Updated in October 2019
Although “dry-type” stigmas are widely regarded as ancestral in angiosperms, the early-divergent family Annonaceae has copious stigmatic exudate. We evaluate three putative functions for this exudate: as a nutritive reward for pollinators; as a pollen germination medium; and as an extragynoecial compitum that enables pollen tube growth between carpels. Stigmatic exudate is fructose dominated (72.2%), but with high levels of glucose and sucrose; the dominance of hexose sugars and the diversity of amino acids observed, including many that are essential for insects, support a nutritive role for pollinators. Sugar concentration in pre-receptive flowers is high (28.2%), falling during the peak period of stigmatic receptivity (17.4%), and then rising again toward the end of the pistillate phase (32.9%). Pollen germination was highest in sugar concentrations <20%. Sugar concentrations during the peak pistillate phase therefore provide optimal osmolarity for pollen hydration and germination; subsequent changes in sugar concentration during anthesis reinforce protogyny (in which carpels mature before stamens), enabling the retention of concentrated exudate into the staminate phase as a pollinator food reward without the possibility of pollen germination. Intercarpellary growth of pollen tubes was confirmed: the exudate therefore also functions as a suprastylar extragynoecial compitum, overcoming the limitations of apocarpy.
Pollen development is a pre-requisite for sexual reproduction of angiosperms, during which various cellular activities are involved. Pollen development accompanies dynamic remodeling of vacuoles through fission and fusion, disruption of which often compromises pollen viability. We previously reported that the Y subunit of adaptor protein 1 (AP1G) mediates synergid degeneration during pollen tube reception. Here, we demonstrate that AP1G is essential for pollen development. AP1G loss-of-function resulted in male gametophytic lethality due to defective pollen development. By ultrastructural analysis and fluorescence labeling, we demonstrate that AP1G loss-of-function compromised dynamic vacuolar remodeling during pollen development and impaired vacuolar acidification of pollen. Results presented here support a key role of vacuoles in gametophytic pollen development.
In angiosperms, initiation of ovule enlargement represents the start of seed development, the molecular mechanism of which is not yet elucidated. It was previously reported that pollen tube contents, rather than double fertilization, can trigger ovule enlargement. However, it remains unclear whether the signal(s) to trigger the initiation of ovule enlargement are from the sperm cells or from the pollen tubes. Recently, we identified a mutant drop1− drop2−, which produces pollen tubes with no sperm cells. Taking advantage of this special genetic material, we conducted pollination assays, and found that the ovules pollinated with drop1− drop2− pollen could initiate the enlargement and exhibited significant enlarged sizes at 36 h after pollination in comparison with those unpollinated ovules. However, the sizes of the ovules pollinated with drop1− drop2− pollen are significantly smaller than those of the ovules pollinated with wild-type pollen. These results demonstrate that the pollen tube, rather than the sperm cells, release the signal to trigger the initiation of ovule enlargement, and that double fertilization is required for further enlargement of the seeds.
Pollen hydration is a critical step that determines pollen germination on the stigma. KINβγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex (SnRK1 complex). In pollen of the Arabidopsis kinβγ mutant, the levels of reactive oxygen species were decreased which lead to compromised hydration of the mutant pollen on the stigma. In this study, we analyzed gene expression in kinβγ mutant pollen by RNA-seq and found the expression of inward shaker K+ channel SPIK was down-regulated in the kinβγ pollen. Furthermore, we showed that the pollen hydration of the Arabidopsis spik mutant was defective on the wild-type stigma, although the mutant pollen demonstrated normal hydration in vitro. Additionally, the defective hydration of spik mutant pollen could not be rescued by the wild-type pollen on the stigma, indicating that the spik mutation deprived the capability of pollen absorption on the stigma. Our results suggest that the Arabidopsis SnRK1 complex regulates SPIK expression, which functions in determining pollen hydration on the stigma.
Lipid and phenolic metabolism are important for pollen exine formation. In Arabidopsis, polyketide synthases (PKSs) are essential for both sporopollenin biosynthesis and exine formation. Here, we characterized the role of a polyketide synthase (OsPKS2) in male reproduction of rice (Oryza sativa). Recombinant OsPKS2 catalyzed the condensation of fatty acyl-CoA with malonyl-CoA to generate triketide and tetraketide α-pyrones, the main components of pollen exine. Indeed, the ospks2 mutant had defective exine patterning and was male sterile. However, the mutant showed no significant reduction in sporopollenin accumulation. Compared with the WT (wild type), ospks2 displayed unconfined and amorphous tectum and nexine layers in the exine, and less organized Ubisch bodies. Like the pksb/lap5 mutant of the Arabidopsis ortholog, ospks2 showed broad alterations in the profiles of anther-related phenolic compounds. However, unlike pksb/lap5, in which most detected phenolics were substantially decreased, ospks2 accumulated higher levels of phenolics. Based on these results and our observation that OsPKS2 is unable to fully restore the exine defects in the pksb/lap5, we propose that PKS proteins have functionally diversified during evolution. Collectively, our results suggest that PKSs represent a conserved and diversified biochemical pathway for anther and pollen development in higher plants.
Two yeast Brix family members Ssf1 and Ssf2, involved in large ribosomal subunit synthesis, are essential for yeast cell viability and mating efficiency. Their putative homologs exist in the Arabidopsis genome; however, their role in plant development is unknown. Here, we show that Arabidopsis thaliana SNAIL1 (AtSNAIL1), a protein sharing high sequence identity with yeast Ssf1 and Ssf2, is critical to mitosis progression of female gametophyte development. The snail1 homozygous mutant was nonviable and its heterozygous mutant was semi-sterile with shorter siliques. The mutation in SNAIL1 led to absence of female transmission and reduced male transmission. Further phenotypic analysis showed that the synchronic development of female gametophyte in the snail1 heterozygous mutant was greatly impaired and the snail1 pollen tube growth, in vivo, was also compromised. Furthermore, SNAIL1 was a nucleolar-localized protein with a putative role in protein synthesis. Our data suggest that SNAIL1 may function in ribosome biogenesis like Ssf1 and Ssf2 and plays an important role during megagametogenesis in Arabidopsis.
