Efficient regeneration from cotyledon protoplasts in Arabidopsis thaliana

Summary. An efficient and fast regeneration system from cotyledon protoplasts was established for Arabidopsis thaliana accessions C24, Columbia, and Wassilewskija. Culture conditions and media compositions were optimised for the development of protoplasts embedded in thin alginate layers. Unexpectedly, the absence of cytokinins had a positive effect on cell development. Moreover, combined adjustment of -naphthylacetic acid and dicamba concentrations resulted in high plating efficiencies of up to 30%, followed by shoot regeneration within only 19 days after protoplast isolation. The protocol is reproducible, efficient, extremely fast, and regenerated plants are fertile. Thus, this cotyledon-based system could prove useful for studying plant cell and molecular biology in A. thaliana.Correspondence and reprints: Zentrum für angewandte Biowissenschaften, Sonnenstrasse 5, 79104 Freiburg, Federal Republic of Germany.Received December 9, 2002; accepted April 13, 2003; published online September 23, 2003     

Quantitative trait loci for floral morphology in Arabidopsis thaliana

A central question in biology is how genes control the expression of quantitative variation. We used statistical methods to estimate genetic variation in eight Arabidopsis thaliana floral characters (fresh flower mass, petal length, petal width, sepal length, sepal width, long stamen length, short stamen length, and pistil length) in a cosmopolitan sample of 15 ecotypes. In addition, we used genome-wide quantitative trait locus (QTL) mapping to evaluate the genetic basis of variation in these same traits in the Landsberg erecta x Columbia recombinant inbred line population. There was significant genetic variation for all traits in both the sample of naturally occurring ecotypes and in the Ler x Col recombinant inbred line population. In addition, broad-sense genetic correlations among the traits were positive and high. A composite interval mapping (CIM) analysis detected 18 significant QTL affecting at least one floral character. Eleven QTL were associated with several floral traits, supporting either pleiotropy or tight linkage as major determinants of flower morphological integration. We propose several candidate genes that may underlie these QTL on the basis of positional information and functional arguments. Genome-wide QTL mapping is a promising tool for the discovery of candidate genes controlling morphological development, the detection of novel phenotypic effects for known genes, and in generating a more complete understanding of the genetic basis of floral development.     

Quantitative trait loci for inflorescence development in Arabidopsis thaliana

Variation in inflorescence development patterns is a central factor in the evolutionary ecology of plants. The genetic architectures of 13 traits associated with inflorescence developmental timing, architecture, rosette morphology, and fitness were investigated in Arabidopsis thaliana, a model plant system. There is substantial naturally occurring genetic variation for inflorescence development traits, with broad sense heritabilities computed from 21 Arabidopsis ecotypes ranging from 0.134 to 0.772. Genetic correlations are significant for most (64/78) pairs of traits, suggesting either pleiotropy or tight linkage among loci. Quantitative trait locus (QTL) mapping indicates 47 and 63 QTL for inflorescence developmental traits in Ler x Col and Cvi x Ler recombinant inbred mapping populations, respectively. Several QTL associated with different developmental traits map to the same Arabidopsis chromosomal regions, in agreement with the strong genetic correlations observed. Epistasis among QTL was observed only in the Cvi x Ler population, and only between regions on chromosomes 1 and 5. Examination of the completed Arabidopsis genome sequence in three QTL regions revealed between 375 and 783 genes per region. Previously identified flowering time, inflorescence architecture, floral meristem identity, and hormone signaling genes represent some of the many candidate genes in these regions.     

Identification of new minisatellites loci in Arabidopsis thaliana

In order to characterize new CG-rich minisatellites present in the Arabidopsis thaliana genome, a genomic library was screened at low stringency with a probe containing nine repeated-units of a minisatellite (CMs1) previously identified. Both minisatellites and minisatellite-like elements were identified. The minisatellites, with a tandemly-repeated structure, all contain the Arabidopsis thaliana-core sequence previously defined (Tourmente et al., 1994). Both minisatellite and minisatellite-like sequences occur in the Arabidopsis genome in low copy and are weakly polymorphic between ecotypes. The genetic mapping of these markers has shown that they are dispersed on the genome. YACs clones of the CIC library carrying these minisatellites and minisatellite-like sequences were identified.Key words: Arabidopsis thaliana, minisatellites, polymorphism      

Chromosomal Mapping of Genes in theRBCS Family in Arabidopsis thaliana

In Arabidopsis thaliana, four genes have been identified inthe RBCS gene family, one being assigned to subfamily RBCS-Aand the other three to subfamily RBCS-B (1B, 2B and 3B). Todetermine the chromosomal location ofthese genes, hybridizationanalysis with CIC YAC high-density filters was carried out forthe RBCS-A gene, and CAPS analysis for the three RBCS-B genes,based on the finding that restriction fragment length polymorphismis present in the upstream region of the gene RBCS-3B. The RBCS-Agene was mapped at 100.8 cM from the top of chromosome 1 andthe three RBCS-B genes at 62.70 cM from the top of chromosome5.     

