Male Sterile Phenotype Research Articles

Floral Developmental Morphology and Biochemical Characteristics of Male Sterile Mutants of Lagerstroemia indica.

Male sterility is a common phenomenon in higher plants and often plays an important role in the selection of superior offspring. 'Xiang Yun' is a mutant of Lagerstroemia indica that does not bear fruit after flowering, and its flowering period is significantly longer than that of normal L. indica. To explore the timing and molecular mechanisms of sterility in 'Xiang Yun', this study determined the period of sterility through anatomical observation and compared the content of nutrients and the activity of antioxidative enzymes at different stages of flower development. Finally, sequence alignment and qPCR were used to analyze the differences in pollen development genes between 'Xiang Yun' and 'Hong Ye'. The results showed that the anthers of 'Xiang Yun' dispersed pollen normally, but the pollen grains could not germinate normally. Observations with scanning electron microscopy revealed that the pollen grains were uneven in size and shriveled in shape. Further observation of anther sections found that abnormal development of the microspores began at the S2 stage, with the callose wall between microspores of 'Xiang Yun' being thicker than that of 'Hong Ye'. In addition, during the flower development of 'Xiang Yun', the contents of soluble sugar, soluble protein, free proline, and triglycerides were deficient to varying degrees, and the activities of POD, SOD, and MDA were lower. Sequence alignment and qPCR showed that there were several mutations in EFD1, TPD1, and DEX1 of 'Xiang Yun' compared with 'Hong Ye', and the expression levels of these genes were abnormally elevated in the later stages of development. Our results clarified the timing and phenotype of male sterility in 'Xiang Yun'. This provides solid and valuable information for further research on the molecular mechanism of sterility in 'Xiang Yun' and the genetic breeding of crape myrtle.

Cell Wall Invertases from Maternal Tissues Modulate Sucrose Flux in Apoplastic Pathways During Rice Anther and Seed Development.

Efficient sucrose transport and metabolism are vital for seed and pollen development in plants. Cell wall invertases (CINs) hydrolyze sucrose into glucose and fructose, maintaining a sucrose gradient in the apoplast of sink tissues. In rice, two CIN isoforms, OsCIN1 and OsCIN2, were identified as being specifically expressed in the anthers but not in pollen. Functional analyses through genetic crosses and mutant characterization showed that oscin1/2 double mutants exhibit a sporophytic male-sterile phenotype and produce shrunken seeds. This suggests that CIN activity is essential for proper pollen development and seed formation in rice. Observation of the progeny genotypes and phenotypes from various genetic crosses revealed that the phenotype of oscin1/2 seeds is determined by the genotype of the maternal tissue, indicating the critical role of CIN function in the apoplast between maternal and filial tissues for sucrose transport and metabolism. The CIN activity in the anthers and seeds of wild-type rice was found to be significantly higher-over 500-fold in the anthers and 5-fold in the seeds-than in the leaves, highlighting the importance of CIN in facilitating the efficient unloading of sucrose. These findings suggest that the fine-tuning of CIN activity in the apoplast, achieved through tissue-specific expression and CIN isoform regulation, plays a key role in determining the carbohydrate distribution across different tissues. Understanding this regulatory mechanism could provide opportunities to manipulate carbohydrate allocation to sink organs, potentially enhancing crop yields.

Unbalanced lipid metabolism in anther, especially the disorder of the alpha-linolenic acid metabolism pathway, leads to cotton male sterility