The specific functions of the genes encoding arginine biosynthesis enzymes in plants are not well characterized. We report the isolation and characterization of Arabidopsis thaliana N-acetylglutamate kinase (NAGK), which catalyzes the second step of arginine biosynthesis. NAGK is a plastid-localized protein and is expressed during most developmental processes in Arabidopsis. Heterologous expression of the Arabidopsis NAGK gene in a NAGK-deficient Escherichia coli strain fully restores bacterial growth on arginine-deficient medium. nagk mutant pollen tubes grow more slowly than wild type pollen tubes and the phenotype is restored by either specifically through complementation by NAGK in pollen, or exogenous supplementation of arginine. nagk female gametophytes are defective in micropylar pollen tube guidance due to the fact that female gametophyte cell fate specification was specifically affected. Expression of NAGK in synergid cells rescues the defect of nagk female gametophytes. Loss-of-function of NAGK results in Arabidopsis embryos not developing beyond the four-celled embryo stage. The embryo-defective phenotype in nagk/NAGK plants cannot be rescued by watering nagk/NAGK plants with arginine or ornithine supplementation. In conclusion, our results reveal a novel role of NAGK and arginine in regulating gametophyte function and embryo development, and provide valuable insights into arginine transport during embryo development.
Both female and male gametophytes harbor companion cells and gametes. MET1, a DNA methyltransferase, is down-regulated in companion cells. However, how MET1 is differentially regulated in gametophytes remains unexplored. ARID1, a transcription factor that is specifically depleted in sperm cells, is occupied by MET1-dependent CG methylation. Here, we show that MET1 confines ARID1 to the vegetative cell of male gametes, but ARID1 conversely represses MET1 in the central cell of female gametes. Compared to the vegetative cell-localization in wild type pollen, ARID1 expands to sperm cells in the met1 mutant. To understand whether MET1-dependent ARID1 inhibition exists during female gametogenesis, we first show that ARID1 is expressed in the megaspore mother cell (MMC), ARID1 but not MET1 is detectable in the central cell at maturity. Interestingly, compared to the absence of MET1 in the central cell and the egg cell of wild type ovules, MET1 significantly accumulates in these two cells in arid1 ovules. Lastly, we show that both ARID1 and MET1 are required for the cell specification of MMC. Collectively, our results uncover a reciprocal dependence between ARID1 and MET1, and provide a clue to further understand how the specification of MMC is likely regulated by DNA methylation.
Hybrids between the indica and japonica subspecies of rice (Oryza sativa) are usually sterile, which hinders utilization of heterosis in the inter-subspecific hybrid breeding. The complex locus Sa comprises two adjacently located genes, SaF and SaM, which interact to cause abortion of pollen grains carrying the japonica allele in japonica-indica hybrids. Here we showed that silencing of SaF or SaM by RNA interference restored male fertility in indica-japonica hybrids with heterozygous Sa. We further used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing to knockout the SaF and SaM alleles, respectively, of an indica rice line to create hybrid-compatible lines. The resultant artificial neutral alleles did not affect pollen viability and other agricultural traits, but did break down the reproductive barrier in the hybrids. We found that some rice lines have natural neutral allele Sa-n, which was compatible with the typical japonica or indica Sa alleles in hybrids. Our results demonstrate that SaF and SaM are required for hybrid male sterility, but are not essential for pollen development. This study provides effective approaches for the generation of hybrid-compatible lines by knocking out the Sa locus or using the natural Sa-n allele to overcome hybrid male sterility in rice breeding. © 2017 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.
In sexual organisms, division of the zygote initiates a new life cycle. Although several genes involved in zygote division are known in plants, how the zygote is activated to start embryogenesis has remained elusive. Here, we showed that a mutation in ZYGOTE-ARREST 3 (ZYG3) in Arabidopsis led to a tight zygote-lethal phenotype. Map-based cloning revealed that ZYG3 encodes the transfer RNA (tRNA) ligase AtRNL, which is a single-copy gene in the Arabidopsis genome. Expression analyses showed that AtRNL is expressed throughout zygotic embryogenesis, and in meristematic tissues. Using pAtRNL::cAtRNL-sYFP-complemented zyg3/zyg3 plants, we showed that AtRNL is localized exclusively in the cytoplasm, suggesting that tRNA splicing occurs primarily in the cytoplasm. Analyses using partially rescued embryos showed that mutation in AtRNL compromised splicing of intron-containing tRNA. Mutations of two tRNA endonuclease genes, SEN1 and SEN2, also led to a zygote-lethal phenotype. These results together suggest that tRNA splicing is critical for initiating zygote division in Arabidopsis.
Grasses display highly diversified inflorescence architectures that differ in the arrangement of spikelets and flowers and determine cereal yields. However, the molecular basis underlying grass inflorescence morphogenesis remains largely unknown. Here we investigate the role of a functionally diversified SEPALLATA MADS-box transcription factor, OsMADS34, in regulating rice (Oryza sativa L.) inflorescence and spikelet development. Microarray analysis showed that, at the very early stages of inflorescence formation, dysfunction of OsMADS34 caused altered expression of 379 genes that are associated with protein modification and degradation, transcriptional regulation, signaling and metabolism activity. Genetic analysis revealed that OsMADS34 controls different aspects of inflorescence structure, branching and meristem activity synergistically with LAX PANICLE1 (LAX1) and FLORAL ORGAN NUMBER4 (FON4), as evidenced by the enhanced phenotypes of osmads34 lax1 and osmads34 fon4 compared with the single mutants. Additionally, double mutant between osmads34 and the sterile lemma defective mutant elongated empty glume (ele) displayed an enhanced phenotype, that is, longer and wider sterile lemmas that were converted into lemma/palea-like organs, suggesting that ELE and OsMADS34 synergistically control the sterile lemma development. OsMADS34 may act together with OsMADS15 in controlling sterile lemma development. Collectively, these findings provide insights into the regulatory function of OsMADS34 in rice inflorescence and spikelet development.
