Evolution of self-compatibility in Arabidopsis by a mutation in the male specificity gene.

Ever since Darwin's pioneering research, the evolution of self-fertilisation (selfing) has been regarded as one of the most prevalent evolutionary transitions in flowering plants. A major mechanism to prevent selfing is the self-incompatibility (SI) recognition system, which consists of male and female specificity genes at the S-locus and SI modifier genes. Under conditions that favour selfing, mutations disabling the male recognition component are predicted to enjoy a relative advantage over those disabling the female component, because male mutations would increase through both pollen and seeds whereas female mutations would increase only through seeds. Despite many studies on the genetic basis of loss of SI in the predominantly selfing plant Arabidopsis thaliana, it remains unknown whether selfing arose through mutations in the female specificity gene (S-receptor kinase, SRK), male specificity gene (S-locus cysteine-rich protein, SCR; also known as S-locus protein 11, SP11) or modifier genes, and whether any of them rose to high frequency across large geographic regions. Here we report that a disruptive 213-base-pair (bp) inversion in the SCR gene (or its derivative haplotypes with deletions encompassing the entire SCR-A and a large portion of SRK-A) is found in 95% of European accessions, which contrasts with the genome-wide pattern of polymorphism in European A. thaliana. Importantly, interspecific crossings using Arabidopsis halleri as a pollen donor reveal that some A. thaliana accessions, including Wei-1, retain the female SI reaction, suggesting that all female components including SRK are still functional. Moreover, when the 213-bp inversion in SCR was inverted and expressed in transgenic Wei-1 plants, the functional SCR restored the SI reaction. The inversion within SCR is the first mutation disrupting SI shown to be nearly fixed in geographically wide samples, and its prevalence is consistent with theoretical predictions regarding the evolutionary advantage of mutations in male components.

Transcriptional characteristics and differences in Arabidopsis stigmatic papilla cells pre- and post-pollination.

Pollination is an important early step in sexual plant reproduction. In Arabidopsis thaliana, sequential pollination events, from pollen adhesion onto the stigma surface to pollen tube germination and elongation, occur on the stigmatic papilla cells. Following successful completion of these events, the pollen tube penetrates the stigma and finally fertilizes a female gametophyte. The pollination events are thought to be initiated and regulated by interactions between papilla cells and pollen. Here, we report the characterization of gene expression profiles of unpollinated (UP), compatible pollinated (CP) and incompatible pollinated (IP) papilla cells in A. thaliana. Based on cell type-specific transcriptome analysis from a combination of laser microdissection and RNA sequencing, 15,475, 17,360 and 16,918 genes were identified as expressed in UP, CP and IP papilla cells, respectively, and, of these, 14,392 genes were present in all three data sets. Differentially expressed gene (DEG) analyses identified 147 and 71 genes up-regulated in CP and IP papilla cells, respectively, and 115 and 46 genes down-regulated. Gene Ontology and metabolic pathway analyses revealed that papilla cells play an active role as the female reproductive component in pollination, particularly in information exchange, signal transduction, internal physiological changes and external morphological modification. This study provides fundamental information on the molecular mechanisms involved in pollination in papilla cells, furthering our understanding of the reproductive role of papilla cells.

Plant Omics Data Center: an integrated web repository for interspecies gene expression networks with NLP-based curation.

Comprehensive integration of large-scale omics resources such as genomes, transcriptomes and metabolomes will provide deeper insights into broader aspects of molecular biology. For better understanding of plant biology, we aim to construct a next-generation sequencing (NGS)-derived gene expression network (GEN) repository for a broad range of plant species. So far we have incorporated information about 745 high-quality mRNA sequencing (mRNA-Seq) samples from eight plant species (Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum, Sorghum bicolor, Vitis vinifera, Solanum tuberosum, Medicago truncatula and Glycine max) from the public short read archive, digitally profiled the entire set of gene expression profiles, and drawn GENs by using correspondence analysis (CA) to take advantage of gene expression similarities. In order to understand the evolutionary significance of the GENs from multiple species, they were linked according to the orthology of each node (gene) among species. In addition to other gene expression information, functional annotation of the genes will facilitate biological comprehension. Currently we are improving the given gene annotations with natural language processing (NLP) techniques and manual curation. Here we introduce the current status of our analyses and the web database, PODC (Plant Omics Data Center; http://bioinf.mind.meiji.ac.jp/podc/), now open to the public, providing GENs, functional annotations and additional comprehensive omics resources.

