Embryo-lethal mutants of Arabidopsis thaliana: Evidence for gametophytic expression of the mutant genes

Summary Normal and aborted seeds from two recessive embryo-lethal mutants (79A and 124D) of Arabidopsis thaliana were shown to be distributed nonrandomly along the length of heterozygous siliques. Significantly more than half of the aborted seeds in these two mutants were located in the top half of the silique, in the region closest to the stigma surface. Segregation ratios (percent aborted seeds) were unusually low at the base of the silique, and slightly higher than expected at the tip. In contrast, aborted seeds from four other embryo-lethal mutants (87A, 123B, 50B, and 71E) were distributed randomly along the length of the silique. These results suggest that the mutant genes in 79A and 124D are expressed during both the gametophytic (n) and sporophytic (2n) phases of development. These two mutants provide further evidence for the hypothesis that many genes expressed prior to fertilization also perform a critical function during growth and development of the sporophyte. Embryo-lethal mutants of Arabidopsis may therefore be useful in future studies of gametophytic gene expression and the regulation of pollen-tube growth in higher plants.     

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.     

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.     

Use of two-dimensional protein-pattern analysis for the characterization of Arabidopsis thaliana mutants

Total proteins extracted from wild-type plants of Arabidopsis thaliana Heyhn, an etiolated mutant, a de-etiolated mutant and a mutant affected in cotyledon morphology, were analyzed by two-dimensional gel electrophoresis. Computer analysis of two-dimensional gels allowed the characterization of the mutants by a set of proteins showing a differential expression when compared with the wild-type plant grown under the same conditions. The overlap between comparisons of the diferent mutants with the wild-type allowed the identification of groups of polypeptides which, since their expression is altered in several mutants, might be involved in certain physiological functions. For example, this approach showed a possible involvement of actin in the elongation process. The simultaneous analysis of the two-dimensional protein patterns of different mutants seems, therefore, to be a promising approach to characterize proteins involved in various physiological functions.Abbreviations 2-D two-dimensional - IEF isoelectrofocusing - M_r relative molecular mass - E17.1 E17 mutant grown in light - E22.1 E22 mutant grown in light - E44.d E44 mutant grown in darkness - WT.d wild-type plants grown in darkness - WT.1 Wild-type plants grown in light We would like to thank our laboratory colleagues J.A. Traas, H. Höfte and D. Bouchez (all from INRA) for useful discussions throughout this work. Also grateful thanks to Mr Zivy (Lab. Génétique des Sytèmes Végétaux. INRA. La Ferme du Moulon, Gif-sur-Yvette, France) for helpful discussions concerning 2-D gel analysis and to I. Small (Lab. Biologie Cellulaire. INRA, Versailles, France) for English text corrections.     

Flavonoid diversity and biosynthesis in seed of Arabidopsis thaliana

Functional characterization of genes involved in the flavonoid metabolism and its regulation requires in-depth analysis of flavonoid structure and composition of seed from the model plant Arabidopsis thaliana. Here, we report an analysis of the diverse and specific flavonoids that accumulate during seed development and maturation in wild types and mutants. Wild type seed contained more than 26 different flavonoids belonging to flavonols (mono and diglycosylated quercetin, kaempferol and isorhamnetin derivatives) and flavan-3-ols (epicatechin monomers and soluble procyanidin polymers with degrees of polymerization up to 9). Most of them are described for the first time in Arabidopsis. Interestingly, a novel group of four biflavonols that are dimers of quercetin-rhamnoside was also detected. Quercetin-3-O-rhamnoside (the major flavonoid), biflavonols, epicatechin and procyanidins accumulated in the seed coat in contrast to diglycosylated flavonols that were essentially observed in the embryo. Epicatechin, procyanidins and an additional quercetin-rhamnoside-hexoside derivative were synthesized in large quantities during seed development, whereas quercetin-3-O-rhamnoside displayed two peaks of accumulation. Finally, 11 mutants affected in known structural or regulatory functions of the pathway and their three corresponding wild types were also studied. Flavonoid profiles of the mutants were consistent with previous predictions based on genetic and molecular data. In addition, they also revealed the presence of new products in seed and underlined the plasticity of this metabolic pathway in the mutants.     

