Redundant role of OsCNGC4 and OsCNGC5 encoding cyclic nucleotide-gated channels in rice pollen germination and tube growth.

In staple crops, such as rice (Oryza sativa L.), pollen plays a crucial role in seed production. However, the molecular mechanisms underlying rice pollen germination and tube growth remain underexplored. Notably, we recently uncovered the redundant expression and mutual interaction of two rice genes encoding cyclic nucleotide-gated channels (CNGCs), OsCNGC4 and OsCNGC5, in mature pollen. Building on these findings, the current study focused on clarifying the functional roles of these two genes in pollen germination and tube growth. To overcome functional redundancy, we produced gene-edited rice plants with mutations in both genes using the CRISPR-Cas9 system. The resulting homozygous OsCNGC4 and OsCNGC5 gene-edited mutants (oscngc4/5) exhibited significantly lower pollen germination rates than the wild type (WT), along with severely reduced fertility. Transcriptome analysis of the double oscngc4/5 mutant revealed downregulation of genes related to receptor kinases, transporters, and cell wall metabolism. To identify the direct regulators of OsCNGC4, which form a heterodimer with OsCNGC5, we screened a yeast two-hybrid library containing rice cDNAs from mature anthers. Subsequently, we identified two calmodulin isoforms (CaM1-1 and CaM1-2), NETWORKED 2 A (NET2A), and proline-rich extension-like receptor kinase 13 (PERK13) proteins as interactors of OsCNGC4, suggesting its roles in regulating Ca2+ channel activity and F-actin organization. Overall, our results suggest that OsCNGC4 and OsCNGC5 may play critical roles in pollen germination and elongation by regulating the Ca2+ gradient in growing pollen tubes.

Rice pollen-specific OsRALF17 and OsRALF19 are essential for pollen tube growth.

Pollen tube growth is essential for successful double fertilization, which is critical for grain yield in crop plants. Rapid alkalinization factors (RALFs) function as ligands for signal transduction during fertilization. However, functional studies on RALF in monocot plants are lacking. Herein, we functionally characterized two pollen-specific RALFs in rice (Oryza sativa) using multiple clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-induced loss-of-function mutants, peptide treatment, expression analyses, and tag reporter lines. Among the 41 RALF members in rice, OsRALF17 was specifically expressed at the highest level in pollen and pollen tubes. Exogenously applied OsRALF17 or OsRALF19 peptide inhibited pollen tube germination and elongation at high concentrations but enhanced tube elongation at low concentrations, indicating growth regulation. Double mutants of OsRALF17 and OsRALF19 (ralf17/19) exhibited almost full male sterility with defects in pollen hydration, germination, and tube elongation, which was partially recovered by exogenous treatment with OsRALF17 peptide. This study revealed that two partially functionally redundant OsRALF17 and OsRALF19 bind to Oryza sativa male-gene transfer defective 2 (OsMTD2) and transmit reactive oxygen species signals for pollen tube germination and integrity maintenance in rice. Transcriptomic analysis confirmed their common downstream genes, in osmtd2 and ralf17/19. This study provides new insights into the role of RALF, expanding our knowledge of the biological role of RALF in regulating rice fertilization.

A myosin XI adaptor, TAPE, is essential for pollen tube elongation in rice.

Pollen tube (PT) elongation is important for double fertilization in angiosperms and affects the seed-setting rate and, therefore, crop productivity. Compared to Arabidopsis (Arabidopsis thaliana L.), information on PT elongation in rice (Oryza sativa L.) is limited by the difficulty in obtaining homozygous mutants. In a screen of T-DNA insertional mutants, we identified a mutant in the Tethering protein of actomyosin transport in pollen tube elongation (TAPE) gene with an unusual segregation ratio by genotyping analysis. A CRISPR/Cas9 knockout mutant of TAPE that produced a short PT was sterile, and TAPE was expressed specifically in pollen grains. TAPE is a homolog of a myosin XI adaptor in Arabidopsis with three tetratricopeptide repeat and Phox and Bem1 protein domains. TAPE showed latrunculin B-sensitive, actin-dependent localization to the endoplasmic reticulum. Yeast two-hybrid screening and transcriptome analysis revealed that TAPE interacted with pollen-specific LIM protein 2b and elongation factor 1-alpha. Loss of TAPE affected transcription of 1,259 genes, especially genes related to cell organization, which were downregulated. In summary, TAPE encodes a myosin XI adaptor essential for rice PT elongation.