Physiological and genetic analysis of CO2-induced breakdown of self-incompatibility in Brassica rapa.

Self-incompatibility (SI) of the Brassicaceae family can be overcome by CO2 gas treatment. This method has been used for decades as an effective means to obtain a large amount of inbred seeds which can then be used for F1 hybrid seed production; however, the molecular mechanism by which CO2 alters the SI pathway has not been elucidated. In this study, to obtain new insights into the mechanism of CO2-induced SI breakdown, the focus was on two inbred lines of Brassica rapa (syn. campestris) with different CO2 sensitivity. Physiological examination using X-ray microanalysis suggested that SI breakdown in the CO2-sensitive line was accompanied by a significant accumulation of calcium at the pollen-stigma interface. Pre-treatment of pollen or pistil with CO2 gas before pollination showed no effect on the SI reaction, suggesting that some physiological process after pollination is necessary for SI to be overcome. Genetic analyses using F1 progeny of a CO2-sensitive × CO2-insensitive cross suggested that CO2 sensitivity is a semi-dominant trait in these lines. Analysis of F2 progeny suggested that CO2 sensitivity could be a quantitative trait, which is controlled by more than one gene. Quantitative trait locus (QTL) analyses identified two major loci, BrSIO1 and BrSIO2, which work additively in overcoming SI during CO2 treatment. No QTL was detected at the loci previously shown to affect SI stability, suggesting that CO2 sensitivity is determined by novel genes. The QTL data presented here should be useful for determining the responsible genes, and for the marker-assisted selection of desirable parental lines with stable but CO2-sensitive SI in F1 hybrid breeding.

Cell type-specific transcriptome of Brassicaceae stigmatic papilla cells from a combination of laser microdissection and RNA sequencing.

Pollination is an early and critical step in plant reproduction, leading to successful fertilization. It consists of many sequential processes, including adhesion of pollen grains onto the surface of stigmatic papilla cells, foot formation to strengthen pollen-stigma interaction, pollen hydration and germination, and pollen tube elongation and penetration. We have focused on an examination of the expressed genes in papilla cells, to increase understanding of the molecular systems of pollination. From three representative species of Brassicaceae (Arabidopsis thaliana, A. halleri and Brassica rapa), stigmatic papilla cells were isolated precisely by laser microdissection, and cell type-specific gene expression in papilla cells was determined by RNA sequencing. As a result, 17,240, 19,260 and 21,026 unigenes were defined in papilla cells of A. thaliana, A. halleri and B. rapa, respectively, and, among these, 12,311 genes were common to all three species. Among the17,240 genes predicted in A. thaliana, one-third were papilla specific while approximately half of the genes were detected in all tissues examined. Bioinformatics analysis revealed that genes related to a wide range of reproduction and development functions are expressed in papilla cells, particularly metabolism, transcription and membrane-mediated information exchange. These results reflect the conserved features of general cellular function and also the specific reproductive role of papilla cells, highlighting a complex cellular system regulated by a diverse range of molecules in these cells. This study provides fundamental biological knowledge to dissect the molecular mechanisms of pollination in papilla cells and will shed light on our understanding of plant reproduction mechanisms.

Time-lapse imaging of self- and cross-pollinations in Brassica rapa.

BACKGROUND AND AIMS: Pollination is an important process in the life cycle of plants and is the first step in bringing together the male and female gametophytes for plant reproduction. While pollination has been studied for many years, accurate knowledge of the morphological aspects of this process is still far from complete. This study therefore focuses on a morphological characterization of pollination, using time-series image analysis of self- and cross-pollinations in Brassica rapa. METHODS: Time-lapse imaging of pollen behaviour during self- and cross-pollinations was recorded for 90 min, at 1 min intervals, using a stereoscopic microscope. Using time-series digital images of pollination, characteristic features of pollen behaviours during self- and cross-pollinations were studied. KEY RESULTS: Pollen exhibited various behaviours in both self- and cross-pollinations, and these were classified into six representative patterns: germination, expansion, contraction, sudden contraction, pulsation and no change. It is noteworthy that in 'contraction' pollen grains shrunk within a short period of 30-50 min, and in 'pulsation' repeated expansion and contraction occurred with an interval of 10 min, suggesting that a dehydration system is operating in pollination. All of the six patterns were observed on an individual stigma with both self- and cross-pollinations, and the difference between self- and cross-pollinations was in the ratios of the different behaviours. With regard to water transport to and from pollen grains, this occurred in multiple steps, before, during and after hydration. Thus, pollination is regulated by a combination of multiple components of hydration, rehydration and dehydration systems. CONCLUSIONS: Regulated hydration of pollen is a key process for both pollination and self-incompatibility, and this is achieved by a balanced complex of hydration, dehydration and nutrient supply to pollen grains from stigmatic papilla cells.