Recent studies have shown that lipid metabolism is a key factor affecting anther development and male fertility. However, how plants regulating the metabolic balance of multiple lipids to ensure proper anther development and male fertility remains unclear. Analyzing lipid molecules related to anther fertility and genes responsible for their biosynthesis is crucial for understanding the physiological significance of lipid metabolism in crop fertility. In this study, we compared the transcriptome and the composition and content of lipids in anthers of two Upland cotton (Gossypium hirsutum) materials, Shida 98 (WT) and its nearly-isogenic male sterile line Shida 98A (MS). Transcriptomics analysis identified many differentially expressed genes between the two materials, with the genes of the alpha-linolenic acid metabolism pathway being the most significantly associated with the male sterility phenotype. Investigations on lipids revealed that the MS anthers over-accumulated free fatty acids (FFAs), phosphatidic acid (PA), mono- and di-galactosyldiacylglycerol (MGDG and DGDG), and had a decreased content of triacylglycerol (TAG), which was closely related to the abnormal metabolism of alpha-linolenic acid (C18:3); therefore, the major lipids containing C18:3-acyl chains, such as PA, MGDG, DGDG and TAG, are proposed to play a major role in cotton anther development. We also showed that an excessive level of MGDG and DGDG caused JA overaccumulation in MS anthers, which in turn inhibited the expression of GhFAD3 and consequently reduced the C18:3 content, presumably via a feedback regulation mechanism, ultimately affecting plant fertility. Together, our results revealed the importance of a balanced lipid metabolism in regulating the development of cotton anther and pollen and consequently male fertility.

GhTKPR1_8 functions to inhibit anther dehiscence and reduce pollen viability in cotton.

Sporopollenin, as the main component of the pollen exine, is a highly resistant polymer that provides structural integrity under unfavourable environmental conditions. Tetraketone α-pyrone reductase 1 (TKPR1) is essential for sporopollenin formation, catalyzing the reduction of tetraketone carbonyl to hydroxylated α-pyrone. The functional role of TKPR1 in male sterility has been reported in flowering plants such as maize, rice, and Arabidopsis. However, the molecular cloning and functional characterization of TKPR1 in cotton remain unaddressed. In this study, we identified 68 TKPR1s from four cotton species, categorized into three clades. Transcriptomics and RT-qPCR demonstrated that GhTKPR1_8 exhibited typical expression patterns in the tetrad stage of the anther. GhTKPR1_8 was localized to the endoplasmic reticulum. Moreover, ABORTED MICROSPORES (GhAMS) transcriptionally activated GhTKPR1_8 as indicated by luciferase complementation tests. GhTKPR1_8-knockdown inhibited anther dehiscence and reduced pollen viability in cotton. Additionally, overexpression of GhTKPR1_8 in the attkpr1 mutant restored its male sterile phenotype. This study offers novel insights into the investigation of TKPR1 in cotton while providing genetic resources for studying male sterility.

Development and Investigation of HRM Markers to Discriminate Two Ogura Cytoplasmic Male Sterility Restorer Genes in Radish

Ogura male sterile cytoplasm is widely used for radish breeding. In this study, high-resolution melting (HRM) markers associated with Rft and Rfo, major restorer-of-fertility genes in Ogura cytoplasmic male sterility (CMS) in radish, were developed. Genetic mapping was carried out using F2 populations derived from crosses between male-sterile Ogura CMS lines and male-fertile lines. Identification of the Rft and Rfo loci was achieved through SNP-based genotyping and linkage grouping. HRM markers were subsequently developed based on flanking sequences of SNPs linked to these loci. For the Rft gene, a set of 117 SNPs was selected within a candidate region on chromosome 5, and 14 HRM markers were successfully developed. Genotyping of F2 showed high correlation between three markers and the phenotype. Regarding the Rfo gene, a set of 27 HRM markers was designed based on flanking sequences of SNPs located on chromosomes 9 and 0. Genotyping in the Rfo segregating population identified a single marker, RSRF27, that accurately distinguished the male sterility phenotype. Validation of the developed markers was performed in populations containing both Rft and Rfo genes, confirming their utility for genotyping and demonstrating that these two genes independently contribute to male sterility recovery. Overall, this study provides HRM markers that can be used for genotyping Rft and Rfo and contributes to a deeper understanding of male sterility restoration mechanisms in Ogura CMS.

Two cytoplasmic male sterility phenotypes in beet (Beta vulgaris L.): implications of their simultaneous onset and divergent paths

Two cytoplasmic male sterility phenotypes in beet (Beta vulgaris L.): implications of their simultaneous onset and divergent paths

Cytoplasmic male sterility and abortive seed traits generated through mitochondrial genome editing coupled with allotopic expression of atp1 in tobacco.

Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Δatp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Δatp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Δatp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Δatp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.

TPLATE complex-dependent endocytosis attenuates CLAVATA1 signaling for shoot apical meristem maintenance.