Mapping genes essential for embryo development in Arabidopsis thaliana.

Summary We have previously isolated and characterized over 90 recessive mutants of Arabidopsis thaliana defective in embryo development. These emb mutants have been shown to differ in lethal phase, extent of abnormal development, and response in culture. We demonstrate in this report the value and efficiency of mapping emb genes relative to visible and molecular markers. Sixteen genes essential for embryo development were mapped relative to visible markers by analyzing progeny of selfed F₁ plants. Embryonic lethals are now the most common type of visible marker included on the linkage map of Arabidopsis. Backcrosses were used in several cases to orient genes relative to adjacent markers. Three genes were located to chromosome arms with telotrisomics by screening for a reduction in the percentage of aborted seeds produced by F₁ plants. A restriction fragment length polymorphism (RFLP) mapping strategy that utilizes pooled EMB/EMB F₂ plants was devised to increase the efficiency of mapping embryonic lethals relative to molecular markers. This strategy was tested by demonstrating that the biol locus of Arabidopsis is within 0.5 cM of an existing RFLP marker. Mapping embryonic lethals with both visible and molecular markers may therefore help to identify large numbers of genes with essential functions in Arabidopsis.     

Isolation and classification of chlorophyll-deficient xantha mutants of Arabidopsis thaliana

Mutant lines of Arabidopsis thaliana that are either blocked at various steps of the biosynthetic pathway of chlorophyll (Chl) or that are disturbed in one of the subsequent steps leading to the assembly of an active photosynthetic membrane were isolated by screening for Chl-deficient xantha (xan) mutants. Only mutants that segregated in a 31 ratio, that contained the same carotenoid spectrum as etiolated wild-type seedlings and less than 2% of the Chl of wild-type control seedlings, and whose Chl content was not affected by the addition of sucrose to the growth medium were selected for a more detailed analysis. As a final test for the classification of the selected mutants, light-grown xan mutants were vacuum-infiltrated and incubated with the common precursor of tetrapyrroles, -aminolevulinic acid (ALA), in the dark. Two major groups of mutants could be distinguished. Some of the mutants were blocked at various steps of the Chl pathway between ALA and protochlorophyllide (Pchlide) and did not accumulate the latter in the dark. The other mutants accumulated Pchlide in the dark regardless of whether exogenous ALA was added. This latter group could be subdivided into mutants with a biochemical lesion in a recently discovered second light-dependent Pchlide reduction step that occurs in green plants and mutants that have blocks in the assembly of Chl protein complexes. In the present work a total of seven different loci could be defined genetically in Arabidopsis that affect the synthesis of Chl and its integration into the growing photosynthetic membrane.Abbreviations ALA -aminolevulinic acid - Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - POR NADPH-Protochlorophyllide oxidoreductase - xan xantha This study was initiated while one of the authors (K.A.) was on sabbatical leave in the laboratory of Dr. C. Somerville (MSU, East Lansing, Mich., USA). We are extremely grateful to Dr. Somerville and his coworkers for advice and support during this time. This research was supported by the Deutsche Forschungsgemeinschaft and the Schweizerischer Nationalfonds.     

Analysis of storage proteins in normal and aborted seeds from embryo-lethal mutants of Arabidopsis thaliana

The major storage proteins isolated from wild-type seeds of Arabidopsis thaliana (L.) Heynh., strain Columbia, were studied by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both the hypocotyl and cotyledons of mature embryos contained abundant 12 S (cruciferin) and 2 S (arabin) proteins that appeared similar in size and subunit composition to the cruciferin (12 S) and napin (1.7 S) seed-storage proteins of Brassica napus. The 12 S protein from Arabidopsis was resolved by SDS-PAGE into two groups of subunits with approximate relative molecular weights of 22–23 kDa (kilodalton) and 30–34 kDa. These polypeptides accumulated late in embryo development, disappeared early in germination, and were not detected in other vegetative or reproductive tissues. Accumulation of the 12 S proteins in aborted seeds from nine embryo-lethal mutants with different patterns of abnormal development was studied to determine the extent of cellular differentiation in arrested embryos from each mutant line. Abundant 12 S proteins were found in arrested embryos from two mutants with late lethal phases, but not in seven other mutants with lethal phases ranging from the globular to the cotyledon stages of embryo development. These results indicate that the accumulation of seed-storage proteins in wild-type embryos of Arabidopsis is closely tied to morphogenetic changes that occur during embryo development. Embryo-lethal mutants may therefore be useful in future studies on the developmental regulation of storage-protein synthesis.Abbreviations kDa kilodalton - M_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Chlorophyll-protein-complexes of thylakoids of wild type and chlorophyll b mutants of Arabidopsis thaliana

