Five gametophytic mutations affecting pollen development and pollen tube growth in Arabidopsis thaliana

Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development. The screening of the Versailles collection of Arabidopsis thaliana T-DNA insertion transformants has allowed us to isolate different mutations affecting male gametophytic functions and viability. Among several mutated lines, five have been extensively studied at the genetic, molecular, and cytological levels. For each mutant, several generations of selfing and outcrossing have been carried out, leading to the conclusion that all these mutations are tagged and affect only the male gametophyte. However, only one out of the five mutations is completely penetrant. A variable number of T-DNA copies has integrated in the mutant lines, although all segregate at one mutated locus. Two mutants could be defined as "early mutants": the mutated genes are presumably expressed during pollen grain maturation and their alteration leads to the production of nonfunctional pollen grains. Two other mutants could be defined as "late mutant" since their pollen is able to germinate but pollen tube growth is highly disturbed. Screening for segregation ratio distortions followed by thorough genetic analysis proved to be a powerful tool for identifying gametophytic mutations of all phases of pollen development.     

The rice SPINDLY gene functions as a negative regulator of gibberellin signaling by controlling the suppressive function of the DELLA protein, SLR1, and modulating brassinosteroid synthesis

SPINDLY (SPY) encodes an O-linked N-acetylglucosamine transferase that is considered to be a negative regulator of gibberellin (GA) signaling through an unknown mechanism. To understand the function of SPY in GA signaling in rice, we isolated a rice SPINDLY homolog (OsSPY) and produced knockdown transgenic plants in which OsSPY expression was reduced by introducing its antisense or RNAi construct. In knockdown plants, the enhanced elongation of lower internodes was correlated with decreased levels of OsSPY expression, similar to the spindly phenotype of Arabidopsis spy mutants, suggesting that OsSPY also functions as a negative factor in GA signaling in rice. The suppressive function of OsSPY in GA signaling was supported by the findings that the dwarfism was partially rescued and OsGA20ox2 (GA20 oxidase) expression was reduced in GA-deficient and GA-insensitive mutants by the knockdown of OsSPY function. The suppression of OsSPY function in a GA-insensitive mutant, gid2, also caused an increase in the phosphorylation of a rice DELLA protein, SLR1, but did not change the amount of SLR1. This indicates that the function of OsSPY in GA signaling is not via changes in the amount or stability of SLR1, but probably involves control of the suppressive function of SLR1. In addition to the GA-related phenotypes, OsSPY antisense and RNAi plants showed increased lamina joint bending, which is a brassinosteroid-related phenotype, indicating that OsSPY may play roles both in GA signaling and in the brassinosteroid pathway.     

Gibberellins are required for seed development and pollen tube growth in Arabidopsis

Gibberellins (GAs) are tetracyclic diterpenoids that are essential endogenous regulators of plant growth and development. GA levels within the plant are regulated by a homeostatic mechanism that includes changes in the expression of a family of GA-inactivating enzymes known as GA 2-oxidases. Ectopic expression of a pea GA 2-oxidase2 cDNA caused seed abortion in Arabidopsis, extending and confirming previous observations obtained with GA-deficient mutants of pea, suggesting that GAs have an essential role in seed development. A new physiological role for GAs in pollen tube growth in vivo also has been identified. The growth of pollen tubes carrying the 35S:2ox2 transgene was reduced relative to that of nontransgenic pollen, and this phenotype could be reversed partially by GA application in vitro or by combining with spy-5, a mutation that increases GA response. Treatment of wild-type pollen tubes with an inhibitor of GA biosynthesis in vitro also suggested that GAs are required for normal pollen tube growth. These results extend the known physiological roles of GAs in Arabidopsis development and suggest that GAs are required for normal pollen tube growth, a physiological role for GAs that has not been established previously.     

