Interactions among Genes Regulating Ovule Development in Arabidopsis Thaliana

The INNER NO OUTER (INO) and AINTEGUMENTA (ANT) genes are essential for ovule integument development in Arabidopsis thaliana. Ovules of ino mutants initiate two integument primordia, but the outer integument primordium forms on the opposite side of the ovule from the normal location and undergoes no further development. The inner integument appears to develop normally, resulting in erect, unitegmic ovules that resemble those of gymnosperms. ino plants are partially fertile and produce seeds with altered surface topography, demonstrating a lineage dependence in development of the testa. ant mutations affect initiation of both integuments. The strongest of five new ant alleles we have isolated produces ovules that lack integuments and fail to complete megasporogenesis. ant mutations also affect flower development, resulting in narrow petals and the absence of one or both lateral stamens. Characterization of double mutants between ant, ino and other mutations affecting ovule development has enabled the construction of a model for genetic control of ovule development. This model proposes parallel independent regulatory pathways for a number of aspects of this process, a dependence on the presence of an inner integument for development of the embryo sac, and the existence of additional genes regulating ovule development.     

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER

The purpose of the experiments described was to identify X chromosome genes functioning mainly or exclusively during oogenesis. Two mutagenesis experiments were carried out with ethyl methane sulfonate. Following treatment inducing 60% lethals, 9% of the treated X chromosomes carried a female sterility mutation which did not otherwise seriously affect viability. Among —95 isolated mutants, 19 were heat-sensitive and 5 cold-sensitive. The mutants have been classified as follows: I (16 mutants; 12 complementation groups): the females laid few or no eggs; the defect concerned either ovulation or oogenesis. II (37 mutants; 18 complementation groups): the female laid morphologically abnormal eggs, often with increased membrane permeability. III A (13 mutants; at least 8 complementation groups): the homozygous females were sterile if mated to mutant males; their progeny (homo- and hemizygous) died at a late embryonic stage (11 mutants), at the larval stage (1 mutant) or at the pupal stage (1 mutant). However fertility was partly restored by breeding to wild-type males as shown by survival of some heterozygous descendants. III B (29 mutants; 22 complementation groups): the fertility of the females was not restored by breeding to a wild-type male. Most of the eggs of 13 of the mutants died at a late stage of embryogenesis. The eggs of the others ceased development earlier or, perhaps, remained unfertilized. The distribution of the number of mutants per complementation group led to an estimation of a total of about 150 X-linked genes involved in female fertility. The females of three mutants, heat-sensitive and totally sterile at 29°, produced at a lower temperature descendants morphologically abnormal or deprived of germ cells. Three other mutants not described in detail showed a reduction in female fertility with many descendants lacking germ cells. A desirable mutant which was not recovered was one with normal fertile females producing descendants which, regardless of their genotype, bore specific morphological abnormalities. The value of the mutants isolated for analysis of the complex processes leading to egg formation and initiation of development is discussed.     

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER with Special Attention to Eggshell Mutants

To study genes that function mainly or exclusively during oogenesis, we have isolated and analyzed female-sterile mutations, with special emphasis on those that affect eggshell formation. Following treatment that induced 61 to 66% lethals, 8.1% of the 1071 X chromosomes tested carried recessive female sterility mutations (87 isolates), and 8.0% carried partial female-sterile mutations (86 isolates), respectively. In addition, three dominant female steriles were recovered. Some of the mutants had very low fecundity, and others laid morphologically normal eggs that failed to develop. A third category included 29 mutants that laid eggs with morphological abnormalities: 26 were female steriles, two were partial female steriles and one was fertile. Mutants of this third category were characterized in some detail and compared with 40 previously isolated mutants that laid similarly abnormal eggs. Approximately 28–31 complementation groups with morphological abnormalities were detected, some of which were large allelic series (11, 9, 7, 6 and 5 alleles). Twenty-four groups were mapped genetically or cytogenetically, and 21 were partially characterized by ultrastructural and biochemical procedures. Of the latter, one group showed clear deficiency of yolk proteins, and nine showed prominent ultrastructural defects in the chorion (at least eight accompanied by deficiencies in characterized chorion proteins). At least six groups with clear-cut effects were found at loci not previously identified with known chorion structural genes.     

