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MOR1 is a member of the MAP215 family of microtubule-associated proteins and is required to establish interphase arrays of cortical microtubules in plant cells1. Here we show that MOR1 binds microtubules in vivo, localizing to both cortical microtubules and to areas of overlapping microtubules in the phragmoplast. Genetic complementation of the cytokinesis-defective gemini pollen 1-1 (gem1-1) mutation with MOR1 shows that MOR1 (which is synonymous with the protein GEM1) is essential in cytokinesis. Phenotypic analysis of gem1-1 and gem1-2, which contains a T-DNA insertion, confirm that MOR1/GEM1 is essential for regular patterns of cytokinesis. Both the gem1-1 and gem1-2 mutations cause the truncation of the MOR1/GEM1 protein. In addition, the carboxy-terminal domain of the protein, which is absent in both mutants, binds microtubules in vitro. Our data show that MOR1/GEM1 has an essential role in the cytokinetic phragmoplast.

1-Aminocyclopropane-1-carboxylic acid (ACC) is a biosynthetic precursor of ethylene, a gaseous plant hormone which controls a myriad of aspects of development and stress adaptation in higher plants. Here, we identified a mutant in Arabidopsis thaliana, designated as ACC-resistant2 (are2), displaying a dose-dependent resistance to exogenously applied ACC. Physiological analyses revealed that mutation of are2 impaired various aspects of exogenous ACC-induced ethylene responses, while not affecting sensitivity to other plant hormones during seedling development. Interestingly, the are2 mutant was normally sensitive to gaseous ethylene, compared with the wild type. Double mutant analysis showed that the ethylene-overproducing mutations, eto1 or eto3, and the constitutive ethylene signaling mutation, ctr1 were epistatic to the are2 mutation. These results suggest that the are2 mutant is not defective in ethylene biosynthesis or ethylene signaling per se. Map-based cloning of ARE2 demonstrated that LYSINE HISTIDINE TRANSPORTER1 (LHT1), encoding an amino acid transporter, is the gene responsible. An uptake experiment with radiolabeled ACC indicated that mutations of LHT1 reduced, albeit not completely, uptake of ACC. Further, we performed an amino acid competition assay and found that two amino acids, alanine and glycine, known as substrates of LHT1, could suppress the ACC-induced triple response in a LHT1-dependent way. Taken together, these results provide the first molecular genetic evidence supporting that a class of amino acid transporters including LHT1 takes part in transport of ACC, thereby influencing exogenous ACC-induced ethylene responses in A. thaliana.

The nature of aberrant gametophytic cell divisions and altered pollen cell fate in the gemini pollen1(gem1) mutant was investigated through ultrastructural analysis. The earliest noticeable defect in gem1 was the appearance of extended membrane profiles at the early bicellular stage. These were replaced by ectopic internal walls, which divided the cytoplasm into twin or multiple cell compartments. Complete or partial internal walls were callosic with highly complex profiles, indicating failed guidance or deregulated cell plate growth. Extended membrane profiles and delayed callose synthesis at division sites further suggested a novel pattern of cell plate assembly ingem1. Multiple cell compartments in gem1adopted vegetative cell fate with regard to lipid body distribution. In the wild type, lipid bodies appear specifically in the vegetative cell, whereas in gem1, lipid bodies accumulated in all cytoplasmic compartments. Our results support the hypothesis that altered pollen cell fate in gem1 results from abnormal inheritance of cell fate determinants as a result of disturbed cytokinesis.

Pollen development and male gametogenesis are critically dependent upon cell polarization leading to a highly asymmetric cell division termed pollen mitosis I. A mutational approach was adopted in Arabidopsis thaliana to identify genes involved these processes. Four independent gemini pollen mutants were isolated which produce divided or twin-celled pollen. The gemini pollen1 mutant was characterized in detail and shown to act gametophytically resulting in reduced transmission through both sexes. gemini pollen1 showed an incompletely penetrant phenotype resulting in equal, unequal and partial divisions at pollen mitosis I. The division planes in gemini pollen1 were shown to be aligned with the polar axis (as in wild type) and evidence was obtained for incomplete nuclear migration, which could account for altered division symmetry. gemini pollen1 also showed division phenotypes consistent with spatial uncoupling of karyokinesis and cytokinesis suggesting that GEMINI POLLEN1 may be required for the localization of phragmoplast activity. Cell fate studies showed that in both equal and unequal divisions a vegetative cell marker gene was activated in both daughter cells. Daughter cells with a range of intermediate or hybrid vegetative/ generative cell fates suggests that cell fate is quantitatively related to cell size. The potential mode of action of GEMINI POLLEN1 and its effects on cell fate are discussed in relation to proposed models of microspore polarity and cell fate determination.