Endocytosis regulates the turnover of cell surface localized receptors, which are crucial for plants to rapidly respond to stimuli. The evolutionary ancient TPLATE complex (TPC) plays an essential role in endocytosis in Arabidopsis plants. Knockout or knockdown of single TPC subunits causes male sterility and seedling lethality phenotypes, complicating analysis of the roles of TPC during plant development. Partially functional alleles of TPC subunits however only cause mild developmental deviations. Here, we took advantage of the partially functional TPLATE allele, WDXM2, to investigate a role for TPC-dependent endocytosis in receptor-mediated signaling. We discovered that reduced TPC-dependent endocytosis confers a hypersensitivity to very low doses of CLAVATA3 peptide signaling. This hypersensitivity correlated with the abundance of the CLAVATA3 receptor protein kinase CLAVATA1 at the plasma membrane. Genetic and biochemical analysis as well as live-cell imaging revealed that TPC-dependent regulation of CLAVATA3-dependent internalization of CLAVATA1 from the plasma membrane is required for shoot stem cell homeostasis. Our findings provide evidence that TPC-mediated endocytosis and degradation of CLAVATA1 is a mechanism to dampen CLAVATA3-mediated signaling during plant development.

Knockout of floral and meiosis genes using CRISPR/Cas9 produces male-sterility in Eucalyptus without impacts on vegetative growth.

Eucalyptus spp. are widely cultivated for the production of pulp, energy, essential oils, and as ornamentals. However, their dispersal from plantings, especially when grown as an exotic, can cause ecological disruptions. To provide new tools for prevention of sexual dispersal by pollen as well as to induce male-sterility for hybrid breeding, we studied the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated knockout of three floral genes in both FT-expressing (early-flowering) and non-FT genotypes. We report male-sterile phenotypes resulting from knockout of the homologs of all three genes, including one involved in meiosis and two regulating early stages of pollen development. The targeted genes were Eucalyptus homologs of REC8 (EREC8), TAPETAL DEVELOPMENT AND FUNCTION 1 (ETDF1), and HECATE3 (EHEC3-like). The erec8 knockouts yielded abnormal pollen grains and a predominance of inviable pollen, whereas the etdf1 and ehec3-like knockouts produced virtually no pollen. In addition to male-sterility, both erec8 and ehec3-like knockouts may provide complete sterility because the failure of erec8 to undergo meiosis is expected to be independent of sex, and ehec3-like knockouts produce flowers with shortened styles and no visible stigmas. When comparing knockouts to controls in wild-type (non-early-flowering) backgrounds, we did not find visible morphological or statistical differences in vegetative traits, including average single-leaf mass, stem volume, density of oil glands, or chlorophyll in leaves. Loss-of-function mutations in any of these three genes show promise as a means of inducing male- or complete sterility without impacting vegetative development.

Industrial chicory genome gives insights into the molecular timetable of anther development and male sterility.

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.

Rice tapetum differentiation is sensitive to downregulation of OsUCH3, a ubiquitin C-terminal hydrolase.