Dominance in self-compatibility between subgenomes of allopolyploid Arabidopsis kamchatica shown by transgenic restoration of self-incompatibility.

The evolutionary transition to self-compatibility facilitates polyploid speciation. In Arabidopsis relatives, the self-incompatibility system is characterized by epigenetic dominance modifiers, among which small RNAs suppress the expression of a recessive SCR/SP11 haplogroup. Although the contribution of dominance to polyploid self-compatibility is speculated, little functional evidence has been reported. Here we employ transgenic techniques to the allotetraploid plant A. kamchatica. We find that when the dominant SCR-B is repaired by removing a transposable element insertion, self-incompatibility is restored. This suggests that SCR was responsible for the evolution of self-compatibility. By contrast, the reconstruction of recessive SCR-D cannot restore self-incompatibility. These data indicate that the insertion in SCR-B conferred dominant self-compatibility to A. kamchatica. Dominant self-compatibility supports the prediction that dominant mutations increasing selfing rate can pass through Haldane's sieve against recessive mutations. The dominance regulation between subgenomes inherited from progenitors contrasts with previous studies on novel epigenetic mutations at polyploidization termed genome shock.

Microstructure of a Brassica rapa genome segment homoeologous to the resistance gene cluster on Arabidopsis chromosome 4.

Genome evolution is a continuous process and genomic rearrangement occurs both within and between species. With the sequencing of the Arabidopsis thaliana genome, comparative genetics and genomics offer new insights into plant biology. The genus Brassica offers excellent opportunities with which to compare genomic synteny so as to reveal genome evolution. During a previous genetic analysis of clubroot resistance in Brassica rapa, we identified a genetic region that is highly collinear with Arabidopsis chromosome 4. This region corresponds to a disease resistance gene cluster in the A. thaliana genome. Relying on synteny with Arabidopsis, we fine-mapped the region and found that the location and order of the markers showed good correspondence with those in Arabidopsis. Microsynteny on a physical map indicated an almost parallel correspondence, with a few rearrangements such as inversions and insertions. The results show that this genomic region of Brassica is conserved extensively with that of Arabidopsis and has potential as a disease resistance gene cluster, although the genera diverged 20 million years ago.

Molecular genetics, physiology and biology of self-incompatibility in Brassicaceae.

Self-incompatibility (SI) is defined as the inability to produce zygotes after self-pollination in a fertile hermaphrodite plant, which has stamens and pistils in the same flower. This structural organization of the hermaphrodite flower increases the risk of self-pollination, leading to low genetic diversity. To avoid this problem plants have established several pollination systems, among which the most elegant system is surely SI. The SI trait can be observed in Brassica crops, including cabbage, broccoli, turnip and radish. To produce hybrid seed of these crops efficiently, the SI trait has been employed in an agricultural context. From another point of view, the recognition reaction of SI during pollen-stigma interaction is an excellent model system for cell-cell communication and signal transduction in higher plants. In this review, we describe the molecular mechanisms of SI in Brassicaceae, which have been dissected by genetic, physiological, and biological approaches, and we discuss the future prospects in relation to associated scientific fields and new technologies.

OryzaExpress: an integrated database of gene expression networks and omics annotations in rice.

Similarity of gene expression profiles provides important clues for understanding the biological functions of genes, biological processes and metabolic pathways related to genes. A gene expression network (GEN) is an ideal choice to grasp such expression profile similarities among genes simultaneously. For GEN construction, the Pearson correlation coefficient (PCC) has been widely used as an index to evaluate the similarities of expression profiles for gene pairs. However, calculation of PCCs for all gene pairs requires large amounts of both time and computer resources. Based on correspondence analysis, we developed a new method for GEN construction, which takes minimal time even for large-scale expression data with general computational circumstances. Moreover, our method requires no prior parameters to remove sample redundancies in the data set. Using the new method, we constructed rice GENs from large-scale microarray data stored in a public database. We then collected and integrated various principal rice omics annotations in public and distinct databases. The integrated information contains annotations of genome, transcriptome and metabolic pathways. We thus developed the integrated database OryzaExpress for browsing GENs with an interactive and graphical viewer and principal omics annotations (http://riceball.lab.nig.ac.jp/oryzaexpress/). With integration of Arabidopsis GEN data from ATTED-II, OryzaExpress also allows us to compare GENs between rice and Arabidopsis. Thus, OryzaExpress is a comprehensive rice database that exploits powerful omics approaches from all perspectives in plant science and leads to systems biology.

