Identification and Functional Characterization of FLOWERING LOCUS T in Platycodon grandiflorus.

Platycodon grandiflorus roots have been used as a foodstuff and traditional medicine for thousands of years in East Asia. In order to increase the root development of P. grandiflorus, cultivators removed the inflorescences, suggesting the possible negative effect of flowering on root development. This indicates that the genetic improvement of P. grandiflorus by late flowering is a potential approach to increase productivity. However, nothing is known about key genes integrating multiple flowering pathways in P. grandiflorus. In order to fill this gap, we identified potential homologs of the FLOWERING LOCUS T (FT) gene in P. grandiflorus. The alignment with other FT members and phylogenetic analysis revealed that the P. grandiflorus FT (PlgFT) protein contains highly conserved functional domains and belongs to the FT-like clade. The expression analysis revealed spatial variations in the transcription of PlgFT in different organs. In addition, the expression level of PlgFT was increased by high temperature but not by photoperiodic light input signals, presumably due to lacking the CONSTANS binding motif in its promoter region. Furthermore, PlgFT induced early flowering upon its overexpression in P. grandiflorus, suggesting the functional role of PlgFT in flowering. Taken together, we functionally characterized PlgFT as a master regulator of P. grandiflorus flowering under inductive high temperature, which will serve as an important target gene for improving the root productivity.

Enhanced production of resveratrol derivatives in tobacco plants by improving the metabolic flux of intermediates in the phenylpropanoid pathway.

The biosynthesis of flavonoids such as anthocyanin and stilbenes has attracted increasing attention because of their potential health benefits. Anthocyanins and stilbenes share common phenylpropanoid precursor pathways. We previously reported that the overexpression of sweetpotato IbMYB1a induced anthocyanin pigmentation in transgenic tobacco (Nicotiana tabacum) plants. In the present study, transgenic tobacco (Nicotiana tabacum SR1) plants (STS-OX and ROST-OX) expressing the RpSTS gene encoding stilbene synthase from rhubarb (Rheum palmatum L. cv. Jangyeop) and the RpSTS and VrROMT genes encoding resveratrol O-methyltransferase from frost grape (Vitis riparia) were generated under the control of 35S promoter. Phenotypic alterations in floral organs, such as a reduction in floral pigments and male sterility, were observed in STS-OX transgenic tobacco plants. However, we failed to obtain STS-OX and ROST-OX plants with high levels of resveratrol compounds. Therefore, to improve the production of resveratrol derivatives in plants, we cross-pollinated flowers of STS-OX or ROST-OX and IbMYB1a-OX transgenic lines (SM and RSM). Phenotypic changes in vegetative and reproductive development of SM and RSM plants were observed. Furthermore, by HPLC and LC-MS analyses, we found enhanced production of resveratrol derivatives such as piceid, piceid methyl ether, resveratrol methyl ether O-hexoside, and 5-methyl resveratrol-3,4'-O-ß-D-diglucopyranoside in SM and RSM cross-pollinated lines. Here, total contents of trans- and cis-piceids ranged from approximately 104-240 µg/g fresh weight in SM (F2). Collectively, we suggest that coexpression of RpSTS and IbMYB1a via cross-pollination can induce enhanced production of resveratrol compounds in plants by increasing metabolic flux into stilbenoid biosynthesis.

Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli.

Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins.

The Arabidopsis PLAT domain protein1 promotes abiotic stress tolerance and growth in tobacco.