Phytochromes mainly function in photoautotrophic organisms to adjust growth in response to fluctuating light signals. The different isoforms of plant phytochromes often display both conserved and divergent roles, presumably to fine-tune plant responses to environmental signals and optimize fitness. Here we describe the distinct, yet partially redundant, roles of phytochromes NaPHYA, NaPHYB1 and NaPHYB2 in a wild tobacco species, Nicotiana attenuata using RNAi-silenced phytochrome lines. Consistent with results reported from other species, silencing the expression of NaPHYA or NaPHYB2 in N. attenuata had mild or no influence on plant development as long as NaPHYB1 was functional; whereas silencing the expression of NaPHYB1 alone strongly altered flowering time and leaf morphology. The contribution of NaPHYB2 became significant only in the absence of NaPHYB1; plants silenced for both NaPHYB1 and NaPHYB2 largely skipped the rosette-stage of growth to rapidly produce long, slender stalks that bore flowers early: hallmarks of the shade-avoidance responses. The phenotyping of phytochrome-silenced lines, combined with sequence and transcript accumulation analysis, suggest the independent functional diversification of the phytochromes, and a dominant role of NaPHYB1 and NaPHYB2 in N. attenuata's vegetative and reproductive development.
In flowering plants, male gametes are delivered to female gametes for double fertilization through pollen tubes. Therefore, pollen tube growth is crucial for double fertilization. Despite its importance to sexual reproduction, genetic mechanisms of pollen tube growth remain poorly understood. In this study, we characterized the receptor-like cytoplasmic protein kinase (RLCK) gene, MARIS (MRI) that plays critical roles in pollen tube growth. MRI is preferentially expressed in pollen grains, pollen tubes and roots. Mutation in MRI by a Ds insertion led to a burst of pollen tubes after pollen germination. Pollen-rescue assay by pollen and pollen tube-specific expression of MRI in the mri-4 mutant showed that loss of MRI function also severely affected root hair elongation. MRI protein interacted with the protein kinase OXIDATIVE SIGNAL INDUCIBLE1 (OXI1) in the in vitro and in vivo assays, which functions in plant defence and root hair development, and was phosphorylated by OXI1 in vitro. Our results suggest that MRI plays important roles in pollen tube growth and may function in root hair elongation through interaction with OXI1.
In flowering plants, pollen tube growth is essential for delivery of male gametes into the female gametophyte or embryo sac for double fertilization. Although many genes have been identified as being involved in the process, the molecular mechanisms of pollen tube growth remains poorly understood. In this study, we identified that the Arabidopsis Transmembrane Protein 18 (AtTMEM18) gene played important roles in pollen tube growth. The AtTMEM18 shares a high similarity with the Transmembrane 18 proteins (TMEM18s) that are conserved in most eukaryotes and may play important roles in obesity in humans. Mutation in the AtTMEM18 by a Ds insertion caused abnormal callose deposition in the pollen grains and had a significant impact on pollen germination and pollen tube growth. AtTMEM18 is expressed in pollen grains, pollen tubes, root tips and other vegetative tissues. The pollen-rescued assays showed that the mutation in AtTMEM18 also caused defects in roots, stems, leaves and transmitting tracts. AtTMEM18-GFP was located around the nuclei. Genetic assays demonstrated that the localization of AtTMEM18 around the nuclei in the generative cells of pollen grains was essential for the male fertility. Furthermore, expression of the rice TMEM18-homologous protein (OsTMEM18) driven by LAT52 promoter could recover the fertility of the Arabidopsis attmem18 mutant. These results suggested that the TMEM18 is important for plant growth in Arabidopsis.
In Arabidopsis, the phytohormone abscisic acid (ABA) plays a vital role in inhibiting seed germination and in post-germination seedling establishment. In the ABA signaling pathway, ABI5, a basic Leu zipper transcription factor, has important functions in the regulation of seed germination. ABI5 protein localizes in nuclear bodies, along with AFP, COP1, and SIZ1, and was degraded through the 26S proteasome pathway. However, the mechanisms of ABI5 nuclear body formation and ABI5 protein degradation remain obscure. In this study, we found that the Arabidopsis CROWDED NUCLEI (CRWN) proteins, predicted nuclear matrix proteins essential for maintenance of nuclear morphology, also participate in ABA-controlled seed germination by regulating the degradation of ABI5 protein. During seed germination, the crwn mutants are hypersensitive to ABA and have higher levels of ABI5 protein compared to wild type. Genetic analysis suggested that CRWNs act upstream of ABI5. The observation that CRWN3 colocalizes with ABI5 in nuclear bodies indicates that CRWNs might participate in ABI5 protein degradation in nuclear bodies. Moreover, we revealed that the extreme C-terminal of CRWN3 protein is necessary for its function in the response to ABA in germination. Our results suggested important roles of CRWNs in ABI5 nuclear body organization and ABI5 protein degradation during seed germination.
In flowering plants, the male and female gametogenesis is a crucial step of sexual reproduction. Although many genes have been identified as being involved in the gametogenesis process, the genetic mechanisms underlying gametogenesis remains poorly understood. We reported here characterization of the gene, ABORTED GAMETOPHYTE 1 (AOG1) that is newly identified as essential for gametogenesis in Arabidopsis thaliana. AOG1 is expressed predominantly in reproductive tissues including the developing pollen grains and ovules. The AOG1 protein shares no significant amino acid sequence similarity with other documented proteins and is located mainly in nuclei of the cells. Mutation in AOG1 caused degeneration of pollen at the uninucleate microspore stage and severe defect in embryo sacs, leading to a significant reduction in male and female fertility. Furthermore, the molecular analyses showed that the aog1 mutant significantly affected the expression of several genes, which are required for gametogenesis. Our results suggest that AOG1 plays important roles in gametogenesis at the stage prior to pollen mitosis I (PMI) in Arabidopsis, possibly through collaboration with other genes.