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.

Spatiogenetic characterization of S receptor kinase (SRK) alleles in naturalized populations of Raphanus sativus L. var. raphanistroides on Yakushima island.

In various coastal areas of Japan, naturalized radish populations are observed. Radish is a cruciferous plant and exhibits self-incompatibility, involving a system controlled by a single locus with multiple S alleles. Although the S allele diversity of radish cultivars and wild radishes has been characterized, the S allele distribution in naturalized populations has not yet been analyzed in relation to the positions of the plants in situ. Here, we show the S allele distribution in naturalized radish populations of Yakushima, a small island in the East China Sea, with positions of the plants. Radish plants were sampled in coastal areas in Yakushima, and their S alleles were detected and characterized. Most of the S alleles had been previously identified in radish cultivars. However, four novel S alleles, which may be unique to Yakushima, were also found. Moreover, seeds in siliques from plants growing in the study areas were sampled, and S allele determination in DNA extracted from these seeds suggested that the plants had exchanged their pollen among their close neighbors. There was also a problem in that the PCR amplification of some SRK alleles was difficult because of their sequence diversity in the naturalized populations, as occurs in cultivars. Our results suggest that the exchange of S alleles between cultivars and naturalized populations occurs and that S alleles in naturalized populations are highly diverse. The methodology established in our study should be applicable to other self-incompatible species to dissect the diversity of S allele distribution in naturalized populations.

UDP-glucose pyrophosphorylase is rate limiting in vegetative and reproductive phases in Arabidopsis thaliana.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the metabolism of UDP-glucose, a precursor for the synthesis of carbohydrate cell wall components, such as cellulose and callose. The Arabidopsis thaliana genome contains two putative genes encoding UGPase, AtUGP1 and AtUGP2. These genes are expressed in all organs. In order to determine the role of UGPase in vegetative and reproductive organs, we employed a reverse genetic approach using the T-DNA insertion mutants, atugp1 and atugp2. Despite a significant decrease in UGPase activity in both the atugp1 and atugp2 single mutants, no decrease in normal growth and reproduction was observed. In contrast, the atugp1/atugp2 double mutant displayed drastic growth defects and male sterility. At the reproductive phase, in the anthers of atugp1/atugp2, pollen mother cells developed normally, but callose deposition around microspores was absent. Genes coding for enzymes at the subsequent steps in the cellulose and callose synthesis pathway were also down-regulated in the double mutant. Taken together, these results demonstrate that the AtUGP1 and AtUGP2 genes are functionally redundant and UGPase activity is essential for both vegetative and reproductive phases in Arabidopsis. Importantly, male fertility was not restored in the double knockout mutant by an application of external sucrose, whereas vegetative growth was comparable in size with that of the wild type. In contrast, an application of external UDP-glucose recovered male fertility in the double mutant, suggesting that control of UGPase in carbohydrate metabolism is different in the vegetative phase as compared with the reproductive phase in A. thaliana.

Novel self-compatible lines of Brassica rapa L. isolated from the Japanese bulk-populations.