The first mitotic division of the haploid microspore in the pathway of pollen development is a striking example of an asymmetric division that leads to different fates in the daughter cells. Recently, new insight into microspore polarity and cell-fate determination in pollen has been obtained in Arabidopsis by applying cell-fate markers in conjunction with a mutational approach. These studies suggest that development into a vegetative cell is the default programme, but that this is repressed in the generative cell as a result of division asymmetry. Gametophytic mutants that affect cell division, division asymmetry and cell fate have highlighted the importance of asymmetrically localized factors in microspore polarity and cell-fate determination in pollen.

Four mutants that show the delayed leaf senescence phenotype were isolated from Arabidopsis thaliana. Genetic analyses revealed that they are all monogenic recessive mutations and fall into three complementation groups, identifying three genetic loci controlling leaf senescence in Arabidopsis. Mutations in these loci cause delay in all senescence parameters examined, including chlorophyll content, photochemical efficiency of photosystem II, relative amount of the large subunit of Rubisco, and RNase and peroxidase activity. Delay of the senescence symptoms was observed during both age-dependent in planta senescence and dark-induced artificial senescence in all of the mutant plants. The results indicate that the three genes defined by the mutations are key genetic elements controlling functional leaf senescence and provide decisive genetic evidence that leaf senescence is a genetically programmed phenomenon controlled by several monogenic loci in Arabidopsis. The results further suggest that the three genes function at a common step of age-dependent and dark-induced senescence processes. It is further shown that one of the mutations is allelic to ein2-1, an ethylene-insensitive mutation, confirming the role of ethylene signal transduction pathway in leaf senescence of 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.

Ethylene controls myriad aspects of plant growth throughout developmental stages in higher plants. It has been well established that ethylene-responsive growth entails extensive crosstalk with other plant hormones, particularly auxin. Here, we report a genetic mutation, named 1-aminocyclopropane carboxylic acid (ACC) resistant root1-1 (are1-1) in Arabidopsis thaliana (L.) Heynh. The CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) encodes a Raf-related protein, functioning as an upstream negative regulator of ethylene signaling in Arabidopsis thaliana. We found that the ctr1-1, a kinase-inactive allele exhibited slightly, but significantly, longer root length, compared to ACC-treated wild-type or ctr1-3, a null allele. Our genetic studies unveiled the existence of are1-1 mutation in the ctr1-1 mutant, as a second-site modifier which confers root-specific ethylene-resistance. Based on well-characterized crosstalk between ethylene and auxin during ethylene-responsive root growth, we performed various physiological analyses. Whereas are1-1 displayed normal sensitivity to synthetic auxins, it showed modest resistance to an auxin transport inhibitor, 1-N-naphthylphthalamic acid. In addition, are1-1 mutant exhibited ectopically altered DR5:GUS activity upon ethylene-treatment. The results implicated the involvement of are1-1 in auxin-distribution, but not in auxin-biosynthesis, -uptake, or -sensitivity. In agreement, are1-1 mutant exhibited reduced gravitropic root growth and defective redistribution of DR5:GUS activity upon gravi-stimulation. Taken together with genetic and molecular analysis, our results suggest that ARE1 defines a novel locus to control ethylene-responsive root growth as well as gravitropic root growth presumably through auxin distribution in Arabidopsis thaliana.

TGF-β1 plays biphasic functions in prostate tumorigenesis, inhibiting cell growth at early stages but promoting malignant progression at later stages. However, the molecular basis for the oncogenic conversion of TGF-β1 function remains largely undefined. Here, we demonstrate that PPARδ is a direct transcription target of TGF-β1 and plays a critical role in oncogenic redirection of TGF-β1 signaling. Blockade of PPARδ induction enhances tumor cell response to TGF-β1-mediated growth inhibition, while its activation promotes TGF-β1-induced tumor growth, migration and invasion. PPARδ-mediated switch of TGF-β1 function is associated with down- and upregulation of Smad and ERK signaling, respectively, and tightly linked to its function to activate ABCA1 cholesterol transporter followed by caveolin-1 (Cav1) induction. Intriguingly, TGF-β1 activation of the PPARδ-ABCA1-Cav1 pathway facilitates degradation of TGF-β receptors (TβRs) and attenuates Smad but enhances ERK response to TGF-β1. Expression of PPARδ and Cav1 is tightly correlated in both prostate tissues and cell lines and significantly higher in cancer vs. normal tissues. Collectively, our study shows that PPARδ is a transcription target of TGF-β1 and contributes to the oncogenic conversion of TGF-β1 function through activation of the ABCA1-Cav1-TβR signaling axis.