OsMTD2-mediated reactive oxygen species (ROS) balance is essential for intact pollen-tube elongation in rice.

The highly specialized haploid male gametophyte-pollen consist of two sperm cells and a large vegetative cell. Successful fertilization requires proper growth timing and rupture of the pollen tube until it delivers sperm cells, which occur immediately after a pollen grain hydrates. Although a tight regulation on polar cell-wall expansion of the pollen tube is fundamentally important, the underlying molecular mechanism remains largely unknown, especially in crop plants. Here, we characterized the function of male-gene transfer defective 2 (OsMTD2) gene in rice (Oryza sativa), which belongs to the plant-specific receptor-like kinase, the CrRLK1L family. We demonstrated that OsMTD2 is an essential male factor participating in pollen-tube elongation based on genetic evidence and physiological observations. Because of unavailability of homozygous mutant via conventional methods, we used CRISPR-Cas9 system to obtain homozygous knockout mutant of OsMTD2. We were able to identify phenotypic changes including male sterility due to early pollen-tube rupture in the mutant. We observed that the production of reactive oxygen species (ROS) was dramatically reduced in mutants of OsMTD2 pollen grain and tubes with defective pectin distribution. Transcriptome analysis of osmtd2-2 versus wild-type anthers revealed that genes involved in defense responses, metabolic alteration, transcriptional and protein modification were highly upregulated in the osmtd2-2 mutant. Through yeast-two-hybrid screening, we found that OsMTD2 kinase interacts with E3 ligase SPL11. Taken together, we propose that OsMTD2 has crucial functions in promoting pollen-tube elongation through cell-wall modification, possibly by modulating ROS homeostasis during pollen-tube growth.

Analysis of sticky generative cell mutants reveals that suppression of callose deposition in the generative cell is necessary for generative cell internalization and differentiation in Arabidopsis.

In flowering plants, double fertilization between male and female gametophytes, which are separated by distance, largely depends on the unique pattern of the male gametophyte (pollen): two non-motile sperm cells suspended within a tube-producing vegetative cell. A morphological screen to elucidate the genetic control governing the strategic patterning of pollen has led to the isolation of a sticky generative cell (sgc) mutant that dehisces abnormal pollen with the generative cell immobilized at the pollen wall. Analyses revealed that the sgc mutation is specifically detrimental to pollen development, causing ectopic callose deposition that impedes the timely internalization and differentiation of the generative cell. We found that the SGC gene encodes the highly conserved domain of unknown function 707 (DUF707) gene that is broadly expressed but is germline specific during pollen development. Additionally, transgenic plants co-expressing fluorescently fused SGC protein and known organelle markers showed that SGC localizes in the endoplasmic reticulum, Golgi apparatus and vacuoles in pollen. A yeast two-hybrid screen with an SGC bait identified a thaumatin-like protein that we named GCTLP1, some homologs of which bind and/or digest ß-1,3-glucans, the main constituent of callose. GCTLP1 is expressed in a germline-specific manner and colocalizes with SGC during pollen development, indicating that GCTLP1 is a putative SGC interactor. Collectively, our results show that SGC suppresses callose deposition in the nascent generative cell, thereby allowing the generative cell to fully internalize into the vegetative cell and correctly differentiate as the germline progenitor, with the potential involvement of the GCTLP1 protein, during pollen development in Arabidopsis.

Physiological Importance of Pectin Modifying Genes During Rice Pollen Development.

