Genetic control of male fertility in Arabidopsis thaliana: structural analysis of premeiotic developmental mutants

We have taken a mutational approach to identify genes important for male fertility in Arabidopsis thaliana and have isolated a number of nuclear male/ sterile mutants in which vegetative growth and female fertility are not altered. Here we describe detailed developmental analyses of four mutants, each of which defines a complementation group and has a distinct developmental end point. All four mutants represent premeiotic developmental lesions. In ms3, tapetum and middle layer hypertrophy result in the degeneration of microsporocytes. In ms4, microspore dyads persist for most of anther development as a result of impaired meiotic division. In ms5, degeneration occurs in all anther cells at an early stage of development. In ms15, both the tapetum and microsporocytes degenerate early in anther development. Each of these mutants had shorter filaments and a greater number of inflorescences than congenic male-fertile plants. The differences in the developmental phenotypes of these mutants, together with the non-allelic nature of the mutations indicate that four different genes important for pollen development, have been identified.     

Anther developmental defects in Arabidopsis thaliana male-sterile mutants

 We identified Arabidopsis thaliana sterility mutants by screening T-DNA and EMS-mutagenized lines and characterized several male-sterile mutants with defects specific for different anther processes. Approximately 44 and 855 sterile mutants were uncovered from the T-DNA and EMS screens, respectively. Several mutants were studied in detail with defects that included the establishment of anther morphology, microspore production, pollen differentiation, and anther dehiscence. Both non-dehiscencing and late-dehiscencing mutants were identified. In addition, pollenless mutants were observed with either apparent meiotic defects and/or abnormalities in cell layers surrounding the locules. Two mutant alleles were identified for the POLLENLESS3 locus which have defects in functional microspore production that lead to the degeneration of cells within the anther locules. pollenless3–1 contains a T-DNA insertion that co-segregates with the mutant phenotype and pollenless3–2 has a large deletion in the POLLENLESS3 gene. The POLLENLESS3 gene has no known counterparts in the GenBank, but encodes a protein containing putative nuclear localization and protein-protein interaction motifs. The POLLENLESS3 gene was shown recently to be the same as MS5, a previously described Arabidopsis thaliana male-sterility mutant. Three genes were identified in the POLLENLESS3 genomic region: GENEY, POLLENLESS3, and β9-TUBULIN. The segment of the Arabidopsis thaliana genome containing the POLLENLESS3 and β9-TUBULIN genes is duplicated and present on a different chromosome. Analysis of the POLLENLESS3 expression pattern determined that the 1.3-kb POLLENLESS3 mRNA is localized specifically within meiotic cells in the anther locules and that POLLENLESS3 mRNA is present only during late meiosis. Received: 15 October 1998 / Revision accepted: 19 November 1998     

Isolation of Arabidopsis thaliana mutants hypersensitive to gamma radiation

A screening method for mutants of Arabidopsis thaliana hypersensitive to -radiation has been devised. Plants grown from ethyl methanesulfonate (EMS)-treated seeds were irradiated at the seedling stage, which is highly radiosensitive due to extensive cell division. Severe growth inhibition of mutant plants by a -ray dose which only slightly affects wild-type plants was the selective criterion. Twelve true-breeding hyper-sensitive lines were isolated from a total of 3394 screened plants. Genetic analysis of five of the lines revealed five new genes, designated RAD1-RAD5. These Arabidopsis RAD mutants are phenotypically similar to mutants in the RAD52 epistasis group of Saccharomyces cerevisiae, which are highly sensitive to ionizing radiation but not hypersensitive to UV light. One possibility is that the Arabidopsis mutants are defective in a nonhomologous or illegitimate recombination mechanism used by plants for repair of chromosome breaks.     