Cytochemical Analysis of Pollen Development in Wild-Type Arabidopsis and a Male-Sterile Mutant

Microsporogenesis has been examined in wild-type Arabidopsis thaliana and the nuclear male-sterile mutant BM3 by cytochemical staining. The mutant lacks adenine phosphoribosyltransferase, an enzyme of the purine salvage pathway that converts adenine to AMP. Pollen development in the mutant began to diverge from wild type just after meiosis, as the tetrads of microspores were released from their callose walls. The first indication of abnormal pollen development in the mutant was a darker staining of the microspore wall due to an incomplete synthesis of the intine. Vacuole formation was delayed and irregular in the mutant, and the majority of the mutant microspores failed to undergo mitotic divisions. Enzyme activities of alcohol dehydrogenase and esterases decreased in the mutant soon after meiosis and were undetectable in mature pollen grains of the mutant. RNA accumulation was also diminished. These results are discussed in relation to the possible role(s) of adenine salvage in pollen development.     

Ovule abortion in Arabidopsis triggered by stress

Environmental stresses frequently decrease plant fertility. In Arabidopsis, the effect of salt stress on reproduction was examined using plants grown in hydroponic medium. Salt stress inhibited microsporogenesis and stamen filament elongation. Because plants grown in hydroponic media can be rapidly and transiently stressed, the minimum inductive treatment to cause ovule abortion could be determined. Nearly 90% of the ovules aborted when roots were incubated for 12 h in a hydroponic medium supplemented with 200 mm NaCl. The anatomical effects of salt stress on maternal organs were distinct from those in the gametophyte. A fraction of cells in the chalaza and integuments underwent DNA fragmentation and programmed cell death. While three-fourths of the gametophytes aborted prior to fertilization, DNA fragmentation was not detected in these cells. Those gametophytes that survived were fertilized and formed embryos. However, very few of these developing embryos formed seeds; most senesced during seed development. Thus, during seed formation, there were multiple points where stress could prematurely terminate plant reproduction. These decreases in fecundity are discussed with respect to the hypothesis of serial adjustment of maternal investment.     

Differential Hypocotyl and Root Development of Arabidopsis Auxin-Insensitive Mutants

Arabidopsis , aux1-7, axr1-3 and axr2-1, grown in a natural sandy soil, without sucrose supplementation. The three mutants showed impaired epidermal cell elongation in the hypocotyls of 15-day-old seedlings, with axr2-1 showing the most marked effects. In addition, the roots of axr2-1 elongated faster and presented a more extended meristematic zone than the other genotypes. Unchanged epidermal cell length in the differentiation zone of axr2-1 relative to the wild-type suggested enhancement of cell proliferation. These alterations may have affected the timing and site of emergence of the root hairs, starting later and further from the root tip than in the other genotypes. Similarly to the wild-type, no root hair growth was initiated in axr2-1 drought-induced short roots, although the epidermis was differentiated into trichoblasts and atrichoblasts. On rehydration of the short roots, hair formation occurred from trichoblasts prior to epidermal cell elongation. Therefore, auxin-insensitivity in the axr2-1 mutant did not result in alterations of the hair-forming process itself. The differential development of axr2-1 seedlings, relative to the other auxin-insensitive mutants, suggested that the AXR2 gene has a complex, regulatory function in multiple hormone signaling. Received 26 July 2000/ Accepted in revised form 28 February 2001     

Gravity-Induced Modifications to Development in Hypocotyls of Arabidopsis Tubulin Mutants

