Genetic control of male fertility in Arabidopsis thaliana: structural analyses of postmeiotic developmental mutants

Seven new male-sterile mutants (ms7–ms13) of Arabidopsis thaliana (L.) Heynh. (ecotype columbia) are described that show a postmeiotic defect of microspore development. In ms9 mutants, microspores recently released from the tetrad appear irregular in shape and are often without exines. The earliest evidence of abnormality in ms12 mutants is degeneration of microspores that lack normal exine sculpturing, suggesting that the MS12 product is important in the formation of pollen exine. Teratomes (abnormally enlarged microsporocytes) are also occasionally present and each has a poorly developed exine. In ms7 mutant plants, the tapetal cytoplasm disintegrates at the late vacuolate microspore stage, apparently causing the degeneration of microspores and pollen grains. With ms8 mutants, the exine of the microspores appears similar to that of the wild type. However, intine development appears impaired and pollen grains rupture prior to maturity. In ms11 mutants, the first detectable abnormality appears at the mid to late vacuolate stage. The absence of fluorescence in the microspores and tapetal cells after staining with 4′,6-diamidino-2-phenylindole (DAPI) and the occasional presence of teratomes indicate degradation of DNA. Viable pollen from ms10 mutant plants is dehisced from anthers but appears to have surface abnormalities affecting interaction with the stigma. Pollen only germinates in high-humidity conditions or during in-vitro germination experiments. Mutant plants also have bright-green stems, suggesting that ms10 belongs to the eceriferum (cer) class of mutants. However, ms10 and cer6 are non-allelic. The ms13 mutant has a similar phenotype to ms10, suggesting is also an eceriferum mutation. Each of these seven mutants had a greater number of flowers than congenic male-fertile plants. The non-allelic nature of these mutants and their different developmental end-points indicate that seven different genes important for the later stages of pollen development have been identified. Received: 14 August 1997 / Accepted: 7 October 1997     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

<Emphasis Type="Italic">pgd1</Emphasis>, an <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> deletion mutant,is defective in pollen germination

An Arabidopsis deletion mutant was fortuitously identified from the alpha population of T-DNA insertional mutants generated at the University of Wisconsin Arabidopsis Knockout Facility. Segregation and reciprocal crosses indicated that the mutant was a gametophytic pollen sterile mutant. Pollen carrying the mutation has the unusual phenotype that it is viable, but cannot germinate. Thus, the mutant was named pollen germination defective mutant 1 (pgd1), based on the pollen phenotype. Flanking sequences of the T-DNA insertion in the pgd1 mutant were identified by thermal asymmetric interlaced (TAIL) PCR. Sequencing of bands from TAIL PCR revealed that the T-DNA was linked to the gene XLG1, At2g23460, at its downstream end, while directly upstream of the T-DNA was a region between At2g22830 and At2g22840, which was 65 genes upstream of XLG1. Southern blotting and genomic PCR confirmed that the 65 genes plus part of XLG1 were deleted in the pgd1 mutant. A 9,177 bp genomic sequence containing the XLG1 gene and upstream and downstream intergenic regions could not rescue the pgd1 pollen phenotype. One or more genes from the deleted region were presumably responsible for the pollen germination defect observed in the pgd1 mutant. Because relatively few mutations have been identified that affect pollen germination independent of any effect on pollen viability, this mutant line provides a new tool for identification of genes specifically involved in this phase of the reproductive cycle.     