Effects of gibberellin mutations on tolerance to apical meristem damage in Arabidopsis thaliana

To examine the role of gibberellin hormones (GAs) in tolerance to apical meristem damage (AMD), we characterized the reaction norms of several GA-deficient and insensitive mutants of Arabidopsis thaliana in response to AMD and compared them to those of the wild type, Landsberg, from which they were derived. We included 'natural' genotypes of A. thaliana--accessions with shorter lab histories--in order to evaluate how representative Landsberg is of other genotypes. The GA mutations did not alter the level of tolerance to AMD, which was consistent with equal compensation for all genotypes. Generally, the reaction norms to AMD did not differ among the GA mutants themselves, or between the GA mutants and Landsberg. The GA mutations did affect the overall phenotypes of the plants, but these effects were not simply related to whether the mutation was early or late in the biochemical pathways. The GA-insensitive mutant was phenotypically different from the GA-deficient mutants and from Landsberg. The natural populations differed significantly from Landsberg, particularly in attributes related to size and inflorescence production, one more example of the need for researchers to be careful when generalizing the results of studies based upon laboratory strains. Our results indicate that early-flowering genotypes of A. thaliana can be remarkably tolerant to AMD, and that GA deficiency/insensitivity does not hinder tolerance to AMD, at least in this genetic background. Moreover, we confirm that mutations at regulatory loci can have noncatastrophic effects on fitness, as recently found by other investigators.     

Release of the repressive activity of rice DELLA protein SLR1 by gibberellin does not require SLR1 degradation in the gid2 mutant

The rice (Oryza sativa) DELLA protein SLR1 acts as a repressor of gibberellin (GA) signaling. GA perception by GID1 causes SLR1 protein degradation involving the F-box protein GID2; this triggers GA-associated responses such as shoot elongation and seed germination. In GA-insensitive and GA biosynthesis mutants, SLENDER RICE1 (SLR1) accumulates to high levels, and the severity of dwarfism is usually correlated with the level of SLR1 accumulation. An exception is the GA-insensitive F-box mutant gid2, which shows milder dwarfism than mutants such as gid1 and cps even though it accumulates higher levels of SLR1. The level of SLR1 protein in gid2 was decreased by loss of GID1 function or treatment with a GA biosynthesis inhibitor, and dwarfism was enhanced. Conversely, overproduction of GID1 or treatment with GA(3) increased the SLR1 level in gid2 and reduced dwarfism. These results indicate that derepression of SLR1 repressive activity can be accomplished by GA and GID1 alone and does not require F-box (GID2) function. Evidence for GA signaling without GID2 was also provided by the expression behavior of GA-regulated genes such as GA-20oxidase1, GID1, and SLR1 in the gid2 mutant. Based on these observations, we propose a model for the release of GA suppression that does not require DELLA protein degradation.     

SEC8, a subunit of the putative Arabidopsis exocyst complex, facilitates pollen germination and competitive pollen tube growth

The exocyst, a complex of eight proteins, contributes to the morphogenesis of polarized cells in a broad range of eukaryotes. In these organisms, the exocyst appears to facilitate vesicle docking at the plasma membrane during exocytosis. Although we had identified orthologs for each of the eight exocyst components in Arabidopsis (Arabidopsis thaliana), no function has been demonstrated for any of them in plants. The gene encoding one exocyst component ortholog, AtSEC8, is expressed in pollen and vegetative tissues of Arabidopsis. Genetic studies utilizing an allelic series of six independent T-DNA mutations reveal a role for SEC8 in male gametophyte function. Three T-DNA insertions in SEC8 cause an absolute, male-specific transmission defect that can be complemented by expression of SEC8 from the LAT52 pollen promoter. Microscopic analysis shows no obvious abnormalities in the microgametogenesis of the SEC8 mutants, and the mutant pollen grains appear to respond to the signals that initiate germination. However, in vivo assays indicate that these mutant pollen grains are unable to germinate a pollen tube. The other three T-DNA insertions are associated with a partial transmission defect, such that the mutant allele is transmitted through the pollen at a reduced frequency. The partial transmission defect is only evident when mutant gametophytes must compete with wild-type gametophytes, and arises in part from a reduced pollen tube growth rate. These data support the hypothesis that one function of the putative plant exocyst is to facilitate the initiation and maintenance of the polarized growth of pollen tubes.     