Cui HH, Liao HZ, Tang Y, Du XY, Chen LQ, Ye D, Zhang XQ (2015) ABORTED GAMETOPHYTE 1 is required for gametogenesis in Arabidopsis. J Integr Plant Biol 57: 1003–1016 doi: 10.1111/jipb.12341
Phosphorus (P) is a major plant nutrient and developing crops with higher P-use efficiency is an important breeding goal. In this context we have conducted a comparative study of irrigated and rainfed rice varieties to assess genotypic differences in colonization with arbuscular mycorrhizal (AM) fungi and expression of different P transporter genes. Plants were grown in three different soil samples from a rice farm in the Philippines. The data show that AM symbiosis in all varieties was established after 4 weeks of growth under aerobic conditions and that, in soil derived from a rice paddy, natural AM populations recovered within 6 weeks. The analysis of AM marker genes (AM1, AM3, AM14) and P transporter genes for the direct Pi uptake (PT2, PT6) and AM-mediated pathway (PT11, PT13) were largely in agreement with the observed root AM colonization providing a useful tool for diversity studies. Interestingly, delayed AM colonization was observed in the aus-type rice varieties which might be due to their different root structure and might confer an advantage for weed competition in the field. The data further showed that P-starvation induced root growth and expression of the high-affinity P transporter PT6 was highest in the irrigated variety IR66 which also maintained grain yield under P-deficient field conditions.
Jeong K, Mattes N, Catausan S, Chin JH, Paszkowski U, Heuer S (2015) Genetic diversity for mycorrhizal symbiosis and phosphate transporters in rice. J Integr Plant Biol 57: 969–979 doi: 10.1111/jipb.12435
Reproductive isolation defines the biological species concept and plays a key role in the formation and maintenance of species. The relative contributions of different isolating stages has been suggested to be closely associated with phylogenetic relatedness. Few studies have focused on the relative contributions of pre- versus post-zygotic mechanisms, and even fewer have been conducted under strict phylogenetic frameworks. Pre- and post-zygotic reproductive isolation stages have been investigated in the sister species Mussaenda kwangtungensis and M. pubescens var. alba. The two species have partly overlapping distribution ranges and flowering times, while the principal pollinators differed strikingly for them, demonstrating strong pre-zygotic isolations. Natural hybrids were detected by simple sequence repeat markers and their maternal parents were identified based on chloroplast gene sequences. Five out of 81 individuals were suggested to be hybrids that fall into the categories F2, BC1, and BC2 by the NewHybrids analysis. Interspecific crossings resulted in significantly reduced fruit set and seed germination rates. Phylogenetic analysis revealed short Kimura-2-parameter distance between M. kwangtungensis and M. pubescens var. alba. These findings strongly supported the hypothesis that for species with a closer phylogenetic relationship, pre-zygotic isolation plays an important part in limiting gene exchange in sympatric areas.
Luo Z, Duan T, Yuan S, Chen S, Bai X, Zhang D (2015) Reproductive isolation between sympatric sister species, Mussaenda kwangtungensis and M. pubescens var.alba. J Integr Plant Biol 57: 859–870 doi: 10.1111/jipb.12325
PECTATE LYASE-LIKE10 (PLL10) was previously identified as one of the differentially expressed genes both in microspores during the late pollen developmental stages and in pistils during the fertilization process in Chinese cabbage (Brassica campestris ssp. chinensis). Here, antisense-RNA was used to study the functions of BcPLL10 in Chinese cabbage. Abnormal pollen was identified in the transgenic lines (bcpll10-4, -5, and -6). In fertilization experiments, fewer seeds were harvested when the antisense-RNA lines were used as pollen donor. In vivo and in vitro pollen germination assays less germinated pollen tubes were observed in bcpll10 lines. Scanning electron microscopy observation verified that the tryphine materials were over accumulated around the pollen surface and sticked them together in bcpll10. Moreover, transmission electron microscopy observation revealed that the internal endintine was overdeveloped and predominantly occupied the intine, and disturbed the normal proportional distribution of the two layers in the non-germinal furrow region; and no obvious demarcation existed between them in the germinal furrow region in the bcpll10 pollen. Collectively, this study presented a novel PLL gene that played an important role during the pollen wall development in B. campestris, which may also possess potential importance for male sterility usage in agriculture.
Jiang J, Yao L, Yu Y, Lv M, Miao Y, Cao J (2014) PECTATE LYASE‐LIKE10 is associated with pollen wall development in Brassica campestris. J Integr Plant Biol 56: 1095–1105. doi: 10.1111/jipb.12209
Reproductive isolation is a fundamental requirement for speciation and includes several sequential stages. Few studies have determined the relative contributions of pre- and post-zygotic reproductive isolation in plants, especially between relative species with clear differentiation in flower form. To investigate the mechanisms responsible for reproductive isolation in sympatric Mussaenda pubescens var. alba and Mussaenda shikokiana (Rubiaceae) in Guangxi Province, China, we made observations of flowering phenology, patterns of insect visitation, and conducted pollination experiments, including artificial hybridization. The two species had overlapping flowering times and were pollinated by overlapping pollinators; however, their relative importance differed significantly with M. pubescens visited more commonly by bees and M. shikokiana more frequently by butterflies. Using vegetative and floral characters and molecular evidence based on nuclear ribosomal internal and external transcribed spacer regions we detected seven naturally occurring hybrids among a sample of approximately 125 individuals. Hybrids were characterized by morphologies that most closely resembled their maternal parents based on chloroplast evidence. Studies of artificially synthesized and natural hybrids demonstrated that hybrid seed had very low germination rates and naturally occurring hybrids exhibited pollen sterility. Post-zygotic reproductive isolating mechanisms play a primary role in limiting gene exchange between co-occurring species and maintaining species integrity in areas of sympatry.