Plant growth and consequently crop yield can be severely compromised by abiotic and biotic stress conditions. Transgenic approaches that resulted in increased tolerance against abiotic stresses often were typically accompanied by adverse effects on plant growth and fitness under optimal growing conditions. Proteins that belong to the PLAT-plant-stress protein family harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and are ubiquitously present in monocot and dicot plant species. Until now, only limited data is available for PLAT-plant-stress family members, which suggested that these proteins in general could promote tolerance towards stress responses. We studied the function of the Arabidopsis PLAT-plant-stress protein AtPLAT1 employing heterologous gain-of-function analysis in tobacco. AtPLAT1 conferred increased abiotic stress tolerance in tobacco, evident by improved tolerance towards cold, drought and salt stresses, and promoted growth, reflected by a faster development under non-stressed conditions. However, the overexpression of AtPLAT1 in tobacco reduced the tolerance towards biotic stress conditions and, therefore, could be involved in regulating the crosstalk between abiotic and biotic stress responses. Thus, we showed that heterologously expressed AtPLAT1 functions as positive regulator of abiotic stress tolerance and plant growth, which could be an important new asset for strategies to develop plants with improved abiotic stress tolerance, without growth and subsequent yield penalties under optimal growth conditions.

GAL4 transactivation-based assay for the detection of selective intercellular protein movement.

Several plant proteins function as intercellular messenger to specify cell fate and coordinate plant development. Such intercellular communication can be achieved by direct, selective, or nonselective (diffusion-based) trafficking through plasmodesmata (PD), the symplasmic membrane-lined nanochannels adjoining two cells. A trichome rescue trafficking assay was reported to allow the detection of protein movement in Arabidopsis leaf tissue using transgenic gene expression. Here, we provide a protocol to dissect the mode of intercellular protein movement in Arabidopsis root. This assay system involves a root ground tissue-specific GAL4/UAS transactivation expression system in combination with fluorescent reporter proteins. In this system, mCherry, a red fluorescent protein, can move cell to cell via diffusion, while mCherry-H2B is tightly cell autonomous. Thus, a protein fused to mCherry-H2B that can move out from the site of synthesis likely contains a selective trafficking signal to impart a cell-to-cell gain-of-trafficking function to the cell-autonomous mCherry-H2B. This approach can be adapted to investigate the cell-to-cell trafficking properties of any protein of interest.

The Arabidopsis PLAT domain protein1 is critically involved in abiotic stress tolerance.

Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty.

RNA-seq analysis of Rubus idaeus cv. Nova: transcriptome sequencing and de novo assembly for subsequent functional genomics approaches.

KEY MESSAGE: Using Illumina sequencing technology, we have generated the large-scale transcriptome sequencing data containing abundant information on genes involved in the metabolic pathways in R. idaeus cv. Nova fruits. Rubus idaeus (Red raspberry) is one of the important economical crops that possess numerous nutrients, micronutrients and phytochemicals with essential health benefits to human. The molecular mechanism underlying the ripening process and phytochemical biosynthesis in red raspberry is attributed to the changes in gene expression, but very limited transcriptomic and genomic information in public databases is available. To address this issue, we generated more than 51 million sequencing reads from R. idaeus cv. Nova fruit using Illumina RNA-Seq technology. After de novo assembly, we obtained 42,604 unigenes with an average length of 812 bp. At the protein level, Nova fruit transcriptome showed 77 and 68 % sequence similarities with Rubus coreanus and Fragaria versa, respectively, indicating the evolutionary relationship between them. In addition, 69 % of assembled unigenes were annotated using public databases including NCBI non-redundant, Cluster of Orthologous Groups and Gene ontology database, suggesting that our transcriptome dataset provides a valuable resource for investigating metabolic processes in red raspberry. To analyze the relationship between several novel transcripts and the amounts of metabolites such as γ-aminobutyric acid and anthocyanins, real-time PCR and target metabolite analysis were performed on two different ripening stages of Nova. This is the first attempt using Illumina sequencing platform for RNA sequencing and de novo assembly of Nova fruit without reference genome. Our data provide the most comprehensive transcriptome resource available for Rubus fruits, and will be useful for understanding the ripening process and for breeding R. idaeus cultivars with improved fruit quality.

De-novo RNA sequencing and metabolite profiling to identify genes involved in anthocyanin biosynthesis in Korean black raspberry (Rubus coreanus Miquel).