Genetic analysis of interspecific incompatibility in Brassica rapa

In interspecific pollination of Brassica rapa stigmas with Brassica oleracea pollen grains, pollen tubes cannot penetrate stigma tissues. This trait, called interspecific incompatibility, is similar to self-incompatibility in pollen tube behaviors of rejected pollen grains. Since some B. rapa lines have no interspecific incompatibility, genetic analysis of interspecific incompatibility was performed using two F₂ populations. Analysis with an F₂ population between an interspecific-incompatible line and a self-compatible cultivar ‘Yellow sarson’ having non-functional alleles of S-locus genes and MLPK, the stigmas of which are compatible with B. oleracea pollen grains, revealed no involvement of the S locus and MLPK in the difference of their interspecific incompatibility phenotypes. In QTL analysis of the strength of interspecific incompatibility, three peaks of LOD scores were found, but their LOD scores were as high as the threshold value, and the variance explained by each QTL was small. QTL analysis using another F₂ population derived from selected parents having the highest and lowest levels of interspecific incompatibility revealed five QTLs with high LOD scores, which did not correspond to those found in the former population. The QTL having the highest LOD score was found in linkage group A02. The effect of this QTL on interspecific incompatibility was confirmed by analyzing backcrossed progeny. Based on synteny of this QTL region with Arabidopsis thaliana chromosome 5, a possible candidate gene, which might be involved in interspecific incompatibility, is discussed.     

Pollen of a Male-Sterile Mutant of Arabidopsis thaliana Isolated from a T-DNA Insertion Pool is Not Effectively Released from The Anther Locule

We have isolated a male-sterile mutant from a pool of T-DNAinsertional lines of Arabidopsis thaliana generated by an inplanta transformation procedure [Chang et al. (1994) Plant J.5: 551]. Pollen in this mutant is not effectively released fromanther locules after cleavage of the stomium. Most mutant pollengrains are round, in contrast to the tricolpate wild-type pollen,and some pollen grains show an abnormal surface structure. Manuallyreleased mutant pollen grains are not fertile and show defectsin pollen tube germination in vitro. Genetic analysis disclosedthat this lesion is due to a single recessive nuclear mutationlocated on chromosome 3 closely linked to the gl1 locus. Themutation locus is tightly linked to the inserted T-DNA. ¹Present address: CSIRO, Division of Plant Industry, GPO Box1600, Canberra, ACT 2601, Australia     

Lipooligosaccharide biosynthesis in Neiseria gonorrhoeae: cloning, identification and characterization of the α1,5 heptosyltransferase I gene (rfaC)

The Escherichia coli arginine repressor (ArgR) is an l -arginine-dependent DNA-binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site-specific recombination at cer and related recombination sites in plasmids. Site-directed mutagenesis was used to isolate two mutants of E. coli ArgR that were defective in arginine binding. Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine-independent manner. Both mutants support Xer site-specific recombination at cer. One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild-type ArgR.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> AtVPS15 is essential for pollen development and germination through modulating phosphatidylinositol 3-phosphate formation

Arabidopsis thaliana phosphatidylinositol 3-kinase (AtVPS34) functions in the development and germination of pollen by catalyzing the biosynthesis of phosphatidylinositol 3-phosphate (PI3P). In yeast, Vps15p is required for the membrane targeting and activity of Vps34. The expression of Arabidopsis thaliana VPS15 (AtVPS15), an ortholog of yeast Vps15, is mainly detected in pollen grains and pollen tubes. To determine its role in pollen development and pollen tube growth, we attempted to isolate the T-DNA insertion mutants of AtVPS15; however, homozygous lines of atvps15 were not obtained from the progeny of atvps15/+ heterozygotes. Genetic analysis revealed that the abnormal segregation is due to the failure of transmission of the atvps15 allele through pollen. Most pollen grains from the atvps15/+ genotype are viable, with normal exine structure and nuclei, but some mature pollen grains are characterized with unusual large vacuoles that are not observed in pollen grains from the wild AtVPS15 genotype. The germination ratio of pollen from the atvps15/+ genotype is about half when compared to that from the wild AtVPS15 genotype. When supplied with PI3P, in vitro pollen germination of the atvps15/+ genotype is greatly improved. Presumably, AtVPS15 functions in pollen development and germination by regulating PI3P biosynthesis in Arabidopsis.     

AtTMEM18 plays important roles in pollen tube and vegetative growth in Arabidopsis

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.     