Chen S, Luo Z, Zhang D (2014) Pre‐and post‐zygotic reproductive isolation between co‐occurring Mussaenda pubescens var. alba and M. shikokiana (Rubiaceae). J Integr Plant Biol 56: 411–419. doi: 10.1111/jipb.12140
Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice (Oryza sativa L.) male sterile mutant. Based on map-based cloning and sequence analysis, we identified a 1,459-bp deletion in an adenosine triphosphate (ATP)-binding cassette (ABC) transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcg15. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 (meiosis II stage) to stage 10 (late microspore stage). Two genes CYP704B2 and WDA1, involved in the biosynthesis of very-long-chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcg15 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther locules. Consistently, histological analysis showed that osabcg15 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice anther development.
Niu BX, He FR, He M, Ren D, Chen LT, Liu YG (2013) The ATP‐binding cassette transporter OsABCG15 is required for anther development and pollen fertility in rice. J. Integr. Plant Biol. 55(8), 710–720.
During early embryogenesis in mammals and higher plants, the maternal-to-zygotic transition (MZT) marks the turnover of developmental control from maternal products to de novo zygotic genome transcripts. Intensive studies in animals indicate that early embryonic development is largely maternally controlled. In recent years, the MZT has drawn the attention of botanists, as it is important for understanding the mechanism of embryogenesis and hybrid vigor. In this study, we present a brief overview of some aspects of the MZT in flowering plants. Based on what we have learned from Nicotiana tabacum, we hypothesize that the MZT occurs before zygotic cell division and that the development of the fertilized egg cell in flowering plants can be divided into two phases: the zygote stage, which is mainly controlled maternally, and the one-celled proembryo stage, in which zygotic genome activation (ZGA) occurs and is required for zygote division.
Xin HP, Zhao J, Sun MX (2012) The maternal-to-zygotic transition in higher plants. J. Integr. Plant Biol. 54(9), 610–615.
Plant mitochondrial genomes (mtDNAs) are large and undergo frequent recombination events. A common phenotype that emerges as a consequence of altered mtDNA structure is cytoplasmic-male sterility (CMS). The molecular basis for CMS remains unclear, but it seems logical that altered respiration activities would result in reduced pollen production. Analysis of tobacco (Nicotiana tabacum) mtDNAs indicated that CMS-associated loci often contain fragments of known organellar genes. These may assemble with organellar complexes and thereby interfere with normal respiratory functions. Here, we analyzed whether the expression of truncated fragments of mitochondrial genes (i.e. atp4, cox1 and rps3) may induce male sterility by limiting the biogenesis of the respiratory machinery. cDNA fragments corresponding to atp4f, cox1f and rps3f were cloned in-frame to a mitochondrial localization signal and a C-termini HA-tag under a tapetum-specific promoter and introduced to tobacco plants by Agrobacterium-mediated transformation. The constructs were then analyzed for their effect on mitochondrial activity and pollen fertility. Atp4f, Cox1f and Rps3f plants demonstrated male sterility phenotypes, which were tightly correlated with the expression of the recombinant fragments in the floral meristem. Fractionation of native organellar extracts showed that the recombinant ATP4f-HA, COX1f-HA and RPS3f-HA proteins are found in large membrane-associated particles. Analysis of the respiratory activities and protein profiles indicated that organellar complex I was altered in Atp4f, Cox1f and Rps3f plants.
Shaya F, Gaiduk S, Keren I, Shevtsov S, Zemah H, Belausov E, Evenor D, Reuveni M, Ostersetzer-Biran O (2012) Expression of mitochondrial gene fragments within the tapetum induce male sterility by limiting the biogenesis of the respiratory machinery in transgenic tobacco. J. Integr. Plant Biol. 54(2), 115–129.
Early embryogenesis is the most fundamental developmental process in biology. Screening of ethyl methanesulfonate (EMS)-mutagenized populations of Arabidopsis thaliana led to the identification of a zygote-lethal mutant embryonic factor 19 (fac19) in which embryo development was arrested at the elongated zygote to octant stage. The number of endosperm nuclei decreased significantly in fac19 embryos. Genetic analysis showed fac19 was caused by a single recessive mutation with typical mendelian segregation, suggesting equal maternal and paternal contributions of FAC19 towards zygotic embryogenesis. Positional cloning showed that FAC19 encodes a putative mitochondrial protein with 16 conserved pentatricopeptide repeat (PPR) motifs. The fac19 mutation caused a conversion from hydrophilic serine located in a previously unknown domain to hydrophobic leucine. Crosses between FAC19/fac19 and the T-DNA insertion mutants in the same gene failed to complement the fac19 defects, confirming the identity of the gene. This study revealed the critical importance of a PPR protein-mediated mitochondrial function in early embryogenesis.
Yu D, Jiang L, Gong H, Liu CM (2012) EMBRYONIC FACTOR 19 encodes a pentatricopeptide repeat protein that is essential for the initiation of zygotic embryogenesis in Arabidopsis. J. Integr. Plant Biol. 54(1), 55–64.
In anther development, tapetal cells take part in complex processes, including endomitosis and apoptosis (programmed cell death). The tapetum provides many of the proteins, lipids, polysaccharides and other molecules necessary for pollen development. Several transcription factors, including DYT1, TDF1, AMS, MS188 and MS1, have been reported to be essential for tapetum development and function in Arabidopsis thaliana. Here, we present a detailed cytological analysis of knockout mutants for these genes, along with an in situ RNA hybridization experiment and double mutant analysis showing that these transcription factors form a genetic pathway in tapetum development. DYT1, TDF1 and AMS function in early tapetum development, while MS188 and MS1 are important for late tapetum development. The genetic pathway revealed in this work facilitates further investigation of the function and molecular mechanisms of tapetum development in Arabidopsis.