Although cell wall dynamics, particularly modification of homogalacturonan (HGA, a major component of pectin) during pollen tube growth, have been extensively studied in dicot plants, little is known about how modification of the pollen tube cell wall regulates growth in monocot plants. In this study, we assessed the role of HGA modification during elongation of the rice pollen tube by adding a pectin methylesterase (PME) enzyme or a PME-inhibiting catechin extract (Polyphenon 60) to in vitro germination medium. Both treatments led to a severe decrease in the pollen germination rate and elongation. Furthermore, using monoclonal antibodies toward methyl-esterified and de-esterified HGA epitopes, it was found that exogenous treatment of PME and Polyphenon 60 resulted in the disruption of the distribution patterns of low- and high-methylesterified pectins upon pollen germination and during pollen tube elongation. Eleven PMEs and 13 PME inhibitors (PMEIs) were identified by publicly available transcriptome datasets and their specific expression was validated by qRT-PCR. Enzyme activity assays and subcellular localization using a heterologous expression system in tobacco leaves demonstrated that some of the pollen-specific PMEs and PMEIs possessed distinct enzymatic activities and targeted either the cell wall or other compartments. Taken together, our findings are the first line of evidence showing the essentiality of HGA methyl-esterification status during the germination and elongation of pollen tubes in rice, which is primarily governed by the fine-tuning of PME and PMEI activities.

MYB81, a microspore-specific GAMYB transcription factor, promotes pollen mitosis I and cell lineage formation in Arabidopsis.

Sexual reproduction in flowering plants relies on the production of haploid gametophytes that consist of germline and supporting cells. During male gametophyte development, the asymmetric mitotic division of an undetermined unicellular microspore segregates these two cell lineages. To explore genetic regulation underlying this process, we screened for pollen cell patterning mutants and isolated the heterozygous myb81-1 mutant that sheds ~50% abnormal pollen. Typically, myb81-1 microspores fail to undergo pollen mitosis I (PMI) and arrest at polarized stage with a single central vacuole. Although most myb81-1 microspores degenerate without division, a small fraction divides at later stages and fails to acquire correct cell fates. The myb81-1 allele is transmitted normally through the female, but rarely through pollen. We show that myb81-1 phenotypes result from impaired function of the GAMYB transcription factor MYB81. The MYB81 promoter shows microspore-specific activity and a MYB81-RFP fusion protein is only expressed in a narrow window prior to PMI. Ectopic expression of MYB81 driven by various promoters can severely impair vegetative or reproductive development, reflecting the strict microspore-specific control of MYB81. Our data demonstrate that MYB81 has a key role in the developmental progression of microspores, enabling formation of the two male cell lineages that are essential for sexual reproduction in Arabidopsis.

PACLOBUTRAZOL-RESISTANCE Gene Family Regulates Floral Organ Growth with Unequal Genetic Redundancy in Arabidopsis thaliana.

A PACLOBUTRAZOL-RESISTANCE (PRE) gene family, consisting of six genes in Arabidopsis thaliana, encodes a group of helix-loop-helix proteins that act in the growth-promoting transcriptional network. To delineate the specific role of each of the PRE genes in organ growth, we took a reverse genetic approach by constructing high order pre loss-of-function mutants of Arabidopsis thaliana. In addition to dwarf vegetative growth, some double or high order pre mutants exhibited defective floral development, resulting in reduced fertility. While pre2pre5 is normally fertile, both pre2pre6 and pre5pre6 showed reduced fertility. Further, the reduced fertility was exacerbated in the pre2pre5pre6 mutant, indicative of the redundant and critical roles of these PREs. Self-pollination assay and scanning electron microscopy analysis showed that the sterility of pre2pre5pre6 was mainly ascribed to the reduced cell elongation of anther filament, limiting access of pollens to stigma. We found that the expression of a subset of flower-development related genes including ARGOS, IAA19, ACS8, and MYB24 was downregulated in the pre2pre5pre6 flowers. Given these results, we propose that PREs, with unequal functional redundancy, take part in the coordinated growth of floral organs, contributing to successful autogamous reproduction in Arabidopsis thaliana.