Identification of Botrytis cinerea susceptibility loci in Arabidopsis thaliana

Botrytis cinerea is a major pathogen of fruit and vegetable crops causing both pre- and post-harvest grey mould. We have analysed 16 Arabidopsis thaliana ecotypes for natural variation in B. cinerea susceptibility. Susceptibility was associated with lower camalexin accumulation, and three ecotypes (Cape Verdi Islands (Cvi-0), Slavice (Sav-0) and Kindalville (Kin-0)) showed differential susceptibility to the two B. cinerea isolates used. Subsequently, to better understand the genetic control of grey mould disease, we assayed the Arabidopsis Landsberg erecta (Ler) x Columbia (Col-0) recombinant inbred population with the two isolates, and identified multiple small-to-medium-effect quantitative trait loci (QTL) governing susceptibility. Interestingly, the QTL for each isolate are distinct, suggesting that different mechanisms govern defence against these two isolates. Two QTL for each isolate exhibited epistatic interactions with specific allele combinations generating heightened B. cinerea susceptibility.     

Cytosolic dehydroascorbate reductase is important for ozone tolerance in Arabidopsis thaliana

Dehydroascorbate reductase (DHAR) is a key component of the ascorbate recycling system. Three functional DHAR genes are encoded in the Arabidopsis genome. Ozone exposure increased the expression of the cytosolic DHAR (cytDHAR) gene alone. We characterized an Arabidopsis mutant with a deficient cytDHAR. The mutant completely lacked cytDHAR activity and was highly ozone sensitive. The amounts of total ascorbate and glutathione were similar in both lines, but the amount of apoplastic ascorbate in the mutant was 61.5% lower. These results indicate that the apoplastic ascorbate, which is generated through the reduction of DHA by cytDHAR, is important for ozone tolerance.     

High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants

A highly efficient and fast Agrobacterium-mediated leaf disc transformation system for the Arabidopsis thaliana L. genotype C24 was developed. This protocol is also amenable to other ecotypes - as could be shown for Landsberg erecta and Wassllewskija. Besides the hygromycin selection also the G418 and kanamycin selection were established. Furthermore the described procedure is appliable not only to leaf explants but also to expanded cotyledons which proved to be an excellent alternative as explant source for transformation experiments.Abbreviations BAP 6-Benzylaminopurine - CIM Callus induction medium - 2.4D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - 2-IP N⁶-,-Dimethylallyladenosine - HPT Hygromycin phosphotransferase - NAA -Naphthaleneacetic acid - NPT II Neomycin phosphotransferase type II - SEM Shoot elongation medium - SIM Shoot induction medium - RIM Root induction medium     

Response of photosynthesis to high light and drought for Arabidopsis thaliana grown under a UV-B enhanced light regime

Arabidopsis thaliana grown in a light regime that included ultraviolet-B (UV-B) radiation (6 kJ m⁻² d⁻¹) had similar light-saturated photosynthetic rates but up to 50% lower stomatal conductance rates, as compared to plants grown without UV-B radiation. Growth responses of Arabidopsis to UV-B radiation included lower leaf area (25%) and biomass (10%) and higher UV-B absorbing compounds (30%) and chlorophyll content (52%). Lower stomatal conductance rates for plants grown with UV-B radiation were, in part, due to lower stomatal density on the adaxial surface. Plants grown with UV-B radiation had more capacity to down regulate photochemical efficiency of photosystem II (PSII) as shown by up to 25% lower φ_PSII and 30% higher non-photochemical quenching of chlorophyll fluorescence under saturating light. These contributed to a smaller reduction in the maximum photochemical efficiency of PSII (F _v/F _m), greater dark-recovery of F _v/F _m, and higher light-saturated carbon assimilation and stomatal conductance and transpiration rates after a four-hour high light treatment for plants grown with UV-B radiation. Plants grown with UV-B were more tolerant to a 12 day drought treatment than plants grown without UV-B as indicated by two times higher photosynthetic rates and 12% higher relative water content. UV-B-grown plants also had three times higher proline content. Higher tolerance to drought stress for Arabidopsis plants grown under UV-B radiation may be attributed to both increased proline content and decreased stomatal conductance. Growth of Arabidopsis in a UV-B-enhanced light regime increased tolerance to high light exposure and drought stress.     