The tapetum is a special tissue within the stamen that supplies nutrients to support microspore differentiation. Many genes have been identified to function in tapetum differentiation (Astrand et al., 2021). Ubiquitin C-terminal hydrolase (UCH) is a type of cysteine protease involved in protein deubiquitination process (Mevissen and Komander, 2017) and plays roles in gonadal transformation and spermatogenesis in animals (Luo et al., 2009; Kwon et al,. 2004; Wang et al., 2006). OsUCH3 (Os02g43760) was highly expressed in early rice stamen cells (Chen et al., 2015), the OsUCH3 expression was first observed in all cells of anther primordium and then gradually concentrated into the germ cells and tapetum cells, and OsUCH3 protein exhibits stronger ubiquitin C-terminal hydrolase enzymatic activity (Wang et al., 2018). These data suggested that OsUCH3 is likely associated with stamen development. To explore whether OsUCH3 plays roles in rice stamen development, we constructed transgenic lines to down-regulate OsUCH3 expression through RNAi vector as described previously with primers (ccACTAGTATGGAGGATGCTCATTCC and TcGGATCCCACAACTTTCGAAAGAGC) (Liu et al., 2005) (Figure 1a–g) and the OsUCH3-targeting CRISPR-Cas9 mutant lines (Figure 1h). CRISPR-Cas9 osuch3 mutant lines exhibited the same male-sterile phenotype as OsUCH3R. In observation of the overall morphology, there was no significant difference at vegetable development (Figure 1a). However, low seed setting rate was observed in the RNAi lines (Figure 1b) and CRISPR-Cas9 osuch3 mutant (Figure 1h). Alexander staining showed that OsUCH3R lines had abnormal pollen (Figure 1d) with the latter being indicated by the significant reduction in pollen grain number (Figure 1e, g). The RT-qPCR results revealed that OsUCH3 expression levels were significantly reduced in OsUCH3R transgenic rice (Figure 1f). To examine whether OsUCH3 affected stamen development, we pollinated OsUCH3R pistils with wild-type pollen grains, which rescued the sterility of OsUCH3R. The result suggested that the low seed setting rate resulted mainly from down-expression of OsUCH3 (Figure 1i). Morphological analysis revealed abnormal tapetum differentiation and locule collapse were observed in the OsUCH3R lines from stage 5 (S5) through 12 (S12) (Figure 1j). This observation suggested that the downregulation of OsUCH3 caused a defect in the tapetum differentiation and stamen development. In contrast to the degeneration of the tapetum that was observed in the WT from stages 7 (S7) to 9 (S9) (Figure 1k–m), the tapetum in the OsUCH3R lines was not properly degenerated during these stages (Figure 1n–p) as revealed by transmission electron microscopy (TEM) analysis. In addition, TEM observations revealed that the secondary cell walls of the endothecium cells were thinner in the OsUCH3R lines than in the wild type (Figure 1p, red arrowhead). We found green fluorescence in wild-type tapetal cells from stages 7 to 9 (Figure 1q-t) using the terminal deoxynucleotidyl transferase-mediated TdT-mediated dUTP Nick-End Labeling (TUNEL) assay (Chen et al., 2018). No such TUNEL signals were detected in OsUCH3R tapetal cells during the same developmental period (Figure 1u–x). These results suggested that DNA degradation during tapetum differentiation after stage 7 was abnormal in OsUCH3R. Numerous genes were known being involved in tapetum differentiation (Chen et al., 2018). GAMYB4 was up-regulated while the expression of YY1, OsCP1 and UDT1 exhibited no change in the OsUCH3R lines (Figure 1y). OsUCH3 is lower expressed in shoot tips and leaf primordia. While no differential expression of SAM genes OsWOX3, the expression of OSH1 and OsWUS was significantly decreased in the OsUCH3R lines (Figure 1y). These changes demonstrated the link between abnormal tapetum differentiation in the OsUCH3R lines with the changes in the expression levels of known tapetum genes. The phenotypes we found in OsUCH3R lines and CRISPR mutants demonstrated that tapetal differentiation is sensitive to the change of expression level of OsUCH3 and protein deubiquitination is an importance factor for proper tapetum differentiation. This finding provided a new opportunity not only in deciphering the regulatory network of tapetum differentiation at protein level, but in manipulating male sterility for agricultural applications. Sequence data from this article can be found in the GenBank/EMBL data library. This work was supported by grants from the Chinese Transgenic Project (grant no. CARS-01-06, 2016ZX08010001 and 2016ZX08009-003) and the National Natural Science Foundation of China (grant no. 31630006). The authors declare no competing interests. D.H.W., Z.H.X. and S.N.B. designed the research and wrote the manuscript. D.H.W. performed most of the experiments. N.L. and S.D.Y. provided an assistance for mutant genotyping and plant material fixation. Z.S.C and Y.N.L provided technical assistance for transgenic plant analyse. Z.H.L helped the semithin sectioning.

A bifunctional selectable marker for wheat transformation contributes to the characterization of male-sterile phenotype induced by a synthetic Ms2 gene.