In vitro morphogenesis of arrested embryos from lethal mutants of Arabidopsis thaliana

Summary Arrested embryos from lethal (emb) mutants of Arabidopsis thaliana were rescued on a nutrient medium designed to promote plant regeneration from immature wild-type cotyledons. The best response was observed with mutant embryos arrested at the heart to cotyledon stages of development. Embryos arrested at a globular stage produced callus but failed to turn green or form normal shoots in culture. Many of the mutant plants produced in culture were unusually pale with abnormal leaves, rosettes, and patterns of reproductive development. Other plants were phenotypically normal except for the presence of siliques containing 100% aborted seeds following self-pollination. These results demonstrate that genes with essential functions during plant embryo development differ in their pattern of expression at later stages of the life cycle. Most of the 15 genes examined in this study were essential for embryogenesis but were required again for subsequent stages of development. Only EMB24 appeared to be limited in function to embryo development. These differences in the response of mutant embryos in culture may facilitate the classification of embryonic lethals and the identification of genes with developmental rather than housekeeping functions.     

An Arabidopsis mutant showing aberrations in male meiosis

A male-sterile mutant, mei-1, of Arabidopsis thaliana is described. In this mutant, instead of a tetrad of four microspores being formed after meiosis, a tetrad consisting of from five to eight microspores is formed. The microspores show a wide range of sizes and of DNA contents. The mutant is female-fertile. This mutant was produced by seed transformation with Agrobacterium and appears to be T-DNA tagged.     

Characterization of two Arabidopsis thaliana glutathione S-transferases

Glutathione S-transferases (GST) are multifunctional proteins encoded by a large gene family, divided on the basis of sequence identity into phi, tau, theta, zeta and lambda classes. The phi and tau classes are present only in plants. GSTs appear to be ubiquitous in plants and are involved in herbicide detoxification and stress response, but little is known about the precise role of GSTs in normal plant physiology and during biotic and abiotic stress response. Two cDNAs representing the two plant classes tau and phi, AtGSTF9 and AtGSTU26, were expressed in vitro and the corresponding proteins were analysed. Both GSTs were able to catalyse a glutathione conjugation to 1-chloro-2,4-dinitrobenzene (CDNB), but they were inactive as transferases towards p-nitrobenzylchloride (pNBC). AtGSTF9 showed activity towards benzyl isothiocyanate (BITC) and an activity as glutathione peroxidase with cumene hydroperoxide (CumHPO). AtGSTU26 was not active as glutathione peroxidase and towards BITC. RT-PCR analysis was used to evaluate the expression of the two genes in response to treatment with herbicides and safeners, chemicals, low and high temperature. Our results reveal that AtGSTU26 is induced by the chloroacetanilide herbicides alachlor and metolachlor and the safener benoxacor, and after exposure to low temperatures. In contrast, AtGSTF9 seems not to be influenced by the treatments employed.     

Screening Arabidopsis for mutants in mineral nutrition

Arabidopsis thaliana is a small herbaceous plant which is used as a model plant for defining the molecular basis of many plant processes. The advantages of this plant for genetic studies are its small, well-characterized genome, a short life cycle, large seed set and small seed size. The analysis of mutants of this plant has proved useful in understanding basic plant processes. To isolate Arabidopsis mutants in mineral nutrition, we have devised a method of screening based on X-ray fluorescence spectrometry (XRFS) analysis of leaves. We have identified three mutants in P and Mn nutrition after screening over 100 000 seedlings. These mutants show either excessive accumulation of P or Mn in shoots or an inabilty to accumulate normal concentrations of P.     

Indole-3-butyric acid synthesis in ecotypes and mutants of Arabidopsis thaliana under different growth conditions