The Korean black raspberry (Rubus coreanus Miquel, KB) on ripening is usually consumed as fresh fruit, whereas the unripe KB has been widely used as a source of traditional herbal medicine. Such a stage specific utilization of KB has been assumed due to the changing metabolite profile during fruit ripening process, but so far molecular and biochemical changes during its fruit maturation are poorly understood. To analyze biochemical changes during fruit ripening process at molecular level, firstly, we have sequenced, assembled, and annotated the transcriptome of KB fruits. Over 4.86 Gb of normalized cDNA prepared from fruits was sequenced using Illumina HiSeq™ 2000, and assembled into 43,723 unigenes. Secondly, we have reported that alterations in anthocyanins and proanthocyanidins are the major factors facilitating variations in these stages of fruits. In addition, up-regulation of F3'H1, DFR4 and LDOX1 resulted in the accumulation of cyanidin derivatives during the ripening process of KB, indicating the positive relationship between the expression of anthocyanin biosynthetic genes and the anthocyanin accumulation. Furthermore, the ability of RcMCHI2 (R. coreanus Miquel chalcone flavanone isomerase 2) gene to complement Arabidopsis transparent testa 5 mutant supported the feasibility of our transcriptome library to provide the gene resources for improving plant nutrition and pigmentation. Taken together, these datasets obtained from transcriptome library and metabolic profiling would be helpful to define the gene-metabolite relationships in this non-model plant.

Evolutionary and molecular analysis of Dof transcription factors identified a conserved motif for intercellular protein trafficking.

· Cell-to-cell trafficking of transcription factors (TFs) has been shown to play an important role in the regulation of plant developmental events, but the evolutionary relationship between cell-autonomous and noncell-autonomous (NCA) TFs remains elusive. · AtDof4.1, named INTERCELLULAR TRAFFICKING DOF 1 (ITD1), was chosen as a representative NCA member to explore this evolutionary relationship. Using domain structure-function analyses and swapping studies, we examined the cell-to-cell trafficking of plant-specific Dof TF family members across Arabidopsis and other species. · We identified a conserved intercellular trafficking motif (ITM) that is necessary and sufficient for selective cell-to-cell trafficking and can impart gain-of-function cell-to-cell movement capacity to an otherwise cell-autonomous TF. The functionality of related motifs from Dof members across the plant kingdom extended, surprisingly, to a unicellular alga that lacked plasmodesmata. By contrast, the algal homeodomain related to the NCA KNOX homeodomain was either inefficient or unable to impart such cell-to-cell movement function. · The Dof ITM appears to predate the evolution of selective plasmodesmal trafficking in the plant kingdom, which may well have acted as a molecular template for the evolution of Dof proteins as NCA TFs. However, the ability to efficiently traffic for KNOX homeodomain (HD) proteins may have been acquired during the evolution of early nonvascular plants.

De novo transcriptome sequencing of Momordica cochinchinensis to identify genes involved in the carotenoid biosynthesis.

The ripe fruit of Momordica cochinchinensis Spreng, known as gac, is featured by very high carotenoid content. Although this plant might be a good resource for carotenoid metabolic engineering, so far, the genes involved in the carotenoid metabolic pathways in gac were unidentified due to lack of genomic information in the public database. In order to expedite the process of gene discovery, we have undertaken Illumina deep sequencing of mRNA prepared from aril of gac fruit. From 51,446,670 high-quality reads, we obtained 81,404 assembled unigenes with average length of 388 base pairs. At the protein level, gac aril transcripts showed about 81.5% similarity with cucumber proteomes. In addition 17,104 unigenes have been assigned to specific metabolic pathways in Kyoto Encyclopedia of Genes and Genomes, and all of known enzymes involved in terpenoid backbones biosynthetic and carotenoid biosynthetic pathways were also identified in our library. To analyze the relationship between putative carotenoid biosynthesis genes and alteration of carotenoid content during fruit ripening, digital gene expression analysis was performed on three different ripening stages of aril. This study has revealed putative phytoene synthase, 15-cis-phytone desaturase, zeta-carotene desaturase, carotenoid isomerase and lycopene epsilon cyclase might be key factors for controlling carotenoid contents during aril ripening. Taken together, this study has also made availability of a large gene database. This unique information for gac gene discovery would be helpful to facilitate functional studies for improving carotenoid quantities.

