flowering phenology and the effect of different temperatures on pollen functionality of 13 apricot cultivars traditionally cultivated in the Campania region (Southern Italy). According to phenological data collected in the field, the studied cultivars were grouped in early, intermediate, and late flowering cultivars. Single flower anthesis was on average 4.9 days in early and intermediate flowering cultivars, whereas late flowering cultivars showed a shorter duration (4.0 days). Pollen of flowers at balloon stage showed a uniformly high viability among all cultivars. To investigate on possible effects of temperature during the effective pollination period (EPP), pollen from the different cultivars was pre-incubated at 5 °C, 15 °C, and 25 °C for 48-h and germinability was then assessed after further 24 h in-vitro germination at the same temperature treatments. The first two temperature values correspond respectively to the minimum and maximum average temperatures of the local area; whereas 25 °C simulated the heat waves recently recorded in the farm during apricot flowering periods. As regard pollen germinability, 15 °C revealed to be the most suitable temperature for apricot pollen to germinate within the EPP. Conversely, 5 °C and 25 °C significantly reduced pollen germination in most cultivars and particularly in intermediate flowering cultivars. Noticeably, a few cultivars showed no difference in pollen germination under the different temperature treatments, preserving high pollen germinability (>70%) even at high temperatures. Overall, our findings highlighted that pollen germination is extremely sensitive to temperature with significant variations among apricot genotypes. Results confirmed that reproductive traits such as pollen germinability represent an important parameter to consider for monitoring fruit production, in the processes of cultivar selection for new orchard plantations and in breeding projects. Moreover, traditional apricot cultivars as those of the Campania region confirmed to be a precious source of genetic diversity possessing a significant pollen resilience to temperature changes.

Proteomic analysis of differential anther development from sterile/fertile lines in Capsicum annuum L.

BackgroundPepper (Capsicum annuum L.) is a major cash crop throughout the world. Male sterility is an important characteristic in crop species that leads to a failure to produce functional pollen, and it has crucial roles in agricultural breeding and the utilization of heterosis.ObjectivesIn this study, we identified many crucial factors and important components in metabolic pathways in anther and pollen development, and elucidated the molecular mechanism related to pollen abortion in pepper.MethodsPepper pollen was observed at different stages to detect the characteristics associated with male sterility and fertility. The phytohormone and oxidoreductase activities were detected in spectrophotometric and redox reaction assays, respectively. Proteins were extracted from male sterile and fertile pepper lines, and identified by TMT/iTRAQ (tandem mass tags/isobaric tags for relative and absolute quantitation) and LC-MS/MS (liquid chromatograph-mass spectrometer) analysis. Differentially abundant proteins (DAPs) were analyzed based on Gene Ontology annotations and the Kyoto Encyclopedia of Genes and Genomes database according to |fold change)| > 1.3 and P value < 0.05. DAPs were quantified in the meiosis, tetrad, and binucleate stages by parallel reaction monitoring (PRM).ResultsIn this study, we screened and identified one male sterile pepper line with abnormal cytological characteristics in terms of pollen development. The peroxidase and catalase enzyme activities were significantly reduced and increased, respectively, in the male sterile line compared with the male fertile line. Phytohormone analysis demonstrated that the gibberellin, jasmonic acid, and auxin contents changed by different extents in the male sterile pepper line. Proteome analysis screened 1,645 DAPs in six clusters, which were mainly associated with the chloroplast and cytoplasm based on their similar expression levels. According to proteome analysis, 45 DAPs were quantitatively identified in the meiosis, tetrad, and binucleate stages by PRM, which were related to monoterpenoid biosynthesis, and starch and sucrose metabolism pathways.ConclusionsWe screened 1,645 DAPs by proteomic analysis and 45 DAPs were related to anther and pollen development in a male sterile pepper line. In addition, the activities of peroxidase and catalase as well as the abundances of phytohormones such as gibberellin, jasmonic acid, and auxin were related to male sterility. The results obtained in this study provide insights into the molecular mechanism responsible for male sterility and fertility in pepper.

RNAi-Mediated Knockdown of Calreticulin3a Impairs Pollen Tube Growth in Petunia.

Pollen tube growth depends on several complex processes, including exo/endocytosis, cell wall biogenesis, intracellular transport, and cell signaling. Our previous results provided evidence that calreticulin (CRT)—a prominent calcium (Ca2+)-buffering molecular chaperone in the endoplasmic reticulum (ER) lumen—is involved in pollen tube formation and function. We previously cloned and characterized the CRT gene belonging to the CRT1/2 subgroup from Petunia hybrida (PhCRT1/2), and found that post-transcriptional silencing of PhCRT1/2 expression strongly impaired pollen tube growth in vitro. Here, we report cloning of a new PhCRT3a homolog; we identified the full-length cDNA sequence and described its molecular characteristics and phylogenetic relationships to other plant CRT3 genes. Using an RNA interference (RNAi) strategy, we found that knockdown of PhCRT3a gene expression caused numerous defects in the morphology and ultrastructure of cultivated pollen tubes, including disorganization of the actin cytoskeleton and loss of cytoplasmic zonation. Elongation of siPhCRT3a pollen tubes was disrupted, and some of them ruptured. Our present data provide the first evidence that PhCRT3a expression is required for normal pollen tube growth. Thus, we discuss relationships between diverse CRT isoforms in several interdependent processes driving the apical growth of the pollen tube, including actomyosin-dependent cytoplasmic streaming, organelle positioning, vesicle trafficking, and cell wall biogenesis.

Diversification of SEC15a and SEC15b isoforms of an exocyst subunit in seed plants is manifested in their specific roles in Arabidopsis sporophyte and male gametophyte.

The exocyst complex is an octameric evolutionarily conserved tethering complex engaged in the regulation of polarized secretion in eukaryotic cells. Here, we focus on the systematic comparison of two isoforms of the SEC15 exocyst subunit, SEC15a and SEC15b. We infer that SEC15 gene duplication and diversification occurred in the common ancestor of seed plants (Spermatophytes). In Arabidopsis, SEC15a represents the main SEC15 isoform in the male gametophyte, and localizes to the pollen tube tip at the plasma membrane. Although pollen tubes of sec15a mutants are impaired, sporophytes show no phenotypic deviations. Conversely, SEC15b is the dominant isoform in the sporophyte and localizes to the plasma membrane in root and leaf cells. Loss-of-function sec15b mutants exhibit retarded elongation of hypocotyls and root hairs, a loss of apical dominance, dwarfed plant stature and reduced seed coat mucilage formation. Surprisingly, the sec15b mutants also exhibit compromised pollen tube elongation in vitro, despite its very low expression in pollen, suggesting a non-redundant role for the SEC15b isoform there. In pollen tubes, SEC15b localizes to distinct cytoplasmic structures. Reciprocally to this, SEC15a also functions in the sporophyte, where it accumulates at plasmodesmata. Importantly, although overexpressed SEC15a could fully complement the sec15b phenotypic deviations in the sporophyte, the pollen-specific overexpression of SEC15b was unable to fully compensate for the loss of SEC15a function in pollen. We conclude that the SEC15a and SEC15b isoforms evolved in seed plants, with SEC15a functioning mostly in pollen and SEC15b functioning mostly in the sporophyte.