An engineered selectable marker combining herbicide resistance and yellow fluorescence contributes to the characterization of male-sterile phenotype in wheat, the severity of which correlates with expression levels of a synthetic Ms2 gene. Genetic transformation of wheat is conducted using selectable markers, such as herbicide and antibiotic resistance genes. Despite their proven effectiveness, they do not provide visual control of the transformation process and transgene status in progeny, which creates uncertainty and prolongs screening procedures. To overcome this limitation, this study developed a fusion protein by combining gene sequences encoding phosphinothricin acetyltransferase and mCitrine fluorescent protein. The fusion gene, introduced into wheat cells by particle bombardment, enabled herbicide selection, and visual identification of primary transformants along with their progeny. This marker was then used to select transgenic plants containing a synthetic Ms2 gene. Ms2 is a dominant gene whose activation in wheat anthers leads to male sterility, but the relationship between the expression levels and the male-sterile phenotype is unknown. The Ms2 gene was driven either by a truncated Ms2 promoter containing a TRIM element or a rice promoter OsLTP6. The expression of these synthetic genes resulted in complete male sterility or partial fertility, respectively. The low-fertility phenotype was characterized by smaller anthers than the wild type, many defective pollen grains, and low seed sets. The reduction in the size of anthers was observed at earlier and later stages of their development. Consistently, Ms2 transcripts were detected in these organs, but their levels were significantly lower than those in completely sterile Ms2TRIM::Ms2 plants. These results suggested that the severity of the male-sterile phenotype was modulated by Ms2 expression levels and that higher levels may be key to activating total male sterility.

The Prmt5-Vasa module is essential for spermatogenesis in Bombyx mori.

In lepidopteran insects, dichotomous spermatogenesis produces eupyrene spermatozoa, which are nucleated, and apyrene spermatozoa, which are anucleated. Both sperm morphs are essential for fertilization, as eupyrene sperm fertilize the egg, and apyrene sperm is necessary for the migration of eupyrene sperm. In Drosophila, Prmt5 acts as a type II arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues in the RNA helicase Vasa. Prmt5 is critical for the regulation of spermatogenesis, but Vasa is not. To date, functional genetic studies of spermatogenesis in the lepidopteran model Bombyx mori has been limited. In this study, we engineered mutations in BmPrmt5 and BmVasa through CRISPR/Cas9-based gene editing. Both BmPrmt5 and BmVasa loss-of-function mutants had similar male and female sterility phenotypes. Through immunofluorescence staining analysis, we found that the morphs of sperm from both BmPrmt5 and BmVasa mutants have severe defects, indicating essential roles for both BmPrmt5 and BmVasa in the regulation of spermatogenesis. Mass spectrometry results identified that R35, R54, and R56 of BmVasa were dimethylated in WT while unmethylated in BmPrmt5 mutants. RNA-seq analyses indicate that the defects in spermatogenesis in mutants resulted from reduced expression of the spermatogenesis-related genes, including BmSxl, implying that BmSxl acts downstream of BmPrmt5 and BmVasa to regulate apyrene sperm development. These findings indicate that BmPrmt5 and BmVasa constitute an integral regulatory module essential for spermatogenesis in B. mori.

A 163-bp insertion in the Capana10g000198 encoding a MYB transcription factor causes male sterility in pepper (Capsicum annuum L.).

Male sterility provides an efficient approach for commercial exploitation of heterosis. Despite more than 20 genic male sterile (GMS) mutants documented in pepper (Capsicum annuum L.), only two causal genes have been successfully identified. Here, a novel spontaneous recessive GMS mutant, designated msc-3, is identified and characterized at both phenotypic and histological levels. Pollen abortion of msc-3 mutant may be due to the delayed tapetum degradation, leading to the non-degeneration of tetrads callosic wall. Then, a modified MutMap method and molecular marker linkage analysis were employed to fine mapping the msc-3 locus, which was delimited to the ~139.91-kb region harboring 10 annotated genes. Gene expression and structure variation analyses indicate the Capana10g000198, encoding a R2R3-MYB transcription factor, is the best candidate gene for the msc-3 locus. Expression profiling analysis shows the Capana10g000198 is an anther-specific gene, and a 163-bp insertion in the Capana10g000198 is highly correlated with the male sterile (MS) phenotype. Additionally, downregulation of Capana10g000198 in male fertile plants through virus-induced gene silencing resulted in male sterility. Finally, possible regulatory relationships of the msc-3 gene with the other two reported pepper GMS genes, msc-1 and msc-2, have been studied, and comparative transcriptome analysis reveals the expression of 16 GMS homologs are significantly downregulated in the MS anthers. Overall, our results reveal that Capana10g000198 is the causal gene underlying the msc-3 locus, providing important theoretical clues and basis for further in-depth study on the regulatory mechanisms of pollen development in pepper.