Two aquaporins,SIP1;1 and PIP1;2, mediate water transport for pollen hydration in the Arabidopsis pistil

Pollination is the crucial initial step that brings together the male and female gametophytes, and occurs at the surface of the stigmatic papilla cell in Arabidopsis thaliana. After pollen recognition, pollen hydration is initiated as a second critical step to activate desiccated mature pollen grains for germination, and thus water transport from pistil to pollen is essential for this process. In this study, we report a novel aquaporin-mediated water transport process in the papilla cell as a control mechanism for pollen hydration. Coupled with a time-series imaging analysis of pollination and a reverse genetic analysis using T-DNA insertion Arabidopsis mutants, we found that two aquaporins, the ER-bound SIP1;1 and the plasma membrane-bound PIP1;2, are key players in water transport from papilla cell to pollen during pollination. In wild type plant, hydration speed reached its maximal value within 5 min after pollination, remained high until 10–15 min. In contrast, sip1;1 and pip1;2 mutants showed no rapid increase of hydration speed, but instead a moderate increase during ∼25 min after pollination. Pollen of sip1;1 and pip1;2 mutants had normal viability without any functional defects for pollination, indicating that decelerated pollen hydration is due to a functional defect on the female side in sip1;1 and pip1;2 mutants. In addition, sip1;1 pip1;2 double knockout mutant showed a similar impairment of pollen hydration to individual single mutants, suggesting that their coordinated regulation is critical for proper water transport, in terms of speed and amount, in the pistil to accomplish successful pollen hydration.     

Precocious pollen germination in Arabidopsis plants with altered callose deposition during microsporogenesis

Pollination is essential for seed reproduction and for exchanges of genetic information between individual plants. In angiosperms, mature pollen grains released from dehisced anthers are transferred to the stigma where they become hydrated and begin to germinate. Pollen grains of wild-type Arabidopsis thaliana do not germinate inside the anther under normal growth conditions. We report two Arabidopsis lines that produced pollen grains able to in situ precociously germinate inside the anther. One of them was a callose synthase 9 (cs9) knockout mutant with a T-DNA insertion in the Callose Synthase 9 gene (CalS9). Male gametophytes carrying a cs9 mutant allele were defective and no homozygous progeny could be produced. Heterozygous mutant plants (cs9/+) produced approximately 50% defective pollen grains with an altered male germ unit (MGU) and aberrant callose deposition in bicellular pollen. Bicellular pollen grains germinated precociously inside the anther. Another line, a transgenic plant expressing callose synthase 5 (CalS5) under the CaMV 35S promoter, also contained abnormal callose deposition during microsporogenesis and displaced MGUs in pollen grains. We also observed that precocious pollen germination could be induced in wild-type plants by incubation with medium containing sucrose and calcium ion and by wounding in the anther. These results demonstrate that precocious pollen germination in Arabidopsis could be triggered by a genetic alteration and a physiological condition.     

Pollen wall development: the associated enzymes and metabolic pathways

Pollen grains are surrounded by a sculpted wall, which protects male gametophytes from various environmental stresses and microbial attacks, and also facilitates pollination. Pollen wall development requires lipid and polysaccharide metabolism, and some key genes and proteins that participate in these processes have recently been identified. Here, we summarise the genes and describe their functions during pollen wall development via several metabolic pathways. A working model involving substances and catalytic enzyme reactions that occur during pollen development is also presented. This model provides information on the complete process of pollen wall development with respect to metabolic pathways.     

Correlation between pollen morphology and pollination mechanisms in the Hydrocharitaceae