Pigment-protein-complexes of two chlorophyll b deficient mutants of Arabidopsis and from the wild type were separated electrophoretically. Light-harvesting proteins were absent in the chlorophyll b free mutant ch¹ and their amount was reduced in the mutant ch² which has a reduced content of chlorophyll b. The ratio of CPa:CP I increased with decreasing chlorophyll b content which indicated that the stoichiometry of photosystem II to photosystem I is not constant.Abbreviations Chl chlorophyll - CPa chlorophyll a-protein - CP I P-700 chlorophyll a-protein - LHCP light-harvesting chlorophyll a/b-protein - PAGE polyacrylamide gel electrophoresis - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Chlorophyll-protein-complexes of thylakoids of wild type and chlorophyll b mutants of Arabidopsis thaliana

Pigment-protein-complexes of two chlorophyll b deficient mutants of Arabidopsis and from the wild type were separated electrophoretically. Light-harvesting proteins were absent in the chlorophyll b free mutant ch¹ and their amount was reduced in the mutant ch² which has a reduced content of chlorophyll b. The ratio of CPa:CP I increased with decreasing chlorophyll b content which indicated that the stoichiometry of photosystem II to photosystem I is not constant.Abbreviations Chl chlorophyll - CPa chlorophyll a-protein - CP I P-700 chlorophyll a-protein - LHCP light-harvesting chlorophyll a/b-protein - PAGE polyacrylamide gel electrophoresis - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

The isolation of apparently homoplastidic mutants induced by a nuclear recessive gene in Arabidopsis thaliana

Summary The nuclear recessive gene, chm1, of Arabidopsis thaliana is a imitator that induces a variety of plastid alterations giving rise to mixed cells and variegated leaves. The variegation is maternally transmitted but chm1 is transmitted in a Mendelian fashion (Rédei 1973; Rédei and Plurad 1973). In order to characterize the different types of plastid alterations induced by chm1, isolating homoplastidic lines, each apparently containing one type of mutant plastid in its cells, was essential since such characterization cannot be carried out on mixed cells. We have used two genetic approaches to isolate several apparently homoplastidic mutant lines by the removal of the mutator from the genetic background, and the maternal transmission of the mutant plastids. The rapidity of obtaining homoplastidic lines in the absence of chm1 indicated a non-stochastic sorting-out of plastids in mixed cells. That each of the chm1-free homoplastidic mutant lines was apparently homoplastidic for one type of mutant plastids was confirmed by electron microscopic observations. Here we report, for the first time, the production of different homoplastidic lines in the absence of the nuclear-mutator gene. Such genetically-stable homogeneous material should be a useful tool for studying the molecular mechanism(s) by which chm1 induces a variety of heritable plastid alterations.     

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.     

Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana

We have examined the cold-induced enhancement of freezing tolerance and expression of cold-regulated (cor) genes in Arabidopsis thaliana (L.) Heynh (Landsberg erecta) and abscisic acid (ABA)-deficient (aba) and ABA-insensitive (abi) mutants derived from it. The results indicate that the abi mutations had no apparent effect on freezing tolerance, while the aba mutations did: cold-acclimated aba mutants were markedly impaired in freezing tolerance compared to wild-type plants. In addition, it was observed that non-frozen leaves from both control and cold-treated aba mutant plants were more ion-leaky than those from corresponding wild-type plants. These data are consistent with previous observations indicating that ABA levels can affect freezing tolerance. Whether ABA has a direct role in the enhancement of freezing tolerance that occurs during cold acclimation, however, is uncertain. Several studies have suggested that ABA might mediate certain changes in gene expression that occur during cold acclimation. Our data indicate that the ABA-induced expression of three ABA-regulated Arabidopsis cor genes was unaffected in the abi2, abi3, and aba-1 mutants, but was dramatically impaired in the abi1 mutant. Cold-regulated expression of all three cor genes, however, was nearly the same in wild-type and abi1 mutant plants. These data suggest that the cold-regulated and ABA-regulated expression of the three cor genes may be mediated through independent control mechanisms.     