Although IBA is a naturally occurring auxin, its role in plant development is still under debate. In this study a set of Arabidopsis mutants was used to analyze the biosynthesis of IBA in vitro. The mutants chosen for this study can be classified as: (1) involvement in auxin metabolism, transport or synthesis (amt1, aux1, ilr1, nit1, rib1, sur1, trp1-100); (2) other hormones possibly involved in the regulation of IBA synthesis (aba1, aba3, eto2, fae1, hls1, jar1); (3) photomorphogenesis (det1, det2, det3); and (4) root architecture (cob1, cob2, scr1). In addition, two transgenic lines overexpressing the IAA glucose synthase (iaglu) gene from maize were analyzed. The ecotypes No-0 and Wassilewskija showed the highest IBA synthetase activity under control conditions, followed by Columbia, Enkheim and Landsberg erecta. In the mutant lines IBA synthetase activity differed in most cases from the wild type, however no particular pattern of up- or down-regulation, which could be correlated to their possible function, was found. For rib1 mutant seedlings it was tested whether reduced IBA synthetase activity correlates with the endogenous IBA levels. Free IBA differed only depending on the culture conditions, but gave no clear correlation with IBA synthetase activity compared to the wild type. Since drought and osmotic stress as well as abscisic acid (ABA) application enhanced IBA synthesis in maize, it was tested whether IBA synthetase from Arabidopsis is also inducible by drought stress conditions. This was confirmed for the two ecotypes Col and Ler which showed different IBA synthetase activity when cultivated with various degrees of drought stress. IBA synthetase was also determined in photomorphogenic mutants under different light regimes. Induction of IBA synthetase in det1 and det3 plants was found under short day plus a red light pulse or in the dark, respectively. The results are discussed with respect to the functions of the mutated genes.     

Growth in vitro of arrested embryos from lethal mutants ofArabidopsis thaliana

Summary Seventeen embryo-lethal mutants ofArabidopsis thaliana with lethal phases ranging from the globular to mature cotyledon stages of development were analyzed by culturing arrested embryos on nutrient media designed to promote either callus formation or the completion of embryo development and the recovery of homozygous mutant plants. Enriched media supplemented with vitamins, amino acids, and nucleosides were used to identify potential auxotrophic mutants. Wild-type embryos produced extensive callus on basal and enriched media supplemented with 2,4-D and kinetin. Numerous roots developed when wildtype callus was grown in the presence of NAA and kinetin. Mutant embryos arrested prior to the heart stage of development formed only a slight amount of callus on basal and enriched media. Arrested embryos from mutants 122G-E and 112A-2A reached a later stage of development and gave the most interesting responses in culture. 122G-E mutant embryos failed to grow on basal media but produced extensive callus and homozygous mutant plants on enriched media. The specific nutrient required for growth of this mutant remains to be determined. Arrested embryos from mutant 112A-2A developed into abnormal plants without roots when placed in culture. Mutant callus also failed to form roots on a variety of root-inducing media. Expression of this mutant gene therefore disrupts development of the root apical meristem during both embryogenesis in vivo and organogenesis in vitro.     

Female meiosis in wild-type Arabidopsis thaliana and in two meiotic mutants

Female meiosis in Arabidopsis has been analysed cytogenetically using an adaptation of a technique previously applied to male meiosis. Meiotic progression was closely correlated with stages of floral development, including the length and morphology of the gynoecium. Meiosis in embryo sac mother cells (EMCs) occurs later in development than male meiosis, in gynoecia that range in size between 0.3 and 0.8 mm. The earliest stages in EMCs coincide with the second division to tetrad stages in pollen mother cells. However, the details of meiotic chromosome behaviour in EMCs correspond closely to the observations we have previously made in male meiosis. In addition, BrdU labelling coupled with an immunolocalisation detection system was used to mark the S phase in cells preceding their entry into prophase I. These techniques allow female meiotic stages of Arabidopsis to be analysed in detail, from the S-phase through to the tetrad stage, and are shown to be equally applicable to the analysis of female meiosis in meiotic mutants. Received: 3 April 2000 / Revision accepted: 2 August 2000     

A comparative ultrastructural analysis of exine pattern development in wild-typeArabidopsis and a mutant defective in pattern formation