Analysis of Arabidopsis transcription factor families revealed extensive capacity for cell-to-cell movement as well as discrete trafficking patterns.

In plants, cell-to-cell communication is pivotal for the orchestration of cell fate determination, organ development, and the integration of whole plant physiology. One of the strategies for intercellular communication uses symplasmic communication channels, called plasmodesmata (PD). These PD establish unique cytoplasmic channels for the intercellular exchange not only of metabolites and small signaling molecules, but also of regulatory proteins and RNAs to allow for local orchestration of development and physiology. A number of non-cell-autonomous transcription factors (NCATFs) have been shown to function in the coordination of specific regulatory networks. To further explore the potential of such NCATFs, a genome-wide screen was performed on the transcription factor (TF) families in Arabidopsis. We here report that, among the 76 TFs examined, 22 were shown to move beyond their sites of transcription in the root apex; these NCATFs belonged to 17 TF families, including homeobox, GRAS, and MYB. Expression studies performed on variously-sized mCherry constructs identified a range of PD size exclusion limits within tissues of the root. In addition, our studies showed that actual protein level was an important factor controlling the range of TF intercellular movement. Interestingly, our studies on CAPRICE movement revealed tissue-specificity with respect to the mode of intercellular trafficking. These findings are discussed with respect to the regulation between cell-autonomous or non-cell-autonomous action.

How to assess the intercellular trafficking of transcription factors.

Non-cell-autonomous (NCA) control of plant development is an emerging field. Transcription factors (TFs) are the most important plant proteins involved in development and cell fate determination. In plants specialized intercellular symplastic channels, called plasmodesmata (PD), facilitate and regulate the NCA action of TFs. NCA-TFs move from cell to cell either selectively or non-selectively depending upon the specific interactions with PD or the pathway proteins. Here we describe different approaches to establish the role of TFs in NCA control of its function and the characteristic movement behavior.

Plasmodesmal receptor-like kinases identified through analysis of rice cell wall extracted proteins.

In plants, plasmodesmata (PD) are intercellular channels that function in both metabolite exchange and the transport of proteins and RNAs. Currently, many of the PD structural and regulatory components remain to be elucidated. Receptor-like kinases (RLKs) belonging to a notably expanded protein family in plants compared to the animal kingdom have been shown to play important roles in plant growth, development, pathogen resistance, and cell death. In this study, cell biological approaches were used to identify potential PD-associated RLK proteins among proteins contained within cell walls isolated from rice callus cultured cells. A total of 15 rice RLKs were investigated to determine their subcellular localization, using an Agrobacterium-mediated transient expression system. Of these six PD-associated RLKs were identified based on their co-localization with a viral movement protein that served as a PD marker, plasmolysis experiments, and subcellular localization at points of wall contact between spongy mesophyll cells. These findings suggest potential PD functions in apoplasmic signaling in response to environmental stimuli and developmental inputs.

Cell-to-cell trafficking of RNA and RNA silencing through plasmodesmata.

Plasmodesmata (PD) are plasma membrane-lined cytoplasmic channels that cross the cell wall and establish symplasmic continuity between neighboring cells in plants. Recently, a wide range of cellular RNAs (including mRNAs and small RNAs (sRNAs)) have been reported to move from cell to cell through PD trafficking pathways. sRNAs are key molecules that function in transcriptional and post-transcriptional RNA silencing, which is a gene expression regulatory mechanism that is conserved among eukaryotes and is important for protection against invading nucleic acids (such as viruses and transposons) and for developmental and physiological regulation. One of the most intriguing aspects of RNA silencing is that it can function either cell autonomously or non-cell autonomously in post-transcriptional RNA silencing pathways. Although the mechanisms underlying cell-to-cell trafficking of RNA and RNA silencing signals are not fully understood, the movement of specific RNAs seems to play a critical role in cell-to-cell and long-distance regulation of gene expression, thereby coordinating growth and developmental processes, gene silencing, and stress responses. In this review, we summarize the current knowledge regarding cell-to-cell trafficking of RNA molecules (including small RNAs), and we discuss potential molecular mechanisms of cell-to-cell trafficking that are mediated by complex networks.