Flowering on time is a critically important for successful reproduction of plants. Here we report an early-flowering mutant in Arabidopsis thaliana, accelerated flowering 1-1D (afl1-1D) that exhibited pleiotropic developmental defects including semi-dwarfism, curly leaf, and increased branching. Genetic analysis showed that afl1-1D mutant is a single, dominant mutant. Chromosomal mapping indicates that AFL1 resides at the middle of chromosome 4, around which no known flowering-related genes have been characterized. Expression analysis and double mutant studies with late flowering mutants in various floral pathways indicated that elevated FT is responsible for the early-flowering of afl1-1D mutant. Interestingly, not only flowering-related genes, but also several floral homeotic genes were ectopically overexpressed in the afl1-1D mutants in both FT-dependent and -independent manner. The degree of histone H3 Lys27-trimethylation (H3K27me3) was reduced in several chromatin including FT, FLC, AG and SEP3 in the afl1-1D, suggesting that afl1-1D might be involved in chromatin modification. In support, double mutant analysis of afl1-1D and lhp1-4 revealed epistatic interaction between afl1-1D and lhp1-4 in regard to flowering control. Taken together, we propose that AFL1 regulate various aspect of development through chromatin modification, particularly associated with H3K27me3 in A. thaliana.

We have isolated a male-sterile mutant from a pool of T-DNA insertional lines of Arabidopsis thaliana generated by an in planta transformation procedure [Chang et al. (1994) Plant J. 5: 551]. Pollen in this mutant is not effectively released from anther locules after cleavage of the stomium. Most mutant pollen grains are round, in contrast to the tricolpate wild-type pollen, and some pollen grains show an abnormal surface structure. Manually released mutant pollen grains are not fertile and show defects in pollen tube germination in vitro. Genetic analysis disclosed that this lesion is due to a single recessive nuclear mutation located on chromosome 3 closely linked to the gll locus. The mutation locus is tightly linked to the inserted T-DNA.

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.

Rice stripe virus (RSV) is a viral disease that seriously impacts rice production in East Asia, most notably in Korea, China, and Japan. Highly RSV-resistant transgenic japonica rice plants were generated using a dsRNAi construct designed to silence the entire sequence region of the RSV-CP gene. Transgenic rice plants were inoculated with a population of viruliferous insects, small brown planthoppers (SBPH), and their resistance was evaluated using ELISA and an infection rate assay. A correlation between the expression of the RSV-CP homologous small RNAs and the RSV resistance of the transgenic rice lines was discovered. These plants were also analyzed by comparing the expression pattern of invading viral genes, small RNA production and the stable transmission of the RSV resistance trait to the T3 generation. Furthermore, the agronomic trait was stably transmitted to the T4 generation of transgenic plants.

Asymmetric cell division is a universal strategy to generate diverse cell types necessary for patterning and proliferation of all eukaryotes. The development of haploid male gametophytes (pollen grains) in flowering plants is a remarkable example in which division asymmetry governs the functional specialization and germline differentiation essential for double fertilization. The male gametophyte is patterned via two mitotic divisions resulting in three highly differentiated daughter cells at maturity, a vegetative cell and two sperm cells. The first asymmetric division segregates a unique male germ cell from an undetermined haploid microspore and is executed in an elaborate sequence of cellular events. However the molecular mechanisms governing the division asymmetry in microspores are poorly understood. Recently we studied the phenotype of sidecar pollen (scp) mutants in detail, and demonstrated a requirement of SCP for both the correct timing and orientation of microspore division. SCP is a microspore-specific member of the LOB/AS2 domain family (LBD27/ASL29) showing that a plant-specific regulator plays a key role in oriented division of polarized microspores. Identification of SCP will serve as a new platform to further explore the largely unknown molecular networks regulating division asymmetry in microspores that establishes the male germline in flowering plants.

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 β-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.

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.