Disposal of chloroplasts with abnormal function into the vacuole in Arabidopsis thaliana cotyledon cells

Summary. Autophagy is a process in which cell membrane rearrangement allows for the sequestration and degradation of part of the cytoplasm. Many protein components of the autophagic mechanism and their corresponding genes have been identified in yeast cells by molecular genetics, and this has enabled researchers to identify homologues of these genes in mammalian and plant systems. Autophagy is involved in the starvation response in which part of the cytoplasm is degraded in order to produce essential substrates to allow the cell to survive during extreme substrate-limiting conditions. However, autophagy may also be important as a quality control mechanism in normal cells. By screening Arabidopsis thaliana T-DNA insert mutants, we isolated an A. thaliana mutant that lacks the AtTIC40 gene and found that the cotyledon cells of this mutant contained undeveloped plastids. Moreover, many toluidine-stained particulate structures were found in the vacuoles of these mutant cells. The images from electron microscopy suggested that some of these particulate structures were partially degraded chloroplasts. Furthermore, oil bodies were found in the cotyledon cells of mutant and wild-type plants, which suggests that the mutant seedlings were not starved under the experimental conditions. These results may indicate that under nutrient-sufficient conditions, plant cells remove abnormal plastids by autophagy and that this mechanism is involved in the quality control of organelles.Present address: BioResource Center, Tsukuba Institute, Institute of Physical and Chemical Research (RIKEN), Tsukuba, Japan.Present address: Genomics Sciences Center, Yokohama Institute, Institute of Physical and Chemical Research (RIKEN), Yokohama, Japan.Correspondence and reprints: School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan.     

Heterogeneous selection at specific loci in natural environments in Arabidopsis thaliana

Genetic variation for quantitative traits is often greater than that expected to be maintained by mutation in the face of purifying natural selection. One possible explanation for this observed variation is the action of heterogeneous natural selection in the wild. Here we report that selection on quantitative trait loci (QTL) for fitness traits in the model plant species Arabidopsis thaliana differs among natural ecological settings and genetic backgrounds. At one QTL, the allele that enhanced the viability of fall-germinating seedlings in North Carolina reduced the fecundity of spring-germinating seedlings in Rhode Island. Several other QTL experienced strong directional selection, but only in one site and seasonal cohort. Thus, different loci were exposed to selection in different natural environments. Selection on allelic variation also depended upon the genetic background. The allelic fitness effects of two QTL reversed direction depending on the genotype at the other locus. Moreover, alternative alleles at each of these loci caused reversals in the allelic fitness effects of a QTL closely linked to TFL1, a candidate developmental gene displaying nucleotide sequence polymorphism consistent with balancing selection. Thus, both environmental heterogeneity and epistatic selection may maintain genetic variation for fitness in wild plant species.     

Chromosomal translocations are a common phenomenon in Arabidopsis thaliana T-DNA insertion lines

Ordered collections of Arabidopsis thaliana lines containing mapped T-DNA insertions have become an important resource for plant scientists performing genetic studies. Previous reports have indicated that T-DNA insertion lines can have chromosomal translocations associated with the T-DNA insertion site, but the prevalence of these rearrangements has not been well documented. To determine the frequency with which translocations are present in a widely-used collection of T-DNA insertion lines, we analyzed 64 independent lines from the Salk T-DNA mutant collection. Chromosomal translocations were detected in 12 of the 64 lines surveyed (19%). Two assays were used to screen the T-DNA lines for translocations: pollen viability and genome-wide genetic mapping. Although the measurement of pollen viability is an indirect screen for the presence of a translocation, all 11 of the T-DNA lines showing an abnormal pollen phenotype were found to contain a translocation when analyzed using genetic mapping. A normal pollen phenotype does not, however, guarantee the absence of a translocation. We observed one T-DNA line with normal pollen that nevertheless had a translocation based on genetic mapping results. One additional phenomenon that we observed through our genetic mapping experiments was that the T-DNA junctions on the 5'- and 3'-sides of a targeted gene can genetically separate from each other in some cases. Two of the lines in our survey displayed this 'T-DNA borders separate' phenomenon. Experimental procedures for efficiently screening T-DNA lines for the presence of chromosomal abnormalities are presented and discussed.     