Hormone-response mutants of Arabidopsis thaliana (L.) Heynh. impaired in somatic embryogenesis

Plant hormones are considered to be the key factors involved in triggering in vitro induced plant morphogenesis, including somatic embryogenesis (SE). Mutants affected in SE and altered in hormonal response therefore provide valuable material for genetic research on in vitro induced plant embryogenesis. The capacity for SE was studied in 27 mutants with defects in response to different plant hormones: auxin, ABA, gibberellin and cytokinin, and evaluated in 2-week-old mutant and wild-type cultures in terms of their efficiency and productivity. SE was induced in vitro via a direct morphogenic pathway, through the culture of immature zygotic embryos on standard solid medium with 5 μM 2,4-D. The majority of the analyzed mutants displayed a significantly impaired capacity for SE; and those affected belonged to several different hormone-defective groups, including forms affected in auxin (axr4), gibberellin (ga) and ABA (abi, hyl1, cpb20, abh1) response. These mutants showed a significant decrease in embryogenic response as manifested by a low efficiency and/or productivity of SE. Additionally, SE efficiency was analyzed for axr4-1 mutant on media supplemented with different auxins while GA₃ and inhibitors of gibberellins (uniconazol P and paclobutrazol), were applied for pkl1-1-mutant. The selected mutants provide a valuable research tool for studying the molecular mechanisms determining the induction of embryogenesis in cultures of somatic tissues. Their usefulness in further studies is discussed.     

Characterization of three male-sterile mutants of Arabidopsis thaliana exhibiting alterations in meiosis

Male-sterile mutants are being studied to deepen our understanding of the complex processes of microsporogenesis and microgametogenesis. Due to difficulties associated with isolating the mutated gene, there is currently very little molecular information on the defects responsible for male sterility. As a first step in utilizing male-sterile mutants to better understand the bio-chemical and molecular processes that control pollen development, we have characterized a number of Arabidopsis thaliana lines that were generated by seed transformation and exhibit male sterility. We report here the identification and characterization of three male-sterile A. thaliana lines, all of which are tagged with T-DNA and show aberrant meiosis. A detailed cytochemical study was conducted on these lines to better understand the timing and nature of each mutation and to investigate how these mutations affect subsequent steps of pollen development. All three mutants undergo apparently normal morphogenesis until the onset of meiosis. In one line (6492) the mutation is most notable at the tetrad stage when up to eight microspores can be seen in each callose-encased tetrad. The resulting mutant microspores are of variable sizes and contain different amounts of DNA. Two other mutants (7219 and 7593) possess many common features, including variable developmental pathways, failure to produce callose, production of vacuolate, coenocytic (multi-nucleate) cells that are surrounded by persistent microsporocyte walls, and asynchronous patterns of development. Unlike the situation in wild-type plants, where developmental stages are correlated with bud length, such correlations are almost impossible with these two mutants. The sporogenous tissue within all three of these mutant lines collapses prior to anthesis.     

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.     