Arabidopsis pollen-specific glycerophosphodiester phosphodiesterase-like genes are essential for pollen tube tip growth

In angiosperms, pollen tube growth is critical for double fertilization and seed formation. Many of the factors involved in pollen tube tip growth are unknown. Here, we report the roles of pollen-specific GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE-LIKE (GDPD-LIKE) genes in pollen tube tip growth. Arabidopsis thaliana GDPD-LIKE6 (AtGDPDL6) and AtGDPDL7 were specifically expressed in mature pollen grains and pollen tubes and green fluorescent protein (GFP)-AtGDPDL6 and GFP-AtGDPDL7 fusion proteins were enriched at the plasma membrane at the apex of forming pollen tubes. Atgdpdl6 Atgdpdl7 double mutants displayed severe sterility that was rescued by genetic complementation with AtGDPDL6 or AtGDPDL7. This sterility was associated with defective male gametophytic transmission. Atgdpdl6 Atgdpdl7 pollen tubes burst immediately after initiation of pollen germination in vitro and in vivo, consistent with the thin and fragile walls in their tips. Cellulose deposition was greatly reduced along the mutant pollen tube tip walls, and the localization of pollen-specific CELLULOSE SYNTHASE-LIKE D1 (CSLD1) and CSLD4 was impaired to the apex of mutant pollen tubes. A rice pollen-specific GDPD-LIKE protein also contributed to pollen tube tip growth, suggesting that members of this family have conserved functions in angiosperms. Thus, pollen-specific GDPD-LIKEs mediate pollen tube tip growth, possibly by modulating cellulose deposition in pollen tube walls.     

Identification of kaonashi mutants showing abnormal pollen exine structure in Arabidopsis thaliana

Exine, the outermost architecture of pollen walls, protects male gametes from the environment by virtue of its chemical and physical stability. Although much effort has been devoted to revealing the mechanism of exine construction, still little is known about it. To identify the genes involved in exine formation, we screened for Arabidopsis mutants with pollen grains exhibiting abnormal exine structure using scanning electron microscopy. We isolated 12 mutants, kaonashi1 (kns1) to kns12, and classified them into four types. The type 1 mutants showed a collapsed exine structure resembling a mutant of the callose synthase gene, suggesting that the type 1 genes are involved in callose wall synthesis. The type 2 mutant showed remarkably thin exine structure, presumably due to defective primexine thickening. The type 3 mutants showed defective tectum formation, and thus type 3 genes are required for primordial tectum formation or biosynthesis and deposition of sporopollenin. The type 4 mutants showed densely distributed baculae, suggesting type 4 genes determine the position of probacula formation. All identified kns mutants were recessive, suggesting that these KNS genes are expressed in sporophytic cells. Unlike previously known exine-defective mutants, most of the kns mutants showed normal fertility. Map-based cloning revealed that KNS2, one of the type 4 genes, encodes sucrose phosphate synthase. This enzyme might be required for synthesis of primexine or callose wall, which are both important for probacula positioning. Analysis of kns mutants will provide new knowledge to help understand the mechanism of biosynthesis of exine components and the construction of exine architecture.     

Misregulation of phosphoinositides in Arabidopsis thaliana decreases pollen hydration and maternal fertility

Phosphoinositides are important lipids involved in membrane identity, vesicle trafficking, and intracellular signaling. In recent years, phosphoinositides have been shown to play a critical role in polarized secretion in plants, as perturbations of phosphoinositide metabolism, through loss of function mutants, result in defects in root hair elongation and pollen tube growth, where polarized secretion occurs rapidly. In the Brassicaceae, responses of stigmatic papillae to compatible pollen are also thought to involve highly regulated secretory events to facilitate pollen hydration and penetration of the pollen tube through the stigmatic surface. We therefore sought to analyze the female sporophyte fertility of the root hair defective4-1 mutant and the PI 4-kinase β1/β2 double mutant, which differentially affect phosphatidylinositol-4-phosphate (PI4P) levels. Stigmas from both mutants supported slower rates of pollen grain hydration, and the fecundity of these mutants was also diminished as a result of failed pollination events. This study therefore concludes that PI4P is integral to appropriate pistil responses to compatible pollen.     

Identification and characterization of gibberellin-insensitive mutants selected from among dwarf mutants of rice