Defect in BrMS1, a PHD-finger transcription factor, induces male sterility in ethyl methane sulfonate-mutagenized Chinese cabbage (Brassica rapa L. ssp. pekinensis).

Male sterility is an ideal character for the female parent in commercial hybrid seed production in Chinese cabbages. We identified three allele male sterile mutants msm2-1/2/3 in progenies of ethyl methane sulfonate mutagenized Chinese cabbage. It was proved that their male sterilities were controlled by a same recessive nuclear gene. Cytological observation showed that the delayed tapetal programmed cell death (PCD) as well as the abnormal pollen exine and intine led to pollen abortion in these mutants. MutMap combined with KASP analyses showed that BraA10g019050.3C, a homologous gene of AtMS1 encoding a PHD-finger transcription factor and regulated pollen development, was the causal gene. A single-nucleotide mutation from G to A occurred at the 2443th base of BrMS1 in msm2-1 which results in premature termination of the PHD-finger protein translation; a single-nucleotide mutation from G to A existed at 1372th base in msm2-2 that makes for frameshift mutation; a single-nucleotide mutation from G to A distributed at 1887th base in msm2-3 which issues in the amino acid changed from Asp to Asn. The three allelic mutations in BrMS1 all led to the male sterile phenotype, which revealed its function in stamen development. Quantitative reverse transcription polymerase chain reaction analysis indicated that BrMS1 specially expressed in the anther at the early stage of pollen development and its expression level was higher in msm2-1/2/3 than that in the wild-type "FT." BrMS1 was located at the nucleus and a length of 12 amino acid residues at the C-terminus had transcriptional activation activity. RNA-seq indicated that the mutation in BrMS1 affected the transcript level of genes related to the tapetum PCD and pollen wall formation, which brought out the pollen abortion. These male sterile mutants we developed provided a novel gene resource for hybrid breeding in Chinese cabbage.

Editing melon eIF4E associates with virus resistance and male sterility.

SummaryThe cap‐binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularization of mRNAs and their subsequent cap‐dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knockout mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single‐nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non‐transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non‐mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male sterility phenotype, while crossing with wild‐type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild‐type. An RNA‐Seq analysis identified critical genes in pollen development that were down‐regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E‐specific mRNA translation initiation is a limiting factor for male gametes formation in melon.

Wheat male-sterile 2 reduces ROS levels to inhibit anther development by deactivating ROS modulator 1

Ms2 is an important dominant male-sterile gene in wheat, but the biochemical function of Ms2 and the mechanism by which it causes male sterility remain elusive. Here, we report the molecular basis underlying Ms2-induced male sterility in wheat. We found that activated Ms2 specifically reduces the reactive oxygen species (ROS) signals in anthers and thereby induces termination of wheat anther development at an early stage. Furthermore, our results indicate that Ms2 is localized in mitochondria, where it physically interacts with a wheat homolog of ROS modulator 1 (TaRomo1). Romo1 positively regulates the ROS levels in humans but has never been studied in plants. We found that single amino acid substitutions in the Ms2 protein that rescue the ms2 male-sterile phenotype abolish the interaction between Ms2 and TaRomo1. Significantly, Ms2 promotes the transition of TaRomo1 proteins from active monomers to inactive oligomers. Taken together, our findings unravel the molecular basis of Ms2-induced male sterility and reveal a regulatory mechanism in which ROS act as essential signals guiding the anther development program in wheat.

Highly Overexpressed AtC3H18 Impairs Microgametogenesis via Promoting the Continuous Assembly of mRNP Granules.