Summary In order to identify factors necessary for the establishment of the reticulate pollen wall pattern, we have characterized a T-DNA tagged mutant ofArabidopsis thaliana that is defective in pattern formation. This study reports the results of an ultrastructural comparison of pollen wall formation in the mutant to wall development in wild-type plants. Pollen wall development in the mutant parallels that of wild-type until the early tetrad stage. At this point in wild-type plants, the microspore plasma membrane assumes a regular pattern of ridges and valleys. Initial sporopollenin deposition occurs on the ridges marking the beginning of probacula formation. In contrast, the plasma membrane in the mutant appears irregular with flattened protuberances and rare invaginations. As a result, the wild-type regular pattern of ridges and valleys is not formed. Sporopollenin is randomly deposited on the plasma membrane and aggregates on the locule wall; it is not anchored to the membrane. Our finding that the mutation blocks the normal invagination of the plasma membrane and disrupts the proper deposition of sporopollenin during wall formation suggests that the mutation could be in a gene responsible for pattern formation. These results also provide direct evidence that the plasma membrane plays a critical role in the establishment of the pollen wall pattern.     

Gene targeting in Arabidopsis thaliana.

Summary Gene targeting of a chromosomally integrated transgene in Arabidopsis thaliana is reported. A chimeric gene consisting of the promoter of the 35S RNA of CaMV, the polyadenylation signal of the octopine synthase gene and the coding region of the bacterial hygromycin phosphotransferase gene (hpt), which was rendered non-functional by deletion of 19 bp, was introduced into the genome of A. thaliana using Agrobacterium-mediated gene transfer. A total of 3.46 x 10⁸ protoplasts isolated from 17 independent transgenic Arabidopsis lines harbouring the defective chimeric hpt gene were transformed via direct gene transfer using various DNA forms containing only the intact coding region of the hpt gene. Out of 150 hygromycin-resistant colonies appearing in the course of these experiments, four were the result of targeted recombination of the incoming DNA with the defective chromosomal locus as revealed by PCR and Southern blot analysis. Comparison with the number of transformants obtained when an hpt gene controlled by a promoter and terminator from the nopaline synthase gene was employed results in a maximal ratio of homologous to non-homologous transformation in A. thaliana of 1 x 10^–4.     

Developmental anatomy of cotyledons and leaves in has mutant of Arabidopsis thaliana

Summary. In this work, we analyzed the developmental anatomy of cotyledons and leaves in the has mutant of Arabidopsis thaliana. It is a recessive T-DNA insertion mutation that causes changes in the size, shape, and tissue organization of the cotyledons and leaves of has plants. Analysis of has cotyledons revealed a prominent decrease in the cell number and an increase in the area of cotyledon cells and intercellular spaces of has plants. At early stages of development, has leaves are fingerlike structures, but later they develop small, lobed blades with rare trichomes. An important characteristic of the mutant leaf anatomy is the absence of mesophyll tissue differentiation. In addition, both cotyledons and leaves display a disrupted pattern of vascular bundles. Furthermore, mutant plants are defective in root and shoot morphology, indicating that the has mutation affects a number of aspects in plant development. Correspondence and reprints: Institute of Botany and “Jevremovac” Botanical Garden, Faculty of Biology, Belgrade University, Takovska 43, 11 000 Belgrade, Serbia.     

New Embryo Lethals in Arabidopsis thaliana: Basic Genetic and Morphological Study

Six different mutations with defects in immature seed development have been identified during screening of a T-DNA collection of Arabidopsis thaliana. The mutations were confirmed to be monogenic and recessive-lethal by genetic analysis. Mutant embryos were blocked in certain steps in the process necessary for embryo viability and development, and therefore they belong to the embryo-lethal class of mutants. The genetic and morphological studies of T-DNA mutations affecting embryo development are presented. The youngest embryos with a defect were observed at the globular stage in the VIII-64 mutation. Externally located cells, precursor of the protoderm, were characterised by abnormal cell division. VIII-41 mutation with a defect at the late globular stage was arrested at the globular-heart stage transition. VIII-111 mutation showed defect at heart stage of embryogenesis with atypical development of cotyledon primordia. The defect was associated with abnormal pattern of cell division constituting the precursor of the shoot apical meristem. In VIII-82 mutation defect in torpedo stage with asymmetric cotyledons was observed. Cotyledon stage of embryos and chlorophyll defect were observed in VIII-75 mutant. Abnormal suspensor consisting of two columns of cells was observed in 280-4-4 mutation. Newly identified embryo-lethals can serve as starting material for more detailed genetic and molecular studies.     