In rice, many dwarf mutants have been isolated and characterized. We have investigated the relationship between dwarfism and the gibberellin (GA)-mediated control of physiological processes. Twenty-three rice cultivars and mutants (9 normal, 3 semi-dwarf, 11 dwarf) were analyzed in terms of two GA-mediated processes, namely, elongation of shoots and production of -amylase activity in the endosperm. As a result, we identified four different groups (groups N, T, D and E). Two-dimensional plotting of the extent of induction of -amylase in the endosperm versus the extent of enhancement of shoot elongation upon treatment with exogenous gibberellic acid (GA₃) provided a useful method for the rapid allocation of large numbers of dwarf mutants of rice to the various groups. Members of group N (normal type), which included all normal cultivars and semi-dwarf mutants, showed a slight increase in elongation of shoots and a remarkable increase in production of -amylase with the application of GA₃ during germination. All of the dwarf mutants were classified as being members of the other three groups. Members of group T (Tan-ginbozu type), including three dwarf mutants, were highly responsive to exogenous GA₃ in terms of elongation of shoots and production of -amylase, with associated lower levels of endogenous GA. In contrast, members of the other three groups, including group N, had normal levels of endogenous GAs. Members of group D (Daikoku type) were only slightly responsive to exogenous GA₃, an indication that they are GA-insensitive mutants. Members of group E (Ebisu type) had responses to GA₃ similar to those of group N, not only in terms of elongation of shoots but also in terms of -amylase production, an indication that they are dwarf mutants that can be considered as neither GA-deficient nor GA-insensitive mutants. We also examined a GA-insensitive mutant selected from among 19 near-isogenic dwarf lines of Shiokari, and we concluded that the d-1 gene is associated with the phenotype of GA-insensitive dwarf mutants.     

Arabidopsis AtVPS15 plays essential roles in pollen germination possibly by interacting with AtVPS34

VPS 15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells.The knowledge about the function of its homologue in plants remains limited.Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis,plays an essential role in pollen germination.Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants.Reciprocal crosses between atvpslS mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes.DAPI staining, Alexander’s stain and scanning electron microscopic analysis showed that atvpsl5 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen,whereas in vitro germination experiments revealed that germination of the pollen grains was defective.GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPSI5 was mainly expressed in pollen grains.Finally,DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34.These results suggest that AtVPS15 is very important for pollen germination,possibly through modulation of the activity of PI3-kinase.     

Analysis of the rice mutant dwarf and gladius leaf 1. Aberrant katanin-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling

Molecular genetic studies of plant dwarf mutants have indicated that gibberellin (GA) and brassinosteroid (BR) are two major factors that determine plant height; dwarf mutants that are caused by other defects are relatively rare, especially in monocot species. Here, we report a rice (Oryza sativa) dwarf mutant, dwarf and gladius leaf 1 (dgl1), which exhibits only minimal response to GA and BR. In addition to the dwarf phenotype, dgl1 produces leaves with abnormally rounded tip regions. Positional cloning of DGL1 revealed that it encodes a 60-kD microtubule-severing katanin-like protein. The protein was found to be important in cell elongation and division, based on the observed cell phenotypes. GA biosynthetic genes are up-regulated in dgl1, but the expression of BR biosynthetic genes is not enhanced. The enhanced expression of GA biosynthetic genes in dgl1 is not caused by inappropriate GA signaling because the expression of these genes was repressed by GA3 treatment, and degradation of the rice DELLA protein SLR1 was triggered by GA3 in this mutant. Instead, aberrant microtubule organization caused by the loss of the microtubule-severing function of DGL1 may result in enhanced expression of GA biosynthetic genes in that enhanced expression was also observed in a BR-deficient mutant with aberrant microtubule organization. These results suggest that the function of DGL1 is important for cell and organ elongation in rice, and aberrant DGL1-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling.     

MARIS plays important roles in Arabidopsis pollen tube and root hair growth

In flowering plants, male gametes are delivered to female gametes for double fertilization through pollen tubes.Therefore, pollen tube growth is crucial for double fertilization. Despite its importance to sexual reproduction, genetic mechanisms of pollen tube growth remain poorly understood.In this study, we characterized the receptor-like cytoplasmic protein kinase(RLCK) gene, MARIS(MRI) that plays critical roles in pollen tube growth. MRI is preferentially expressed in pollen grains, pollen tubes and roots. Mutation in MRI by a Ds insertion led to a burst of pollen tubes after pollen germination. Pollen-rescue assay by pollen and pollen tubespecific expression of MRI in the mri-4 mutant showed that loss of MRI function also severely affected root hair elongation. MRI protein interacted with the protein kinase OXIDATIVE SIGNAL INDUCIBLE1(OXI1) in the in vitro and in vivo assays, which functions in plant defence and root hair development, and was phosphorylated by OXI1 in vitro. Our results suggest that MRI plays important roles in pollen tube growth and may function in root hair elongation through interaction with OXI1.