Self-incompatibility (SI) in Brassicaceae is sporophytically controlled by a single S-locus with multi allelic variety. The male S determinant, SP11/SCR (S-locus protein 11/S-locus cysteine-rich protein), is a small cysteine-rich protein, and the female S determinant, SRK (S-locus receptor kinase), functions as a receptor for SP11 at the surface of stigma papilla cells. Although a few of the following downstream factors in the SP11-SRK signaling cascade have been identified, a comprehensive understanding of the SI mechanism still remains unexplained in Brassicaceae. Analysis of self-compatible (SC) mutants is significant for understanding the molecular mechanism in SI reactions, thus we screened SC lines from a variety of Japanese bulk-populations of B. rapa vegetables. Two lines, TSC4 and TSC28, seem to have disruptions in the SI signaling cascade, while the other line, TSC2, seems to have a deficiency in a female S determinant, SRK. In TSC4 and TSC28, known SI-related factors, i.e. SRK, SP11, MLPK (M-locus protein kinase), THL (thioredoxin-h-like), and ARC1 (arm repeat containing 1), were expressed normally, and their expression levels were comparable with those in SI lines. On a B. rapa genetic linkage map, potential SC genes in TSC4 and TSC28 were mapped on linkage groups A3 and A1, respectively, whereas MLPK, ARC1, and THL were mapped on A3, A4, and A6, respectively. Although potential SC genes of TSC4 and MLPK were on the same linkage group, their positions were apparently independent. These results indicate that the SC genes of TSC4 and TSC28 are independent from the S-locus or known SI-related genes. Thus, the SC lines selected here have mutations in novel factors of the SI signaling cascade, and they will contribute to fill pieces in a signal transduction pathway of the SI system in Brassicaceae.

Morphological and gene expression analysis under cool temperature conditions in rice anther development.

Cool temperature conditions are known to lead to pollen sterility in rice. Pollen sterility is an agriculturally important phenomenon because it imparts a large influence directly on rice yield. However, cool temperature stress tolerance varies among rice cultivars and avoidance of cool temperature stress is difficult by practical method of agriculture. In this study using two rice cultivars, Hitomebore (high tolerance) and Sasanishiki (low tolerance), we analyzed morphological features and gene expression profiles, under cool temperature stress, in anther development of rice. Hitomebore was given cool temperature stress (19 degrees C) at flowering stage, and showed 87.3% seed fertility. Meanwhile, the seed fertility decreased to 41.7% in the case of Sasanishiki. A transverse section of Hitomebore anther revealed that the degradation of the tapetum started at the uninucleate microspore stage, and the tapetum had completely vanished at mature stage. The tapetum provides nutrients for pollen development, and its degradation occurs at a late stage in pollen development. In contrast, degradation of the tapetum did not occur at the uninucleate microspore stage in Sasanishiki, and the tapetum was clearly intact at mature stage, suggesting that tapetum degradation is critical for accurate pollen development and cool temperature tolerance correlated with the degree of tapetum degeneration. In gene expression analysis of anther, 356 genes that showed different expression levels between two cultivars at cool temperatures were found. These genes will lead to understanding the mechanism of cool temperature stress response in rice pollen development and the identification of genes involved in accurate tapetum degradation.

Double-Locking Mechanism of Self-Compatibility in Arabidopsis thaliana: The Synergistic Effect of Transcriptional Depression and Disruption of Coding Region in the Male Specificity Gene.

Self-compatibility in Arabidopsis thaliana represents the relatively recent disruption of ancestral obligate cross pollination, recognized as one of the prevalent evolutionary pathways in flowering plants, as noted by Darwin. Our previous study found that inversion of the male specificity gene (SP11/SCR) disrupted self-incompatibility, which was restored by overexpressing the SCR with the reversed inversion. However, SCR in A. thaliana has other mutations aside from the pivotal inversion, in both promoter and coding regions, with probable effects on transcriptional regulation. To examine the functional consequences of these mutations, we conducted reciprocal introductions of native promoters and downstream sequences from orthologous loci of self-compatible A. thaliana and self-incompatible A. halleri. Use of this inter-species pair enabled us to expand the scope of the analysis to transcriptional regulation and deletion in the intron, in addition to inversion in the native genomic background. Initial analysis revealed that A. thaliana has a significantly lower basal expression level of SCR transcripts in the critical reproductive stage compared to that of A. halleri, suggesting that the promoter was attenuated in inducing transcription in A. thaliana. However, in reciprocal transgenic experiments, this A. thaliana promoter was able to restore partial function if coupled with the functional A. halleri coding sequence, despite extensive alterations due to the self-compatible mode of reproduction in A. thaliana. This represents a synergistic effect of the promoter and the inversion resulting in fixation of self-compatibility, primarily enforced by disruption of SCR. Our findings elucidate the functional and evolutionary context of the historical transition in A. thaliana thus contributing to the understanding of the molecular events leading to development of self-compatibility.

The AtXTH28 gene, a xyloglucan endotransglucosylase/hydrolase, is involved in automatic self-pollination in Arabidopsis thaliana.