We investigated the roles of cortical microtubules in gravity-induced modifications to the development of stem organs by analyzing morphology and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis (Arabidopsis thaliana) tubulin mutants, tua3(D205N), tua4(S178Δ), and tua6(A281T), cultivated under 1g and hypergravity (300g) conditions. Hypocotyls of tubulin mutants were shorter and thicker than the wild type even at 1g, and hypergravity further suppressed elongation and stimulated expansion. The degree of such changes was clearly smaller in tubulin mutants, in particular in tua6. Hypocotyls of tubulin mutants also showed either left-handed or right-handed helical growth at 1g, and the degree of twisting phenotype was intensified under hypergravity conditions, especially in tua6. Hypergravity induced reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells of wild-type hypocotyls. In tubulin mutants, especially in tua6, the percentage of cells with longitudinal microtubules was high even at 1g, and it was further increased by hypergravity. The twisting phenotype was most obvious at cells 10 to 12 from the top, where reorientation of cortical microtubules from transverse to longitudinal directions occurred. Moreover, the left-handed helical growth mutants (tua3 and tua4) had right-handed microtubule arrays, whereas the right-handed mutant (tua6) had left-handed arrays. There was a close correlation between the alignment angle of epidermal cell files and the alignment of cortical microtubules. Gadolinium ions, blockers of mechanosensitive ion channels (mechanoreceptors), suppressed the twisting phenotype in tubulin mutants under both 1g and 300g conditions. Microtubule arrays in tubulin mutants were oriented more transversely by gadolinium treatment, irrespective of gravity conditions. These results support the hypothesis that cortical microtubules play an essential role in maintenance of normal growth phenotype against the gravitational force, and suggest that mechanoreceptors are involved in modifications to morphology and orientation of microtubule arrays by 1g gravity and hypergravity in tubulin mutants.The direction of cell expansion is important for determining the shape of whole plant body. Cortical microtubules are assumed to be responsible for anisotropic expansion of plant cells (Wasteneys and Galway, 2003; Lloyd and Chan, 2004; Mathur, 2004; Baskin, 2005; Paredez et al., 2008). The prevailing view is that cortical microtubule arrays direct or constrain the movement of the cellulose synthase complexes and thus align nascent cellulose microfibrils in the same direction in the innermost layer of the cell wall (Baskin, 2001), although some other mechanisms may also be involved (Baskin, 2001; Sugimoto et al., 2003; Wasteneys, 2004).It is evident that orientation of cortical microtubules plays an essential role in creating the distinct shape of higher plant organs, even if there is uncertainty over the mechanism by which microtubules influence morphogenesis. The importance of cortical microtubule arrays for anisotropic growth has been documented by pharmacological studies and experiments with helical growth mutants of Arabidopsis (Arabidopsis thaliana). Mutants on α- and β-tubulins as well as microtubule-associated proteins show either left-handed or right-handed helical growth (Thitamadee et al., 2002; Nakajima et al., 2004; Sedbrook et al., 2004; Shoji et al., 2004). The rapidly elongating cells of these mutants skew consistently either to the right or to the left and exhibit cortical microtubule arrays that form shallow helices with fixed handedness (Thitamadee et al., 2002; Abe and Hashimoto, 2005; Ishida et al., 2007). Cortical microtubule arrays in the left-handed helical growth mutants form right-handed helix, whereas those in right-handed helical growth mutants form left-handed helix (Thitamadee et al., 2002; Abe and Hashimoto, 2005; Ishida et al., 2007). These results indicate that dysfunctional cortical microtubules are arranged in helical arrays and affect the direction of cell expansion.The gravitational force is one of the environmental factors that determine the plant body shape. Under hypergravity conditions produced by centrifugation, plants generally have a shorter and thicker body (Soga et al., 2006). Namely, hypergravity modifies growth anisotropy. In Arabidopsis hypocotyls, the expression of most α- and β-tubulin genes was up-regulated by hypergravity (Yoshioka et al., 2003; Matsumoto et al., 2007). In protoplasts of Brassica hypocotyls, hypergravity stimulated the regeneration of cortical microtubules into parallel arrays (Skagen and Iversen, 1999), and in azuki bean (Vigna angularis) epicotyls it increased the percentage of cells with longitudinal cortical microtubules (Soga et al., 2006). The reorientation of cortical microtubules from transverse to longitudinal directions may be involved in modifications by hypergravity to growth anisotropy.The aim of this study was to clarify the roles of cortical microtubules in gravity-induced modifications to development of stem organs. For this purpose, we examined the changes in growth, morphology, and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis amino acid substitution mutants in α-tubulin structure, tua3, tua4, and tua6, grown under 1g and 300g conditions. We have reported the possible involvement of mechanosensitive ion channels (mechanoreceptors) in hypergravity-induced modifications to growth and cell wall properties (Soga et al., 2004, 2005, 2006). Thus, we also examined the effect of blockers of mechanoreceptors on helical growth and orientation of cortical microtubule arrays in the tubulin mutants.     