Application of rice microspore-preferred promoters to manipulate early pollen development in Arabidopsis: a heterologous system.

KEY MESSAGE: Rice microspore-promoters. Based on microarray data analyzed for developing anthers and pollen grains, we identified nine rice microspore-preferred (RMP) genes, designated RMP1 through RMP9. To extend their biotechnological applicability, we then investigated the activity of RMP promoters originating from monocotyledonous rice in a heterologous system of dicotyledonous Arabidopsis. Expression of GUS was significantly induced in transgenic plants from the microspore to the mature pollen stages and was driven by the RMP1, RMP3, RMP4, RMP5, and RMP9 promoters. We found it interesting that, whereas RMP2 and RMP6 directed GUS expression in microspore at the early unicellular and bicellular stages, RMP7 and RMP8 seemed to be expressed at the late tricellular and mature pollen stages. Moreover, GUS was expressed in seven promoters, RMP3 through RMP9, during the seedling stage, in immature leaves, cotyledons, and roots. To confirm microspore-specific expression, we used complementation analysis with an Arabidopsis male-specific gametophytic mutant, sidecar pollen-2 (scp-2), to verify the activity of three promoters. That mutant shows defects in microspore development prior to pollen mitosis I. These results provide strong evidence that the SIDECAR POLLEN gene, driven by RMP promoters, successfully complements the scp-2 mutation, and they strongly suggest that these promoters can potentially be applied for manipulating the expression of target genes at the microspore stage in various species.

Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage.

Microspore production using endogenous developmental programs has not been well studied. The main limitation is the difficulty in identifying genes preferentially expressed in pollen grains at early stages. To overcome this limitation, we collected transcriptome data from anthers and microspore/pollen and performed meta-expression analysis. Subsequently, we identified 410 genes showing preferential expression patterns in early developing pollen samples of both japonica and indica cultivars. The expression patterns of these genes are distinguishable from genes showing pollen mother cell or tapetum-preferred expression patterns. Gene Ontology enrichment and MapMan analyses indicated that microspores in rice are closely linked with protein degradation, nucleotide metabolism, and DNA biosynthesis and regulation, while the pollen mother cell or tapetum are strongly associated with cell wall metabolism, lipid metabolism, secondary metabolism, and RNA biosynthesis and regulation. We also generated transgenic lines under the control of the promoters of eight microspore-preferred genes and confirmed the preferred expression patterns in plants using the GUS reporting system. Furthermore, cis-regulatory element analysis revealed that pollen specific elements such as POLLEN1LELAT52, and 5659BOXLELAT5659 were commonly identified in the promoter regions of eight rice genes with more frequency than estimation. Our study will provide new sights on early pollen development in rice, a model crop plant.

Analysis of gemini pollen 3 mutant suggests a broad function of AUGMIN in microtubule organization during sexual reproduction in Arabidopsis.

In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by two rounds of mitotic division. Haploid microspores undergo polar nuclear migration and asymmetric division at pollen mitosis I to segregate the male germline, followed by division of the germ cell to generate a pair of sperm cells. We previously reported two gemini pollen (gem) mutants that produced twin-celled pollen arising from polarity and cytokinesis defects at pollen mitosis I in Arabidopsis. Here, we report an independent mutant, gem3, with a similar division phenotype and severe genetic transmission defects through pollen. Cytological analyses revealed that gem3 disrupts cell division during male meiosis, at pollen mitosis I and during female gametophyte development. We show that gem3 is a hypomorphic allele (aug6-1) of AUGMIN subunit 6, encoding a conserved component in the augmin complex, which mediates microtubule (MT)-dependent MT nucleation in acentrosomal cells. We show that MT arrays are disturbed in gem3/aug6-1 during male meiosis and pollen mitosis I using fluorescent MT-markers. Our results demonstrate a broad role for the augmin complex in MT organization during sexual reproduction, and highlight gem3/aug6-1 mutants as a valuable tool for the investigation of augmin-dependent MT nucleation and dynamics in plant cells.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD)10 interacts with SIDECAR POLLEN/LBD27 to control pollen development in Arabidopsis.