Zhu J, Lou Y, Xu X, Yang ZN (2011) A genetic pathway for tapetum development and function in Arabidopsis. J. Integr. Plant Biol. 53(11), 892–900.
Glucose appears to have an antagonistic relationship with ethylene and ethylene and polyamines appear to play antagonistic roles in the abortion of seeds and fruits. Moreover, ethylene, spermidine, and spermine share a common biosynthetic precursor. The synchronous changes of them and the relationships with kernel set are currently unclear. Here, we stimulated maize (Zea mays L.) apical kernel set and studied their changes at 4, 8, 12, and 16 d after pollination (DAP). The status of the apical kernels changed from abortion to set, showing a pattern similar to that of the middle kernels, with slow decrease in glucose and rapid decline in ethylene production, and a sharp increase in spermidine and spermine after four DAP. Synchronous changes in ethylene and spermidine were also observed. However, the ethylene production decreased slowly in the aborted apical kernels, the glucose and polyamines concentrations were lower. Ethephon application did not block the change from abortion to set for the setting apical kernels. These data indicate that the developmental change may be accompanied by an inhibition of adequate glucose to ethylene synthesis and subsequent promotion of spermidine and spermine synthesis, and adequate carbohydrate supply may play a key role in the developmental process.
Feng HY, Wang ZM, Kong FN, Zhang MJ, Zhou SL (2011) Roles of carbohydrate supply and ethylene, polyamines in maize kernel set. J. Integr. Plant Biol. 53(5), 388–398.
LEUNIG (LUG) and LEUNIG_HOMOLOG (LUH) encode two closely related Arabidopsis proteins, belonging to the Gro/TLE family of transcriptional co-repressors. These two genes were previously shown to exhibit partially overlapping functions in embryo and flower development. In this report, the role of both LUH and LUG on seed mucilage extrusion was examined. Seed mucilage extrusion occurs after the seeds are imbibed, serving as functional aid in seed hydration, germination, and dispersal. While luh-1 mutants exhibited strong defects in seed mucilage extrusion, lug-3 mutants exhibited a minor phenotype in mucilage extrusion. Further characterization indicates that luh-1 does not exhibit any obvious defect in seed epidermal cell differentiation, mucilage synthesis, or mucilage deposition, suggesting a specific role of LUH in mucilage extrusion. This seed mucilage phenotype of luh-1 is identical to that of mucilage modified 2 (mum2) mutants. MUM2 encodes a β-galactosidase required for the modification of the mucilage. Quantitative reverse transcription polymerase chain reaction of RNA extracted from siliques detected a slight decrease of MUM2 mRNA in the luh-1 mutant compared to the wild type. Together, LUH and possibly LUG may specifically regulate mucilage extrusion by promoting the expression of genes required for mucilage maturation.
Bui M, Lim N, Sijacic P, Liu Z (2011) LEUNIG_HOMOLOG and LEUNIG regulate seed mucilage extrusion in Arabidopsis. J. Integr. Plant Biol. 53(5), 399–408.
In higher plants, male reproductive development is a complex biological process that includes cell division and differentiation, cell to cell communication etc., while the mechanism underlying plant male reproductive development remains less understood. GAMYB encodes a gibberellins acid (GA) inducible transcription factor that is required for the early anther development in rice (Oryza sativa L.). Here, we report the isolation and characterization of a new allele gamyb-4 with a C base deletion in the second exon (+2308), causing a frame shift and premature translational termination. Histological analysis showed that gamyb-4 developed abnormal enlarged tapetum and could not undergo normal meiosis. To understand the regulatory role of GAMYB, we carried out quantitative reverse transcription- polymerase chain reaction analysis and comparison of microarray data. These results revealed that the expression of TDR (TAPETUM DEGENERATION RETARDATION), a tapetal cell death regulator, was downregulated in gamyb-4 and udt1 (undeveloped tapetum1). While the GAMYB expression was not obviously changed in tdr and udt1-1, and no apparent expression fold change of UDT1 in tdr and gamyb-4, suggesting that TDR may act downstream of GAMYB and UDT1, and GAMYB and UDT1 work in parallel to regulate rice early anther development. This work is helpful in understanding the regulatory network in rice anther development.
Liu Z, Bao W, Liang W, Yin J, Zhang D (2010) Identification of gamyb-4 and analysis of the regulatory role of GAMYB in rice anther development. J. Integr. Plant Biol. 52(7), 670–678.
The diverse functions of phosphoenolpyruvate carboxylase (PEPCase; EC 184.108.40.206) in C3 plants are not as well understood as in C4 plants. To investigate the functions of PEPCase in C3 plants, rapeseed (Brassica napus L.) PEPCase gene (referred to as BNPE15) was silenced by the RNA interference (RNAi) technique. Under normal growth conditions, no significant difference in lipid content and fatty acid composition were found between wild-type (WT) and transgenic rapeseed plants. However, when these plants were subjected to osmotic stress induced by osmoticum polyethylene glycol (PEG-6000), membrane permeability and membrane lipid peroxidization in roots and leaves of transgenic plants were higher than those of WT plants. It suggested that transgenic plants are more susceptible to osmotic stress than WT plants. Taken together, the results showed that the suppression of PEPCase by RNAi leads to susceptibility to osmotic stress in rapeseed, and PEPCase is involved in the response of C3 plants to environmental stress.
Chen M, Tang Y, Zhang J, Yang M, Xu Y (2010) RNA interference-based suppression of phosphoenolpyruvate carboxylase results in susceptibility of rapeseed to osmotic stress. J. Integr. Plant Biol. 52(6), 585–592.