Proteome study of the phloem sap of pumpkin using multidimensional protein identification technology.

The phloem is the major transport route for both small substances and large molecules, such as proteins and RNAs, from their sources to sink tissues. To investigate the proteins present in pumpkin phloem sap, proteome analysis using multidimensional protein identification technology was carried out. Pumpkin phloem peptides obtained by liquid chromatography/mass spectrometry/mass spectrometry were searched against pumpkin protein data derived from the National Center for Biotechnology Information. A total of 47 pumpkin phloem proteins were identified. The identified proteins mainly corresponded to enzymes involved in gibberellin biosynthesis, antioxidation processes, or defense mechanisms. Interestingly, seven enzymes required for gibberellin biosynthesis were identified for the first time by this proteomics approach. In summary, the new phloem proteins identified in this study provide strong evidence for stress and defense signaling and new insights regarding the role of gibberellin in the developmental programming of higher plants through the phloem.

Comprehensive proteome analysis of lettuce latex using multidimensional protein-identification technology.

Commercially, lettuce (Lactuca sativa) is one of the most important leafy vegetables. Lettuce produces a milky latex of variable chemical compositions within its laticifers. As a step toward understanding the main physiological roles of this latex in higher plants, we embarked on its proteomic analysis. We investigated 587 latex proteins that were identified from the lettuce latex using multidimensional protein-identification technology. A bioinformatics analysis showed that the most frequently encountered proteins in the latex were organellar proteins from plastids and mitochondria, followed by nucleic and cytoplasmic proteins. Functional classification of the identified proteins showed that proteins related to metabolism, cell rescue, defense, and virulence were the most abundant in lettuce latex. Furthermore, numerous resistance proteins of lettuce and viral proteins were present in the latex suggesting for the first time a possible function of the lettuce latex in defense or pathogenesis. To the knowledge of the authors, this is the first large-scale proteome analysis of lettuce latex.

The Arabidopsis callose synthase gene GSL8 is required for cytokinesis and cell patterning.

Cytokinesis is the division of the cytoplasm and its separation into two daughter cells. Cell plate growth and cytokinesis appear to require callose, but direct functional evidence is still lacking. To determine the role of callose and its synthesis during cytokinesis, we identified and characterized mutants in many members of the GLUCAN SYNTHASE-LIKE (GSL; or CALLOSE SYNTHASE) gene family in Arabidopsis (Arabidopsis thaliana). Most gsl mutants (gsl1-gsl7, gsl9, gsl11, and gsl12) exhibited roughly normal seedling growth and development. However, mutations in GSL8, which were previously reported to be gametophytic lethal, were found to produce seedlings with pleiotropic defects during embryogenesis and early vegetative growth. We found cell wall stubs, two nuclei in one cell, and other defects in cell division in homozygous gsl8 insertional alleles. In addition, gsl8 mutants and inducible RNA interference lines of GSL8 showed reduced callose deposition at cell plates and/or new cell walls. Together, these data show that the GSL8 gene encodes a putative callose synthase required for cytokinesis and seedling maturation. In addition, gsl8 mutants disrupt cellular and tissue-level patterning, as shown by the presence of clusters of stomata in direct contact and by islands of excessive cell proliferation in the developing epidermis. Thus, GSL8 is required for patterning as well as cytokinesis during Arabidopsis development.

Proteomic analysis of the secretome of rice calli.