Mapping quantitative trait loci associated with selenate tolerance in Arabidopsis thaliana

Selenium is essential for many organisms, but is toxic at higher levels. To investigate the genetic basis of selenate tolerance in Arabidopsis thaliana, quantitative trait loci (QTL) associated with selenate tolerance in accessions Landsberg erecta and Columbia were mapped using recombinant inbred lines (RILs). The selenate tolerance index (TI(D10) = root growth + 30 microm selenate/root growth control x 100%) was fourfold higher for parental line Col-4 (59%) than for parent Ler-0 (15%). Among the 96 F8 RILs, TI(D10) ranged from 11 to 75% (mean 37%). Using composite interval mapping, three QTL were found on chromosomes 1, 3 and 5, which together explained 24% of variation in TI(D10) and 32% of the phenotypic variation for the difference in root length +/- Se (RL(D10)). Highly significant epistatic interactions between the QTL and markers on chromosome 2 explained additional variation for both traits. Potential candidate genes for Se tolerance in each of the QTL regions are discussed. These results offer insight into the genetic basis of selenate tolerance, and may be useful for identification of selenate-tolerance genes.     

Organization of protein complexes under photomorphogenic UV-B in Arabidopsis

Low-fluence and long-wavelength UV-B light promotes photomorphogenic development in Arabidopsis. CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) is a positive regulator in this pathway while it is a negative regulator of the traditional photomorphogenesis triggered by far-red and visible light. We have recently reported the mechanism by which the switch of COP1 function is accomplished in distinct light contexts. In response to photomorphogenic UV-B, the photoactivated UV RESISTANCE LOCUS 8 (UVR8) associates with the COP1- SUPRESSOR OF PHYA (SPA) core complexes, resulting in the physical and functional disassociation of COP1-SPA from the CULLIN4-DAMAGED DNA BINDING PROTEIN 1 (CUL4-DDB1) E3 scaffold. These UV-B dependent UVR8-COP1-SPA complexes promote the stability and activity of ELONGATED HYPOCOTYL 5 (HY5), and eventually cause COP1 to switch from repressing to promoting photomorphogenesis. In addition, it is possible that CUL4-DDB1 might simultaneously recruit alternative DDB1 BINDING WD40 (DWD) proteins to repress this UV-B-specific signaling. Further investigation is required, however, to verify this hypothesis. Overall, the coordinated organization of various protein complexes facilitates an efficient and balanced UV-B signaling.     

Light-stimulated cotyledon expansion in the blu3 and hy4 mutants of Arabidopsis thaliana.

Cotyledon expansion in response to blue light was compared for wild-type Arabidopsis thaliana (L.) Heynh. and the mutants blu3 and hy4, which show reduced inhibition of hypocotyl growth in blue light. White, blue, and red light stimulated cotyledon expansion in both intact and excised cotyledons of wild-type seedlings (ecotypes No-0, WS, Co-0, La-er). Cotyledons on intact blu3 and hy4 seedlings did not grow as well as those on the wild type in response to blue light, but pretreatment of blu3 seedlings with low fluence rates of red light increased their responsiveness to blue light. Excision of cotyledons alleviated the mutant phenotype so that both mutant and wild-type cotyledons grew equally well in blue light. The loss of the mutant cotyledon phenotype upon excision indicates that the blu3 and hy4 lesions affect cotyledon expansion indirectly via a whole-plant response to light. Furthermore, the ability of excised, mutant cotyledons to grow normally in blue light shows that this growth response to blue light is mediated by a photosystem other than the ones impaired by the blu3 and hy4 lesions.     

Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana

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

Joint genetic and network analyses identify loci associated with root growth under NaCl stress in Arabidopsis thaliana

Plants have evolved a series of tolerance mechanisms to saline stress, which perturbs physiological processes throughout the plant. To identify genetic mechanisms associated with salinity tolerance, we performed linkage analysis and genome‐wide association study (GWAS) on maintenance of root growth of Arabidopsis thaliana in hydroponic culture with weak and severe NaCl toxicity. The top 200 single‐nucleotide polymorphisms (SNPs) determined by GWAS could cumulatively explain approximately 70% of the variation observed at each stress level. The most significant SNPs were linked to the genes of ATP‐binding cassette B10 and vacuolar proton ATPase A2. Several known salinity tolerance genes such as potassium channel KAT1 and calcium sensor SOS3 were also linked to SNPs in the top 200. In parallel, we constructed a gene co‐expression network to independently verify that particular groups of genes work together to a common purpose. We identify molecular mechanisms to confer salt tolerance from both predictable and novel physiological sources and validate the utility of combined genetic and network analysis. Additionally, our study indicates that the genetic architecture of salt tolerance is responsive to the severity of stress. These gene datasets are a significant information resource for a following exploration of gene function.