Microscopic imaging of fluorescent reporters for key meristem regulators in live tissues is emerging as a powerful technique, enabling researchers to observe dynamic spatial and temporal distribution of hormonal and developmental regulators in living cells. Aided by time-lapse microphotography, new types of imaging acquisition and analysis software, and computational modeling, we are gaining significant insights into shoot apical meristem (SAM) behavior and function. This review is focused on summarizing recent advances in the understanding of SAM organization, development, and behavior derived from live-imaging techniques. This includes the revelation of mechanical forces in microtubule-controlled anisotropic growth, the role of the CLV-WUS network in the specification of peripheral zone and central zone cells, the multiple feedback loops involving cytokinin in controlling WUS expression, auxin dynamics in determining the position of new primordia, and, finally, sequence of regulatory events leading to de novo assembly of shoots from callus in culture. Future studies toward formulating "digital SAM" that incorporates multi-dimensional data ranging from images of SAM morphogenesis to a genome-scale expression map of SAM will greatly enhance our ability to understand, predict, and manipulate SAM, containing the stem cells that give rise to all above ground parts of a plant.
Sijacic P, Liu Z (2010) Novel insights from live-imaging in shoot meristem development. J. Integr. Plant Biol. 52(4), 393–399.
Sialyltransferases (SiaTs) exist widely in vertebrates and play important roles in a variety of biological processes. In plants, several genes have also been identified to encode the proteins that share homology with the vertebrate SiaTs. However, very little is known about their functions in plants. Here we report the identification and characterization of a novel Arabidopsis gene, MALE GAMETOPHYTE DEFECTIVE 2 (MGP2) that encodes a sialyltransferase-like protein. MGP2 was expressed in all tissues including pollen grains and pollen tubes. The MGP2 protein was targeted to Golgi apparatus. Knockout of MGP2 significantly inhibited the pollen germination and retarded pollen tube growth in vitro and in vivo, but did not affect female gametophytic functions. These results suggest that the sialyltransferase-like protein MGP2 is important for normal pollen germination and pollen tube growth, giving a novel insight into the biological roles of the sialyltransferase-like proteins in plants.
Deng Y, Wang W, Li WQ, Xia C, Liao HZ, Zhang XQ, Ye D (2010) MALE GAMETOPHYTE DEFECTIVE 2 (MGP2), encoding a sialyltransferase-like protein, is required for normal pollen germination and pollen tube growth in Arabidopsis. J. Integr. Plant Biol. 52(9), 829–843.
RNA helicases are adenosine tri‐phosphatases that unwind the secondary structures of RNAs and are required in almost any aspect of RNA metabolism. They are highly conserved from prokaryotic to eukaryotic organisms. However, their precise roles in plant physiology and development remain to be clarified. Here we report that the mutation in the gene SLOW WALKER3 (SWA3) results in the slow and retarded progression of mitosis during megagametogenesis in arabidopsis. SWA3 is a putative RNA helicase of DEAD-box subfamily. Mutant megagametophyte development is arrested at four- or eight-nucleate stages, furthermore, one of the synergids in about half of the mutant embryo sacs displays abnormal polarity, with its nucleus locating at the chalazal end, instead of the micropylar end in the wild-type. Transmission of the mutation through female gametophytes is severely reduced in swa3. However, a small portion of mutant embryo sacs are able to develop into mature and functional female gametophytes when pollination was postponed. The SWA3 in arabidopsis is a homologue of Dbp8 in yeast. Dbp8 interacts with Efs2 and is essential for biogenesis of 18S rRNA in yeast. Our data suggest that SWA3, may form complex with AtEfs2 and take roles in ribosomal biogenesis as RNA helicase during megagametogenesis in Arabidopsis.
LiuM, Shi DQ, Yuan L, Liu J, YangWC(2010) SLOW WALKER3, encoding a putative DEAD-box RNA helicase, is essential for female gametogenesis in Arabidopsis. J. Integr. Plant Biol. 52(9), 817–828.
Double fertilization is a key process of sexual reproduction in higher plants. The role of calcium in the activation of female sex cells through fertilization has recently received a great deal of attention. The establishment of a Ca2+-imaging technique for living, single, female sex cells is a difficult but necessary prerequisite for evaluating the role of Ca2+ in the transduction of external stimuli, including the fusion with the sperm cell, to internal cellular processes. The present study describes the use of Fluo-3 for reporting the Ca2+ signal in isolated, single, female sex cells, egg cells and central cells, of tobacco plants. A suitable loading protocol was optimized by loading the cells at pH 5.6 with 2 μM Fluo-3 for 30 min at 30◦C. Under these conditions, several key factors related to in vitro fertilization were also investigated in order to test their possible effects on the [Ca2+]cyt of the female sex cells. The results indicated that the bovine serum albumin-fusion system was superior to the polyethlene glycol-fusion system for detecting calcium fluctuations in female sex cells during fertilization. The central cell was fertilized with the sperm cell in bovine serum albumin; however, no evident calcium dynamic was detected, implying that a transient calcium rise might be a specific signal for egg cell fertilization.
Peng X-B, Sun M-X, Yang H-Y (2009). Comparative detection of calcium fluctuations in single female sex cells of tobacco to distinguish calcium signals triggered by in vitro fertilization. J. Integr. Plant Biol. 51(8), 782–791.
Photoperiod-sensitive genic male-sterile (PSGMS) rice (Oryza sativa L.), a natural mutant found in the rice cultivar Nongken 58, is very useful for the development of hybrid rice cultivars. Despite its widespread use in breeding programs, the initial stage of the abortive development of PSGMS rice and the possible cytological mechanisms of pollen abortion have not been determined. In the present study, a systematic cytological comparison of the anther development of PSGMS rice with its normal fertile counterpart is conducted. The results show that pollen abortion in PSGMS rice first occurs before the pollen mother cell (PMC) stage, and continues during the entire process of pollen development until pollen degradation. The abortive process was closely associated with the abnormal behavior of the tapetum. Although tapetum degeneration in PSGMS rice initiates already at the PMC stage, it proceeds slowly and does not complete until the breakdown of the pollen. Such cytological observations were supported by the results of the TUNEL (TdT mediated dUTP Nick End Labeling) assay, which detects DNA fragmentation resulting from programmed cell death (PCD), indicating that the premature tapetum degeneration is in the process of PCD.