Ethylene-responsive factors (ERFs), within a subgroup of the AP2/ERF transcription factor family, are involved in diverse plant reactions to biotic or abiotic stresses. Here, we report that overexpression of an ERF gene from Brassica rapa ssp. pekinensis (BrERF4) led to improved tolerance to salt and drought stresses in Arabidopsis. It also significantly affected the growth and development of transgenic plants. We detected that salt-induced expressions of a transcriptional repressor gene, AtERF4, and some Ser/Thr protein phosphatase2C genes, ABI1, ABI2 and AtPP2CA, were suppressed in BrERF4-overexpressing Arabidopsis plants. Furthermore, BrERF4 was induced by treatment with ethylene or methyljasmonate, but not by abscisic acid or NaCl in B. rapa. These results suggest that BrERF4 is activated through a network of different signaling pathways in response to salinity and drought.

BACKGROUND: Recently, it was reported that the 4-week exposure of rats to a concrete building environment under cool temperatures (11.7–12.1°C) had adverse effects on the general health parameters. This study examined the potential effects of concrete and hwangto building environments on pregnant dams and embryo-fetal development in Sprague-Dawley rats. METHODS: Groups of 10 mated females were exposed to polycarbonate (control), concrete, or hwangto cages from gestational days (GD) 0 to 20 under cool temperatures (11.9–12.5°C). All the females underwent a caesarean section on GD 20, and their fetuses were examined for any morphological abnormalities. RESULTS: The temperatures in the cages were similar in all groups but the relative humidity in the concrete and hwangto groups were higher than in the control group. The concentration of volatile organic compounds in the hwangto group during the study period was lower than in the control group. In the concrete group, maternal effects manifested as an increase in the incidence of clinical signs, a lower body weight, and a decrease in the thymus and ovary weights. Developmental effects included increased post-implantation loss and decreased litter size. In contrast, in the hwangto group, there were no exposure-related adverse effects on the maternal and developmental parameters. CONCLUSIONS: Overall, the exposure of pregnant rats to a concrete building environment under cool temperatures has adverse effects on the clinical signs, body weight, organ weight, and embryo-fetal development. On the other hand, exposure to a hwangto building environment does not have any adverse effects on pregnant dams or on embryo-fetal development in rats. Birth Defects Res (Part B), 2008. © 2008 Wiley-Liss, Inc.

This study investigated the potential adverse effects of tert-butyl acetate (TBAc) on maternal toxicity and embryo-fetal development after maternal exposure of pregnant rats from gestational days 6 through 19. TBAc was administered to pregnant rats by gavage at 0, 400, 800, and 1,600 mg/kg/day. All dams were subjected to a Caesarean section on day 20 of gestation, and their fetuses were examined for any morphological abnormalities. At 1,600 mg/kg, maternal toxicity manifested as increases in the incidence of clinical signs and death, lower body weight gain and food intake, increases in the weights of adrenal glands and liver, and a decrease in thymus weight. Developmental toxicity included a decrease in fetal weight, an increase in the incidence of skeletal variation, and a delay in fetal ossification. At 800 mg/kg, only a minimal developmental toxicity, including an increase in the incidence of skeletal variation and a delay in fetal ossification, were observed. In contrast, no adverse maternal or developmental effects were observed at 400 mg/kg. These results show that a 14-day repeated oral dose of TBAc is embryotoxic at a maternally toxic dose (i.e., 1,600 mg/kg/day) and is minimally embryotoxic at a nonmaternally toxic dose (i.e., 800 mg/kg/day) in rats. However, no evidence for the teratogenicity of TBAc was noted in rats. It is concluded that the developmental findings observed in the present study are secondary effects to maternal toxicity. Under these experimental conditions, the no-observed-adverse-effect level of TBAc is considered to be 800 mg/kg/day for dams and 400 mg/kg/day for embryo-fetal development.

This study was carried out to investigate the potential subchronic inhalation toxicity of dimethyl disulfide (DMDS) via whole-body exposure in F344 rats. Groups of 10 rats of each sex were exposed to DMDS vapor by whole-body exposure at concentrations of 0, 5, 25, or 125 ppm for 6 h/day, 5 days/wk for 13 wk. All the rats were sacrificed at the end of treatment period. During the test period, clinical signs, mortality, body weights, food consumption, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross findings, organ weights, and histopathology were examined. At 25 ppm, a decrease in the body weight gain, food intake, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) was observed in the males, but not in the females. However, at 125 ppm, a decrease in the body weight gain, food intake, and thymus weight and an increase in the weights of adrenal glands were observed in both genders. Serum biochemical investigations revealed a decrease in the AST, ALT, BUN, creatine phosphokinase (CPK), and triglyceride levels and an increase in the glucose level. In contrast, no treatment-related effects were observed in the 5 ppm group. The toxic potency of DMDS was slightly higher in males than that in females. In these experimental conditions, the target organ was not determined in rats. The no-observed-adverse-effect concentration (NOAEC) was found to be 5 ppm, 6 h/day for male rats and 25 ppm, 6 h/day for female rats.