During male gametophyte development in Arabidopsis thaliana, the microspores undergo an asymmetric division to produce a vegetative cell and a generative cell, which undergoes a second division to give rise to two sperm cells. SIDECAR POLLEN/LATERAL ORGAN BOUNDARIES DOMAIN (LBD) 27 plays a key role in the asymmetric division of microspores. Here we provide molecular genetic evidence that a combinatorial role of LBD10 with LBD27 is crucial for male gametophyte development in Arabidopsis. Expression analysis, genetic transmission and pollen viability assays, and pollen development analysis demonstrated that LBD10 plays a role in the male gametophyte function primarily at germ cell mitosis. In the mature pollen of lbd10 and lbd10 expressing a dominant negative version of LBD10, LBD10:SRDX, aberrant microspores such as bicellular and smaller tricellular pollen appeared at a ratio of 10-15% with a correspondingly decreased ratio of normal tricellular pollen, whereas in lbd27 mutants, 70% of the pollen was aborted. All pollen in the lbd10 lbd27 double mutants was aborted and severely shrivelled compared with that of the single mutants, indicating that LBD10 and LBD27 are essential for pollen development. Gene expression and subcellular localization analyses of LBD10:GFP and LBD27:RFP during pollen development indicated that posttranscriptional and/or posttranslational controls are involved in differential accumulation and subcellular localization of LBD10 and LBD27 during pollen development, which may contribute in part to combinatorial and distinct roles of LBD10 with LBD27 in microspore development. In addition, we showed that LBD10 and LBD27 interact to form a heterodimer for nuclear localization.

Evaluation of rice promoters conferring pollen-specific expression in a heterologous system, Arabidopsis.

Promoters can direct gene expression specifically to targeted tissues or cells. Effective with both crop species and model plant systems, these tools can help researchers overcome the practical obstacles associated with transgenic protocols. Here, we identified promoters that allow one to target the manipulation of gene expression during pollen development. Utilizing published transcriptomic databases for rice, we investigated the promoter activity of selected genes in Arabidopsis. From various microarray datasets, including those for anthers and pollen grains at different developmental stages, we selected nine candidate genes that showed high levels of expression in the late stages of rice pollen development. We named these Oryza sativa late pollen-specific genes. Their promoter regions contained various cis-acting elements that could be responsible for anther-/pollen-specific expression. Promoter::GUS-GFP reporters were constructed and introduced into Arabidopsis plants. Histochemical GUS staining revealed that six of the nine rice promoters conferred strong GUS expression that was restricted to the anthers in Arabidopsis. Further analysis showed that although the GUS signals were not detected at the unicellular stage, they strengthened in the bicellular or tricellular stages, peaking at the mature pollen stage. This paralleled their transcriptomic profiles in rice. Based on our results, we proposed that these six rice promoters, which are active in the late stages of pollen formation in the dicot Arabidopsis, can aid molecular breeders in generating new varieties of a monocot plant, rice.

Arabidopsis Fused kinase TWO-IN-ONE dominantly inhibits male meiotic cytokinesis.

Arabidopsis Fused kinase TWO-IN-ONE (TIO) controls phragmoplast expansion through its interaction with the Kinesin-12 subfamily proteins that anchor the plus ends of interdigitating microtubules in the phragmoplast midzone. Previous analyses of loss-of-function mutants and RNA interference lines revealed that TIO positively controls both somatic and gametophytic cell cytokinesis; however, knowledge of the full spectrum of TIO functions during plant development remains incomplete. To characterize TIO functions further, we expressed TIO and a range of TIO variants under control of the TIO promoter in wild-type Arabidopsis plants. We discovered that TIO-overexpressing transgenic lines produce enlarged pollen grains, arising from incomplete cytokinesis during male meiosis, and show sporophytic abnormalities indicative of polyploidy. These phenotypes arose independently in TIO variants in which either gametophytic function or the ability of TIO to interact with Kinesin-12 subfamily proteins was abolished. Interaction assays in yeast showed TIO to bind to the AtNACK2/TETRASPORE, and plants doubly homozygous for kinesin-12a and kinesin-12b knockout mutations to produce enlarged pollen grains. Our results show TIO to dominantly inhibit male meiotic cytokinesis in a dosage-dependent manner that may involve direct binding to a component of the canonical NACK-PQR cytokinesis signaling pathway.