Identification of Arabidopsis thaliana variants with differential glyphosate responses

To facilitate future investigations of glyphosate-resistance mechanisms, three approaches were taken to obtain Arabidopsis thaliana variants that differed in glyphosate response. Recurrent selection by spraying with sub-lethal glyphosate concentrations was performed with Columbia-0 seedlings. After seven cycles of treatment, no resistance was found. A population of 800,000 ethylmethanesulfonate-mutagenized M(2) seedlings was screened on agar containing 0.2mM glyphosate, a lower concentration than that previously used in other studies, and no resistant mutants were recovered. Seventy-two Arabidopsis ecotypes were screened with glyphosate and a range of responses was observed. In a follow-up experiment on a subset of these ecotypes, reduction of seed yield by 11.5 g/ha glyphosate (about 1% the typical field use rate) ranged among ecotypes from 0% to >90%, relative to untreated controls. However, even the least sensitive ecotypes were severely injured by relatively low glyphosate rates. Overall, attempts to select Arabidopsis seedlings that were significantly glyphosate-resistant were unsuccessful and consistent with previous reports. Arabidopsis ecotypes identified with differential glyphosate responses could be used for further studies though the inherently high sensitivity of Arabidopsis to glyphosate could limit their utility in studying glyphosate-resistance mechanisms.     

Expression Patterns of Duplicate Genes in the Developing Root in Arabidopsis thaliana

Data on gene expression in the development of the root in Arabidopsis thaliana were used to test for expression profile differences among multi-gene families and to examine the extent to which expression differences accompanied coding sequences divergence within families. Significant differences among families were observed on two principal axes, accounting for over 80% of the variance in the expression data. The number of synonymous nucleotide substitutions per synonymous site (d_S) and the number of nonsynonymous nucleotide substitutions per nonsynonymous site (d_N) were estimated between the members of two-member families (N=428) and between phylogenetically independent sister pairs (N=190) of sequences within larger families. Ribosomal proteins and a few other proteins were exceptional in showing highly divergent expression patterns in spite of very low levels of amino acid sequence divergence, as indicated by the low d_N relative to d_S. However, the majority of gene duplicates showed relatively high levels of amino acid sequence divergence without appreciable change in expression pattern in the cell types analyzed. Reviewing Editor:Dr. Manyuan Long     

Expression of three members of the calcium-dependent protein kinase gene family in Arabidopsis thaliana

Calcium-dependent protein kinases (CDPKs) belong to a unique family of enzymes containing a single polypeptide chain with a kinase domain at the amino terminus and a putative calcium-binding EF hands structure at the carboxyl terminus. From Arabidopsis thaliana, we have cloned three distinct cDNA sequences encoding CDPKs, which were designated as atcdpk6, atcdpk9 and atcdpk19. The full-length cDNA sequences for atcdpk6, atcdpk9 and atcdpk19 encode proteins with a molecular weight of 59343, 55376 and 59947, respectively. Recombinant atCDPK6 and atCDPK9 proteins were fully active as kinases whose activities were induced by Ca²⁺. Biochemical studies suggested the presence of an autoinhibitory domain in the junction between the kinase domain and the EF hands structure. Serial deletion of the four EF hands of atCDPK6 demonstrated that the integrity of the four EF hands was crucial to the Ca²⁺ response. All the three atcdpk genes were ubiquitously expressed in the plant as demonstrated by RNA gel blot experiments. Comparison of the genomic sequences suggested that the three cdpk genes have evolved differently. Using antibodies against atCDPK6 and atCDPK9 for immunohistochemical experiments, CDPKs were found to be expressed in specific cell types in a temporally and developmentally regulated manner.