Single-Channel Characteristics of Wild-Type IKs Channels and Channels formed with Two MinK Mutants that Cause Long QT Syndrome

I_Ks channels are voltage dependent and K⁺ selective. They influence cardiac action potential duration through their contribution to myocyte repolarization. Assembled from minK and KvLQT1 subunits, I_Ks channels are notable for a heteromeric ion conduction pathway in which both subunit types contribute to pore formation. This study was undertaken to assess the effects of minK on pore function. We first characterized the properties of wild-type human I_Ks channels and channels formed only of KvLQT1 subunits. Channels were expressed in Xenopus laevis oocytes or Chinese hamster ovary cells and currents recorded in excised membrane patches or whole-cell mode. Unitary conductance estimates were dependent on bandwidth due to rapid channel “flicker.” At 25 kHz in symmetrical 100-mM KCl, the single-channel conductance of I_Ks channels was ∼16 pS (corresponding to ∼0.8 pA at 50 mV) as judged by noise-variance analysis; this was fourfold greater than the estimated conductance of homomeric KvLQT1 channels. Mutant I_Ks channels formed with D76N and S74L minK subunits are associated with long QT syndrome. When compared with wild type, mutant channels showed lower unitary currents and diminished open probabilities with only minor changes in ion permeabilities. Apparently, the mutations altered single-channel currents at a site in the pore distinct from the ion selectivity apparatus. Patients carrying these mutant minK genes are expected to manifest decreased K⁺ flux through I_Ks channels due to lowered single-channel conductance and altered gating.     

Analysis of Vascular Development in the hydra Sterol Biosynthetic Mutants of Arabidopsis

Background

The control of vascular tissue development in plants is influenced by diverse hormonal signals, but their interactions during this process are not well understood. Wild-type sterol profiles are essential for growth, tissue patterning and signalling processes in plant development, and are required for regulated vascular patterning.

Methodology/Principal Findings

Here we investigate the roles of sterols in vascular tissue development, through an analysis of the Arabidopsis mutants hydra1 and fackel/hydra2, which are defective in the enzymes sterol isomerase and sterol C-14 reductase respectively. We show that defective vascular patterning in the shoot is associated with ectopic cell divisions. Expression of the auxin-regulated AtHB8 homeobox gene is disrupted in mutant embryos and seedlings, associated with variably incomplete vascular strand formation and duplication of the longitudinal axis. Misexpression of the auxin reporter proIAA2∶GUS and mislocalization of PIN proteins occurs in the mutants. Introduction of the ethylene-insensitive ein2 mutation partially rescues defective cell division, localization of PIN proteins, and vascular strand development.

Conclusions

The results support a model in which sterols are required for correct auxin and ethylene crosstalk to regulate PIN localization, auxin distribution and AtHB8 expression, necessary for correct vascular development.     