Arabidopsis Fused kinase and the Kinesin-12 subfamily constitute a signalling module required for phragmoplast expansion.

The conserved Fused kinase plays vital but divergent roles in many organisms from Hedgehog signalling in Drosophila to polarization and chemotaxis in Dictyostelium. Previously we have shown that Arabidopsis Fused kinase termed TWO-IN-ONE (TIO) is essential for cytokinesis in both sporophytic and gametophytic cell types. Here using in vivo imaging of GFP-tagged microtubules in dividing microspores we show that TIO is required for expansion of the phragmoplast. We identify the phragmoplast-associated kinesins, PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B, as TIO-interacting proteins and determine TIO-Kinesin-12 interaction domains and their requirement in male gametophytic cytokinesis. Our results support the role of TIO as a functional protein kinase that interacts with Kinesin-12 subfamily members mainly through the C-terminal ARM repeat domain, but with a contribution from the N-terminal kinase domain. The interaction of TIO with Kinesin proteins and the functional requirement of their interaction domains support the operation of a Fused kinase signalling module in phragmoplast expansion that depends upon conserved structural features in diverse Fused kinases.

Endoplasmic reticulum- and Golgi-localized phospholipase A2 plays critical roles in Arabidopsis pollen development and germination.

The phospholipase A(2) (PLA(2)) superfamily of lipolytic enzymes is involved in a number of essential biological processes, such as inflammation, development, host defense, and signal transduction. Despite the proven involvement of plant PLA(2)s in many biological functions, including senescence, wounding, elicitor and stress responses, and pathogen defense, relatively little is known about plant PLA(2)s, and their genes essentially remain uncharacterized. We characterized three of four Arabidopsis thaliana PLA(2) paralogs (PLA(2)-ß, -γ, and -δ) and found that they (1) are expressed during pollen development, (2) localize to the endoplasmic reticulum and/or Golgi, and (3) play critical roles in pollen development and germination and tube growth. The suppression of PLA(2) using the RNA interference approach resulted in pollen lethality. The inhibition of pollen germination by pharmacological PLA(2) inhibitors was rescued by a lipid signal molecule, lysophosphatidyl ethanolamine. Based on these results, we propose that plant reproduction, in particular, male gametophyte development, requires the activities of the lipid-modifying PLA(2)s that are conserved in other organisms.

Antioxidant activity and inhibition of lipid peroxidation in germinating seeds of transgenic soybean expressing OsHGGT.

Tocochromanols are potent lipid-soluble antioxidants and essential nutrients for human health. Genetic engineering techniques were used to develop soybeans with enhanced vitamin E levels, including tocotrienols, which are not found in soybean. The gene encoding rice homogentisate geranylgeranyl transferase (HGGT) was overexpressed in soybeans using seed-specific and constitutive promoters. The association between abundance of vitamin E isomers and antioxidant activity was investigated during seed germination. With the exception of ß-tocotrienol, all vitamin E isomers were detected in germinating seeds expressing OsHGGT. The antioxidant properties of germinating seed extracts were determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radicals and lipid peroxidation (TBARS). Compared with intact wild-type seeds, transgenic seeds showed increases in radical scavenging of 5.4-17 and 23.2-35.3% in the DPPH and ABTS assays, respectively. Furthermore, the lipid peroxidation levels were 2.0-4.5-fold lower in germinating seeds from transgenic lines than in wild-type seeds. Therefore, it appears that the antioxidant potential of transgenic oil-producing plants such as soybean, sunflower, and corn may be enhanced by overexpressing OsHGGT during seed germination.