Shi Y, Zhao S, Yao J (2009). Premature tapetum degeneration: a major cause of abortive pollen development in photoperiod sensitive genic male sterility in rice. J. Integr. Plant Biol. 51(8), 774-781
In flowering plants, male gametes are delivered to female gametophytes by pollen tubes. Although it is important for sexual plant reproduction, little is known about the genetic mechanism that controls pollen germination and pollen tube growth. Here we report the identification and characterization of two novel mutants, gnom-like 2-1 (gnl2-1) and gnl2-2 in Arabidopsis thaliana, in which the pollen grains failed to germinate in vitro and in vivo. GNL2 encodes a protein homologous to the adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factors, GNOM and GNL1 that are involved in endosomal recycling and endoplasmic reticulum-Golgi vesicular trafficking. It was prolifically expressed in pollen grains and pollen tubes. The results of the present study suggest that GNL2 plays an important role in pollen germination.
Jia DJ, Cao X, Wang W, Tan XY, Zhang XQ, Chen LQ, Ye D (2009). GNOM-LIKE 2, encoding anadenosine diphosphate-ribosylation factor- guanine nucleotide exchange factor protein homologous to GNOM and GNL1, is essential for pollen germination in Arabidopsis. J. Integr. Plant Biol. 51(8), 762–773.
Calcium, an ubiquitous second messenger, plays an essential and versatile role in cellular signaling. The diverse function of calcium signals is achieved by an excess of calcium sensors. Plants possess large numbers of calcium sensors, most of which have not been functionally characterized. To identify physiologically relevant calcium sensors in a specific cell type, we conducted a genome-wide functional survey in pollen tubes, for which spatiotemporal calcium signals are well-characterized and required for polarized tip growth. Pollen-specific members of calmodulin (CaM), CaM-like (CML), calcium-dependent protein kinase (CDPK) and calcineurin B-like protein (CBL) families were tagged with green fluorescence protein (GFP) and their localization patterns and overexpression phenotypes were characterized in tobacco pollen tubes. We found that several fusion proteins showed distinct overexpression phenotypes and subcellular localization patterns. CDPK24-GFP was localized to the vegetative nucleus and the generative cell/sperms. CDPK32-GFP caused severe growth depolarization. CBL2-GFP and CBL3-GFP exhibited dynamic patterns of subcellular localization, including several endomembrane compartments, the apical plasma membrane (PM), and cytoskeleton-like structures in pollen tubes. Their overexpression also inhibited pollen tube elongation and induced growth depolarization. These putative calcium sensors are excellent candidates for the calcium sensors responsible for the regulation of calcium homeostasis and calciumdependent tip growth and growth oscillation in pollen tubes.
Zhou L, Fu Y, Yang Z (2009). A genome-wide functional characterization of Arabidopsis regulatory calcium sensors in pollen tubes. J. Integr. Plant Biol. 51(8), 751–761.
Actin cytoskeleton undergoes rapid reorganization in response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions in plant cell biology. The pollen tube is a well characterized actin-based cell morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding themolecularmechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and unexpected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.
Chen N, Qu X, Wu Y, Huang S (2009). Regulation of actin dynamics in pollen tubes: control of actin polymer level. J. Integr. Plant Biol. 51(8), 740–750.
Although pollen tube growth is a prerequisite for higher plant fertilization and seed production, the processes leading to pollen tube emission and elongation are crucial for understanding the basic mechanisms of tip growth. It was generally accepted that pollen tube elongation occurs by accumulation and fusion of Golgi-derived secretory vesicles (SVs) in the apical region, or clear zone, where they were thought to fuse with a restricted area of the apical plasma membrane (PM), defining the apical growth domain. Fusion of SVs at the tip reverses outside cell wall material and provides new segments of PM. However, electron microscopy studies have clearly shown that the PM incorporated at the tip greatly exceeds elongation and a mechanism of PM retrieval was already postulated in the mid-nineteenth century. Recent studies on endocytosis during pollen tube growth showed that different endocytic pathways occurred in distinct zones of the tube, including the apex, and led to a new hypothesis to explain vesicle accumulation at the tip; namely, that endocytic vesicles contribute substantially to V-shaped vesicle accumulation in addition to SVs and that exocytosis does not involve the entire apical domain. New insights suggested the intriguing hypothesis that modulation between exo- and endocytosis in the apex contributes to maintain PM polarity in terms of lipid/protein composition and showed distinct degradation pathways that could have different functions in the physiology of the cell. Pollen tube growth in vivo is closely regulated by interaction with style molecules. The study of endocytosis and membrane recycling in pollen tubes opens new perspectives to studying pollen tube-style interactions in vivo.
Moscatelli A, Idilli AI (2009). Pollen tube growth: a delicate equilibrium between secretory and endocytic pathways. J. Integr. Plant Biol. 51(8), 727–739.
The pistil, the female reproductive organ of plants, is a key player in the success of sexual plant reproduction. Ultimately, the production of fruits and seeds depends on the proper pistil development and function. Therefore, the identification and characterization of pistil expressed genes is essential for a better understanding and manipulation of the plant reproduction process. For studying the function of pistil expressed genes, transgenic and/or mutant plants for the genes of interest are used. The present article provides a review of methods already exploited to analyze sexual reproductive success. It intends to supply useful information and to guide future experiments in the study of genes affecting the pistil development and function.
Calixto CPG, Goldman GH, Goldman MHS (2009). Analyses of sexual reproductive success in transgenic and/or mutant plants. J. Integr. Plant Biol. 51(8), 719-726.