Summary The FUSED (FU) Ser/Thr protein kinase family has a key role in the hedgehog signaling pathway known to control cell proliferation and patterning in fruit flies and humans [1, 2]. The genomes of Arabidopsis thaliana and rice each encode a single Fu ortholog, but their role is unknown. Here, we show that cytokinesis-defective mutants, which we named two-in-one ( tio ), result from mutations in Arabidopsis Fu . Phenotypic analysis of tio mutants reveals an essential role for TIO in conventional modes of cytokinesis in plant meristems and during male gametogenesis. TIO also has a key role in nonconventional modes of cytokinesis (cellularization) during female gametogenesis. We demonstrate that TIO is tightly localized to the midline of the nascent phragmoplast and remains associated with the expanding phragmoplast ring. These data reveal the evolution of a divergent role for the Fu kinase family as an essential phragmoplast-associated protein that functions in different cell type-specific modes of cytokinesis in plants.

Aim: To determine whether in utero and neonatal exposure to a 60 Hz extremely low frequency electromagnetic field (EMF) results in spermatotoxicity and reproductive dysfunction in the F1 offspring of rats. Methods: Age-matched, pregnant Sprague–Dawley rats were exposed continuously (21 h/day) to a 60 Hz EMF at field strengths of 0 (sham control), 5, 83.3 or 500 µT from day 6 of gestation through to day 21 of lactation. The experimentally generated magnetic field was monitored continuously (uninterrupted monitoring over the period of the study) throughout the study. Results: No exposure-related changes were found in exposed or sham-exposed animals with respect to the anogenital distance, preputial separation, testis weight, testicular histology, sperm count, daily sperm production, sperm motility, sperm morphology and reproductive capacity of F1 offspring. Conclusion: Exposure of Sprague– Dawley rats to a 60 Hz EMF at field strengths of up to 500 µT from day 6 of gestation to day 21 of lactation did not produce any detectable alterations in offspring spermatogenesis and fertility. (Asian J Androl 2005 Jun; 7: 189–194)

Gametophytic cytokinesis is essential for the development and function of the male and female gametophytes. We have previously described the isolation and characterisation of the gemini pollen 1 (gem1) that acts gametophytically to disturb asymmetric division and cytokinesis at pollen mitosis I in Arabidopsis. Here we describe the genetic and cytological analysis of an independent gametophytic mutant, gem2, with similar characteristics to gem1, but which maps to a different genetic locus. gem2 shows reduced genetic transmission through both male and female gametes and leads to the production of divided or twin-celled pollen. Developmental analysis revealed that gem2 does not affect karyokinesis at pollen mitosis I, but leads to repositioning of the cell plate and partial or complete failure of cytokinesis, resulting in symmetrical divisions or binucleate pollen grains respectively. Symmetrical divisions lead to altered pollen cell fate with both sister cells displaying vegetative cell fate. Moreover, we demonstrate that the predominant female defect in gem2 is a lack of cellularization of the embryo sac during megagametogenesis. GEM2 therefore defines an independent genetic locus that is involved in the correct specification of both male and female gametophytic cytokinesis.

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.

As a strategy for the identification of T-DNA-tagged gametophytic mutants, we have used T-DNA insertional mutagenesis based on screening for distorted segregation ratios by antibiotic selection. Screening of ~1000 transgenic Arabidopsis families led to the isolation of eight lines showing reproducible segregation ratios of ~1:1, suggesting that these lines are putative gametophytic mutants caused by T-DNA insertion at a single locus. Genetic analysis of T-DNA transmission through reciprocal backcrosses with wild type showed severe reductions in genetic transmission of the T-DNA through the male and/or female gametes. Direct evidence for mutant phenotypes in these lines was investigated by DAPI staining of mature pollen grains and by the analysis of seed set and embryo sac morphology in cleared ovules. One line, termed limpet pollen, showed a novel pollen phenotype in that the generative cell failed to migrate inward after pollen mitosis I, such that the generative or sperm cells remained against the pollen wall. Two other lines, andarta and tistrya, were defective in female transmission and showed an early arrest of embryo sac development with the viable megaspore not initiating the nuclear division cycles. These data demonstrate the efficacy of a segregation ratio distortion strategy for the identification of T-DNA-tagged gametophytic mutants in Arabidopsis.