The SIDECAR POLLEN gene encodes a microspore-specific LOB/AS2 domain protein required for the correct timing and orientation of asymmetric cell division.

Cellular patterning and differentiation in plants depend on the balance of asymmetric and symmetric divisions. Patterning of the male gametophyte (pollen grains) in flowering plants requires asymmetric division of the microspore followed by a symmetric division of the germ cell to produce three highly differentiated cells: a single vegetative cell and two sperm cells. In Arabidopsis sidecar pollen (scp) mutants a proportion of microspores first divide symmetrically, and then go on to produce 'four-celled' pollen with an extra vegetative cell; however, details of the timing and origin of phenotypic defects in scp and the identity of the SCP gene have remained obscure. Comparative analysis of the original hypomorphic scp-1 allele and a T-DNA-induced null allele, scp-2, revealed that in the absence of SCP, microspores undergo normal nuclear positioning, but show delayed entry into mitosis, increased cell expansion and alterations in the orientation of nuclear division. We identified the SCP gene to encode a male gametophyte-specific LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES 2-like (LBD/ASL) protein that is expressed in microspore nuclei in a tightly regulated phase-specific manner. Therefore, our study demonstrates that the correct patterning of male gametophyte depends on the activity of a nuclear LBD/ASL family protein that is essential for the correct timing and orientation of asymmetric microspore division.

The tobacco MAP215/Dis1-family protein TMBP200 is required for the functional organization of microtubule arrays during male germline establishment.

The haploid microspore division during pollen development in flowering plants is an intrinsically asymmetric division which establishes the male germline for sexual reproduction. Arabidopsis gem1 mutants lack the male germline as a result of disturbed microspore polarity, division asymmetry, and cytokinesis and represent loss-of-function mutants in MOR1/GEM1, a plant orthologue of the conserved MAP215/Dis1 microtubule associated protein (MAP) family. This provides genetic evidence for the role of MAP215/Dis1 in the organization of gametophytic microtubule arrays, but it has remained unknown how microtubule arrays are affected in gem1 mutant microspores. Here, novel male gametophytic microtubule-reporter Nicotiana tabacum plants were constructed, expressing a green fluorescent protein-alpha-TUBULIN fusion protein (GFP-TUA6) under the control of a microspore-specific promoter. These plants allow effective visualization of all major male gametophytic microtubule arrays and provide useful tools to study the regulation of microtubule arrays by MAPs and other effectors. Depletion of TMBP200, a tobacco homologue of MOR1/GEM1 in gametophytic microtubule-reporter plants using microspore-targeted RNA interference, induced defects in microspore polarity, division asymmetry and cytokinesis that were associated with striking defects in phragmoplast position, orientation, and structure. Our observations further reveal a requirement for TMBP200 in gametophytic spindle organization and a novel role in spindle position and orientation in polarized microspores. These results provide direct evidence for the function of MAP215/Dis1 family protein TMBP200 in the organization of microtubule arrays critical for male germline formation in plants.

A genotype-specific pollen gene associated with self-incompatibility in Lycopersicon peruvianum.

Self-incompatibility is a genetically controlled process used to prevent self-pollination. We report here the characterization of pollen cDNA clones of Lycopersicon peruvianum, and the identification of a genotype-specific pollen factor involved in self-incompatibility. To identify the latter, differential mRNA display RT-PCR was performed on pollen cDNAs from S12Sa and S11Sa genotypes. We isolated four cDNA fragments expressed preferentially in S12Sa pollen, and screened a cDNA library from S12Sa pollen with the four cDNA fragments to isolate the corresponding full length cDNAs. One of the four isolated cDNAs encoded part of an actin depolymerizing factor protein that we named LpADF. LpADF is highly homologous to actin depolymerizing factors of Arabidopsis thaliana, Lilium longiflorum, and Zea mays. RNA blot analysis revealed that LpADF is only expressed in mature pollen of the S12Sa genotype, and is therefore a candidate pollen factor in the gametophyte self-incompatibility system of L. peruvianum.