Plant CCCH zinc-finger proteins form a large family of regulatory proteins function in many aspects of plant growth, development and environmental responses. Despite increasing reports indicate that many CCCH zinc-finger proteins exhibit similar subcellular localization of being localized in cytoplasmic foci, the underlying molecular mechanism and the connection between this specific localization pattern and protein functions remain largely elusive. Here, we identified another cytoplasmic foci-localized CCCH zinc-finger protein, AtC3H18, in Arabidopsis thaliana. AtC3H18 is predominantly expressed in developing pollen during microgametogenesis. Although atc3h18 mutants did not show any abnormal phenotype, possibly due to redundant gene(s), aberrant AtC3H18 expression levels caused by overexpression resulted in the assembly of AtC3H18-positive granules in a dose-dependent manner, which in turn led to male sterility phenotype, highlighting the importance of fine-tuned AtC3H18 expression. Further analyzes demonstrated that AtC3H18-positive granules are messenger ribonucleoprotein (mRNP) granules, since they can exhibit liquid-like physical properties, and are associated with another two mRNP granules known as processing bodies (PBs) and stress granules (SGs), reservoirs of translationally inhibited mRNAs. Moreover, the assembly of AtC3H18-positive granules depends on mRNA availability. Combined with our previous findings on the AtC3H18 homologous genes in Brassica campestris, we concluded that appropriate expression level of AtC3H18 during microgametogenesis is essential for normal pollen development, and we also speculated that AtC3H18 may act as a key component of mRNP granules to modulate pollen mRNAs by regulating the assembly/disassembly of mRNP granules, thereby affecting pollen development.

Identification of a Male Sterile Candidate Gene in Lilium x formolongi and Transfer of the Gene to Easter Lily (L. longiflorum) via Hybridization.

Pollen-free varieties are advantageous in promoting cut-flower production. In this study, we identified a candidate mutation which is responsible for pollen sterility in a strain of Lilium × formolongi, which was originally identified as a naturally occurred male-sterile plant in a seedling population. The pollen sterility occurred due to the degradation of pollen mother cells (PMCs) before meiotic cell division. Genetic analysis suggested that the male-sterile phenotype is attributed to one recessive locus. Transcriptome comparison between anthers of sterile and fertile plants in a segregated population identified a transcript that was expressed only in pollen-fertile plants, which is homologous to TDF1 (DEFECTIVE in TAPETAL DEVELOPMENT and FUNCTION1) in Arabidopsis, a gene encoding a transcription factor AtMYB35 that is known as a key regulator of pollen development. Since tdf1 mutant shows male sterility, we assumed that the absence transcript of the TDF1-like gene, named as LflTDF1, is the reason for pollen sterility observed in the mutant. A 30 kbp-long nanopore sequence read containing LflTDF1 was obtained from a pollen-fertile accession. PCR analyses using primers designed from the sequence suggested that at least a 30kbp-long region containing LflTDF1 was deleted or replaced by unknown sequence in the pollen-sterile mutant. Since the cross between L. × formolongi and Easter lily (L. longiflorum) is compatible, we successfully introgressed the male-sterile allele, designated as lfltdf1, to Easter lily. To our knowledge, this is the first report of molecular identification of a pollen-sterile candidate gene in lily. The identification and marker development of LflTDF1 gene will assist pollen-free lily breeding of Easter lilies and other lilies.

Application of CRISPR/CasΦ2 System for Genome Editing in Plants.

CRISPR/Cas system has developed a new technology to modify target genes. In this study, CasΦ2 is a newly Cas protein that we used for genome modification in Arabidopsis and tobacco. PDS and BRI1 of marker genes were chosen for targeting. CasΦ2 has the function to cleave pre-crRNA. In the presence of 10 mM Mg2+ irons concentration, sgRNA3 type guided CasΦ2 to edit target gene and generate mutation, and a mutant seedling of AtBRI1 gene with an expected male sterile phenotype was obtained. In the process of tobacco transformation, the gene editing activity of CasΦ2 can be activated by 100 nM Mg2+ irons concentration, and sgRNA1 type guided CasΦ2 to edit target gene. Mutant seedlings of NtPDS gene with an expected albino were obtained. The results indicate that CasΦ2 can effectively edit target genes under the guidance of different sgRNA type in the presence of Mg2+ ions. Together, our results verify that the CRISPR/CasΦ2 system is an effective and precise tool for genome editing in plants.