Successful automatic self-pollination in flowering plants is dependent on the correct development of reproductive organs. In the stamen, the appropriate growth of the filament, which largely depends on the mechanical properties of the cell wall, is required to position the anther correctly close to the stigma at the pollination stage. Xyloglucan endotransglucosylase/hydrolases (XTHs) are a family of enzymes that mediate the construction and restructuring of xyloglucan cross-links, thereby controlling the extensibility or mechanical properties of the cell wall in a wide variety of plant tissues. Our reverse genetic analysis has revealed that a loss-of-function mutation of an Arabidopsis XTH family gene, AtXTH28, led to a decrease in capability for self-pollination, probably due to inhibition of stamen filament growth. Our results also suggest that the role of AtXTH28 in the development of the stamen is not functionally redundant with its closest paralog, AtXTH27. Thus, our finding indicates that AtXTH28 is specifically involved in the growth of stamen filaments, and is required for successful automatic self-pollination in certain flowers in Arabidopsis thaliana.

Genetic and tissue-specific RNA-sequencing analysis of self-compatible mutant TSC28 in Brassica rapa L. toward identification of a novel self-incompatibility factor.

Self-incompatibility (SI) is a sophisticated system for pollen selectivity to prevent pollination by genetically identical pollen. In Brassica, it is genetically controlled by a single, highly polymorphic S-locus, and the male and female S-determinant factors have been identified as S-locus protein 11 (SP11)/S-locus cysteine-rich protein (SCR) and S-locus receptor kinase (SRK), respectively. However, the overall molecular system and identity of factors in the downstream cascade of the SI reaction remain unclear. Previously, we identified a self-compatible B. rapa mutant line, TSC28, which has a disruption in an unidentified novel factor of the SI signaling cascade. Here, in a genetic analysis of TSC28, using an F2 population from a cross with the reference B. rapa SI line Chiifu-401, the causal gene was mapped to a genetic region of DNA containing markers BrSA64 and ACMP297 in B. rapa chromosome A1. By fine mapping using an F2 population of 1,034 plants, it was narrowed down to a genetic region between DNA markers ACMP297 and BrgMS4028, with physical length approximately 1.01 Mbp. In this genomic region, 113 genes are known to be located and, among these, we identified 55 genes that were expressed in the papilla cells. These are candidates for the gene responsible for the disruption of SI in TSC28. This list of candidate genes will contribute to the discovery of a novel downstream factor in the SP11-SRK signaling cascade in the Brassica SI system.

Separated transcriptomes of male gametophyte and tapetum in rice: validity of a laser microdissection (LM) microarray.

In flowering plants, the male gametophyte, the pollen, develops in the anther. Complex patterns of gene expression in both the gametophytic and sporophytic tissues of the anther regulate this process. The gene expression profiles of the microspore/pollen and the sporophytic tapetum are of particular interest. In this study, a microarray technique combined with laser microdissection (44K LM-microarray) was developed and used to characterize separately the transcriptomes of the microspore/pollen and tapetum in rice. Expression profiles of 11 known tapetum specific-genes were consistent with previous reports. Based on their spatial and temporal expression patterns, 140 genes which had been previously defined as anther specific were further classified as male gametophyte specific (71 genes, 51%), tapetum-specific (seven genes, 5%) or expressed in both male gametophyte and tapetum (62 genes, 44%). These results indicate that the 44K LM-microarray is a reliable tool to analyze the gene expression profiles of two important cell types in the anther, the microspore/pollen and tapetum.

Various spatiotemporal expression profiles of anther-expressed genes in rice.

The male gametophyte and tapetum play different roles during anther development although they are differentiated from the same cell lineage, the L2 layer. Until now, it has not been possible to delineate their transcriptomes due to technical difficulties in separating the two cell types. In the present study, we characterized the separated transcriptomes of the rice microspore/pollen and tapetum using laser microdissection (LM)-mediated microarray. Spatiotemporal expression patterns of 28,141 anther-expressed genes were classified into 20 clusters, which contained 3,468 (12.3%) anther-enriched genes. In some clusters, synchronous gene expression in the microspore and tapetum at the same developmental stage was observed as a novel characteristic of the anther transcriptome. Noteworthy expression patterns are discussed in connection with gene ontology (GO) categories and gene annotations, which are related to important biological events in anther development, such as pollen maturation, pollen germination, pollen tube elongation and pollen wall formation.