The pollen morphology of 11 genera and 11 species of the Hydrocharitaceae and one species of the Najadaceae was studied using scanning and transmission electron microscopies, and the exine structures and sculptures are discussed in relation to pollination mechanisms and the molecular phylogeny. The pollen grains of the Hydrocharitaceae are spherical, inaperturate, and form monads or tetrads, while those of the Najadaceae are elliptical, inaperturate, and form monads. The entomophilous genera Egeria, Blyxa, Ottelia, Stratiotes, and Hydrocharis share pollen grains that have projections like spines or bacula. The anemophilous genus Limnobium has reticulate pollen grains. The hypohydrophilous genera Thalassia and Najas are characterized by pollen grains with reduced exine structures. The pollen-epihydrophilous genera Elodea and Hydrilla have tightly arranged small spinous pollen grains, and the male flower-epihydrophilous genera Enhalus and Vallisneria have reduced reticulate or gemmate exines. Character state reconstruction of the exine structures and sculptures using a molecular phylogenetic tree suggests that variation in the exine is generally correlated with the pollination mechanism; the selective pressures acting on the pollination mechanisms have reduced the exine structure in hypohydrophilous plants and resulted in various exine sculptures that are adapted to the different pollination mechanisms in entomophilous, anemophilous, and pollen-epihydrophilous plants.     

Fine mapping of an Arabidopsis thaliana male sterile mutant EC2-157

An Arabidopsis thaliana male sterile mutant EC2-157 has been isolated using an EMS mutagenesis strategy. Genetic analysis indicated that it was controlled by a single recessive gene called ms157. No pollen grains have been observed in mutant anthers. ms157 Has been mapped to a region of 74kb located in BAC clone T6K22 on chromosome IV using a map-based cloning strategy. As no male sterile genes have been reported in this region, ms157 could be a novel gene related to fertility. The further molecular cloning and functional analysis on this gene should facilitate our understanding of A. thaliana another development. __________ Translated from Journal of Shanghai Normal University (Natural Sciences), 2005, 34(1): 58–63 [译自: 上海师范大学学报 (自然科学版), 2005, 34(1): 58–63]     

gaMS-1: A gametophytic male sterile mutant in maize

Several pollen-specific genes from different species have been isolated and characterized at the molecular level, but the precise role of most of them is unknown. Mutant analysis represents a direct approach to uncovering gene function, but the paucity of available mutants affecting pollen development and/or function and the poor characterization of the known mutants have so far limited the exploitation of this approach. Here we present the cytological characterization ofgametophytic male sterile-1 (gaMS-1), a maize mutant that we identified in a program of transposon insertion mutagenesis for the production of mutations in gametophytically acting genes involved in microsporogenesis.gaMS-1 is expressed during or immediately after the first microspore division and leads to the production of immature, nonfunctional pollen grains. The mutation appears to affect the events leading to the developmental switch that follows the first microspore mitosis.     

AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube guidance

Reproductive isolation is a prerequisite to form and maintain a new species. Multiple prezygotic and postzygotic reproductive isolation barriers have been reported in plants. In the model plant, Arabidopsis thaliana conspecific pollen tube precedence controlled by AtLURE1/PRK6-mediated signaling has been recently reported as a major prezygotic reproductive isolation barrier. By accelerating emergence of own pollen tubes from the transmitting tract, A. thaliana ovules promote self-fertilization and thus prevent fertilization by a different species. Taking advantage of a septuple atlure1null mutant, we now report on the role of AtLURE1/PRK6-mediated signaling for micropylar pollen tube guidance. Compared with wild-type (WT) ovules, atlure1null ovules displayed remarkably reduced micropylar pollen tube attraction efficiencies in modified semi-in vivo A. thaliana ovule targeting assays. However, when prk6 mutant pollen tubes were applied, atlure1null ovules showed micropylar attraction efficiencies comparable to that of WT ovules. These findings indicate that AtLURE1/PRK6-mediated signaling regulates micropylar pollen tube attraction in addition to promoting emergence of own pollen tubes from the transmitting tract. Moreover, semi-in vivo ovule targeting competition assays with the same amount of pollen grains from both A. thaliana and Arabidopsis lyrata showed that A. thaliana WT and xiuqiu mutant ovules are mainly targeted by own pollen tubes and that atlure1null mutant ovules are also entered to a large extent by A. lyrata pollen tubes. Taken together, we report that AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube attraction representing an additional prezygotic isolation barrier.

A modified ovule targeting assay revealed that AtLURE1/PRK6-mediated signaling promotes micropylar guidance of Arabidopsis thaliana pollen tubes while discriminating tubes of related Arabidopsis lyrata.