胚珠发育的分子基础

胚珠作为胚囊的携带者,在植物的生殖过程中起重要作用。胚珠是种子的前身,它在受精后发育成种子。近年来通过诱变已创造出一些胚珠和胚囊发育异常的突变体,如 sin1, bell, ovm2, ovm3。这几个突变体的表现型不但是珠被发育异常,而且胚囊不能形成或发育异常,最终结果是雌性不育。同时,已分别从蝶兰和矮牵牛的胚珠中分离出一批胚珠发育特异的基因,其中有关MADS Box基因在胚珠形成和发育中的作用研究得比较清楚,基因转化工作证实胚珠的分化和形成受一类新的MADS Box基因控制。     

Light-Stimulated Apical Hook Opening in Wild-Type Arabidopsis thaliana Seedlings

Apical hook opening and cotyledon unfolding are characteristic responses that occur during deetiolation of dicotyledonous seedlings. Light-stimulated apical hook opening and cotyledon unfolding in etiolated Arabidopsis thaliana seedlings appears to involve the activities of multiple photosensory systems. Red, far-red, and blue light are all effective in stimulating these responses in Arabidopsis. Stimulation of hook opening by red light and low fluence blue light is inductive, far-red reversible, and exhibits reciprocity, as is characteristic of many low fluence-dependent phytochrome-mediated responses. Far-red and high-fluence blue light appear to stimulate hook opening and cotyledon unfolding through high-irradiance-response systems during long-term light treatments. Although a phytochrome high-irradiance-response system presumably mediates the responses in far-red light, the responses to high-fluence blue light may be mediated by a blue light-specific photosensory system.     

Ovule initiation: the essential step controlling offspring number in Arabidopsis

Seed is the offspring of angiosperms. Plants produce large numbers of seeds to ensure effective reproduction and survival in varying environments. Ovule is a fundamentally important organ and is the precursor of the seed. In Arabidopsis and other plants characterized by multi-ovulate ovaries, ovule initiation determines the maximal ovule number, thus greatly affecting seed number per fruit and seed yield. Investigating the regulatory mechanism of ovule initiation has both scientific and economic significance. However, the genetic and molecular basis underlying ovule initiation remains unclear due to technological limitations. Very recently, rules governing the multiple ovules initiation from one placenta have been identified, the individual functions and crosstalk of phytohormones in regulating ovule initiation have been further characterized, and new regulators of ovule boundary are reported, therefore expanding the understanding of this field. In this review, we present an overview of current knowledge in ovule initiation and summarize the significance of ovule initiation in regulating the number of plant offspring, as well as raise insights for the future study in this field that provide potential routes for the improvement of crop yield.     

Leafy Cotyledon Mutants of Arabidopsis

We have previously described a homeotic leafy cotyledon (lec) mutant of Arabidopsis that exhibits striking defects in embryonic maturation and produces viviparous embryos with cotyledons that are partially transformed into leaves. In this study, we present further details on the developmental anatomy of mutant embryos, characterize their response to abscisic acid (ABA) in culture, describe other mutants with related phenotypes, and summarize studies with double mutants. Our results indicate that immature embryos precociously enter a germination pathway after the torpedo stage of development and then acquire characteristics normally restricted to vegetative parts of the plant. In contrast to other viviparous mutants of maize (vp1) and Arabidopsis (abi3) that produce ABA-insensitive embryos, immature lec embryos are sensitive to ABA in culture. ABA is therefore necessary but not sufficient for embryonic maturation in Arabidopsis. Three other mutants that produce trichomes on cotyledons following precocious germination in culture are described. One mutant is allelic to lec1, another is a fusca mutant (fus3), and the third defines a new locus (lec2). Mutant embryos differ in morphology, desiccation tolerance, pattern of anthocyanin accumulation, presence of storage materials, size and frequency of trichomes on cotyledons, and timing of precocious germination in culture. The leafy cotyledon phenotype has therefore allowed the identification of an important network of regulatory genes with overlapping functions during embryonic maturation in Arabidopsis.