The cell wall and extracellular matrix in higher plants include secreted proteins that play critical roles in a wide range of cellular processes, such as structural integrity and biogenesis. Compared with the intensive cell wall proteomic studies in Arabidopsis, the list of cell wall proteins identified in monocot species is lacking. Therefore, we conducted a large-scale proteomic analysis of secreted proteins from rice. Highly purified secreted rice proteins were obtained from the medium of a suspension of callus culture and were analyzed with multidimensional protein identification technology (MudPIT). As a result, we could detect a total of 555 rice proteins by MudPIT analysis. Based on bioinformatic analyses, 27.7% (154 proteins) of the identified proteins are considered to be secreted proteins because they possess a signal peptide for the secretory pathway. Among the 154 identified proteins, 27% were functionally categorized as stress response proteins, followed by metabolic proteins (26%) and factors involved in protein modification (24%). Comparative analysis of cell wall proteins from Arabidopsis and rice revealed that one third of the secreted rice proteins overlapped with those of Arabidopsis. Furthermore, 25 novel rice-specific secreted proteins were found. This work presents the large scale of the rice secretory proteome from culture medium, which contributes to a deeper understanding of the rice secretome.

A non-cell-autonomous mechanism for the control of plant architecture and epidermal differentiation involves intercellular trafficking of BREVIPEDICELLUS protein.

Intercellular trafficking of maize KNOTTED1 and its homologous KNOTTED1-related homeobox (KNOX) proteins has been reported; however, little is known about the functional significance of KNOX trafficking in plant development. In this study, we showed that intercellular movement of BREVIPEDICELLUS (BP or KNAT1), the closest Arabidopsis homologue of KNOTTED1, is tissue-specific and takes place through a selective pathway. When BP was fused to a red fluorescent mCherry construct, it could move from the mesophyll to epidermal cells of leaves, although it could not move out from the cortex/endodermis of roots. Using a trichome rescue-trafficking assay, we also showed that BP fusion could confer gain-of-trafficking function to the cell-autonomous GLABROUS1 (GL1) protein. In the wild type, BP transcripts are expressed in the sub-epidermal cortical cell layers of the inflorescence stem and pedicel. However, bp mutant phenotypes include defects in epidermal cell differentiation suggesting a non-cell-autonomous function. Expression of a GFP:BP fusion under the control of a BP promoter specific to the stem cortex layers resulted in epidermal GFP fluorescence suggesting its movement from subepidermis to epidermis. Here, we provide evidence from complementation analyses using cell autonomous or non-cell-autonomous BP fusions that the intercellular trafficking of BP protein is important for plant architecture and epidermal differentiation.

Arabidopsis glucan synthase-like 10 functions in male gametogenesis.

Callose or beta-1,3-glucan performs multiple functions during male and female gametophyte development. Callose is synthesized by 12 members of the glucan synthase-like (GSL) gene family in Arabidopsis thaliana. To elucidate the biological roles of Arabidopsis GSL family members during sexual development, we initiated a reverse genetic approach with T-DNA insertional mutagenesis lines. We screened T-DNA insertion lines for all members of the GSL gene family and detected homozygous mutant seedlings for all members except GSL10. Three independent alleles in GSL10, gsl10-1, gsl10-3 and gsl10-4 showed distorted segregation (1:1:0) of T-DNA inserts rather than Mendelian segregation (1:2:1). By genetic analysis through reciprocal cross, we determined that gsl10 pollen could not be transmitted to descendent. The mutant pollen of GSL10/gsl10 plants at tetrad and microspore stages were not different from that of wild type, suggesting that GSL10 is not essential for normal microspore growth. Analysis of GSL10/gsl10 hemizygous pollen during development revealed abnormal function in asymmetric microspore division. gsl10 mutant microspores failed to enter into mitosis. Unlike the previously described functions of GSL1, GSL2 and GSL5, GSL10 involves an independent process of pollen development at the mitotic division stage.