Mutants of Arabidopsis thaliana with altered phototropism

Thirty five strains of Arabidopsis thaliana (L.) Heynh. have been identified with altered phototropic responses to 450-nm light. Four of these mutants have been more thoroughly characterized. Strain JK224 shows normal gravitropism and second positive phototropism. However, while the amplitude for first positive phototropism is the same as that in the wild-type, the threshold and fluence for the maximum response in first positive phototropism are shifted to higher fluence by a factor of 20–30. This mutant may represent an alteration in the photoreceptor pigment for phototropism. Strain JK218 exhibits no curvature to light at any fluence from 1 mol·m^-2 to 2700 mol·m^-2, but shows normal gravitropism. Strain JK345 shows no first positive phototropism, and reduced gravitropism and second positive phototropism. Strain JK229 shows no measurable first positive phototropism, but normal gravitropism and second positive phototropism. Based on these data, it is suggested that: 1. gravitropism and phototropism contain at least one common element; 2. first positive and second positive phototropism contain at least one common element; and 3. first positive phototropism can be substantially altered without any apparent alteration of second positive phototropism.Abbreviation WT wild-type     

Photomorphogenic mutants of Arabidopsis thaliana reveal activities of multiple photosensory systems during light-stimulated apical-hook opening

Fluence rate-response curves were generated for red-, far-red-, and blue-light-stimulated apical-hook opening in seedlings of several photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. Compared to wild-type plants, hook opening was reduced in the phytochrome-deficient hy1, hy2, and hy6 mutants in red and far-red light at all fluence rates tested, and in low-fluence blue light, but was normal under high-irradiance blue light. In contrast, the blue-light-response mutants (blu1, blu2, and blu3) lacked the high-irradiance-dependent hook-opening response in blue light while hook opening was normal in low-fluence blue light and in red and farred light at all fluence rates tested. Hook opening in the phytochrome-B-deficient hy3 mutant was similar to wild type in all light conditions tested. The effects of the different mutations on light-induced hook opening indicate that a phytochrome(s) other than phytochrome B mediates hook opening stimulated by red, far-red and lowfluence blue light, while a blue-light-absorbing photoreceptor mediates the blue-light-sensitive high-irradiance response. Although the phytochrome and blue-light photosensory systems appear to work independently for the most part, some of their signal-transduction components may interact since the hy4, and hy5 mutants showed reduced hook-opening responses under conditions dependent on the phytochrome and blue-light-photosensory systems.We thank Jeff Young and Brian Parks for their many helpful suggestions during the progress of this research. This work was supported by National Science Foundation Grant No. DCB-9106697.     

Co-action between phytochrome B and HY4 in Arabidopsis thaliana

A combination of physiological and genetic approaches was used to investigate whether phytochromes and blue light (BL) photoreceptors act in a fully independent manner during photomorphogenesis of Arabidopsis thaliana (L.) Heynh. Wild-type seedlings and phyA, phyBand hy4 mutants were daily exposed to 3 h BL terminated with either a red light (R) or a far-red light (FR) pulse. In wild-type and phyA-mutant seedlings, BL followed by an R pulse inhibited hypocotyl growth and promoted cotyledon unfolding. The effects of BL were reduced if exposure to BL was followed by an FR pulse driving phytochrome to the R-absorbing form (Pr). In the wild type, the effects of R versus FR pulses were small in seedlings not exposed to BL. Thus, maximal responses depended on the presence of both BL and the FR-absorbing form of phytochrome (Pfr) in the subsequent dark period. Impaired responses to BL and to R versus FR pulses were observed in phyB and hy4 mutants. Simultaneous irradiation with orange light indicated that BL, perceived by specific BL photoreceptors (i.e. not by phytochromes), required phytochrome B to display a full effect. These results indicate interdependent co-action between phytochrome B and BL photoreceptors, particularly the HY4 gene product. No synergism between phytochrome A (activated by continuous or pulsed FR) and BL photoreceptors was observed.Abbreviations BL blue light - D darkness - FR far-redlight - FRc continuous FR - Pfr FR-absorbing form of phytochrome - Pfr/P proportion of phytochrome as Pfr - phyA phytochrome A - phyB phytochrome B - R red light - WT wild type We thank Professors R.E. Kendrick and M. Koornneef (Wageningen Agricultural University, The Netherlands), Professor J. Chory (Salk Institute, Calif., USA) and the Arabidopsis Biological Resource Center (Ohio State University, Ohio, USA) for their kind provision of the original seed batches. This work was financially supported by CONICET, Universidad de Buenos Aires (AG 040) and Fundación Antorchas (A-12830/1 0000/9)