Complementation of anArabidopsis thaliana biotin auxotroph with anEscherichia coli biotin biosynthetic gene

TheArabidopsis thaliana biotin auxotrophbio1 was rendered prototrophic by transformation with a chimeric transgene containing theEscherichia coli bioA gene driven by a constitutive promoter. ThebioA gene encodes the biotin biosynthetic enzyme 7,8-diaminopelargonic acid aminotransferase. Unlike the untransformed control plants, transgenic plants expressing the bacterial transgene synthesized biotin and grew to maturity without biotin-deficiency symptoms. These findings demonstrate thatbio1/bio1 mutant plants are defective in the gene encoding 7,8-diaminopelargonic acid aminotransferase.     

SUCROSE TRANSPORTER 5 supplies Arabidopsis embryos with biotin and affects triacylglycerol accumulation

The Arabidopsis SUC5 protein represents a classical sucrose/H⁺ symporter. Functional analyses previously revealed that SUC5 also transports biotin, an essential co‐factor for fatty acid synthesis. However, evidence for a dual role in transport of the structurally unrelated compounds sucrose and biotin in plants was lacking. Here we show that SUC5 localizes to the plasma membrane, and that the SUC5 gene is expressed in developing embryos, confirming the role of the SUC5 protein as substrate carrier across apoplastic barriers in seeds. We show that transport of biotin but not of sucrose across these barriers is impaired in suc5 mutant embryos. In addition, we show that SUC5 is essential for the delivery of biotin into the embryo of biotin biosynthesis‐defective mutants (bio1 and bio2). We compared embryo and seedling development as well as triacylglycerol accumulation and fatty acid composition in seeds of single mutants (suc5, bio1 or bio2), double mutants (suc5 bio1 and suc5 bio2) and wild‐type plants. Although suc5 mutants were like the wild‐type, bio1 and bio2 mutants showed developmental defects and reduced triacylglycerol contents. In suc5 bio1 and suc5 bio2 double mutants, developmental defects were severely increased and the triacylglycerol content was reduced to a greater extent in comparison to the single mutants. Supplementation with externally applied biotin helped to reduce symptoms in both single and double mutants, but the efficacy of supplementation was significantly lower in double than in single mutants, showing that transport of biotin into the embryo is lower in the absence of SUC5.     

Embryo-lethal mutants of Arabidopsis thaliana: analysis of mutants with a wide range of lethal phases

Summary Embryo-lethal mutants of Arabidopsis thaliana were isolated by treating mature seeds with an aqueous solution of ethyl methanesulfonate (EMS), screening the resulting M-1 plants for siliques containing 25% aborted seeds following self-pollination, and verifying the presence of induced mutations in subsequent generations. Thirty-two recessive lethals with a Mendelian pattern of inheritance were examined in detail. Developmental arrest of mutant embryos ranged from the zygotic stage of embryogenesis in mutant 53D-4A to the linear and curled cotyledon stages of development in mutants 112A-2A and 130B-A-2. These lethal phases did not change significantly when plants were grown at 18 °C rather than at 24 °C. Differences between mutant lines were found in the color of arrested embryos and aborted seeds, the percentage and distribution of aborted seeds in heterozygous siliques, the size of arrested embryos, and the extent of abnormal development. Unusual mutant phenotypes included the presence of unusually large suspensors, distorted and fused cotyledons, reduced hypocotyls, and arrested embryos without distinct cotyledons or hypocotyl tissue. The isolation of eight new mutants with a non-random distribution of aborted seeds in heterozygous siliques provides further evidence that many of the genes that control early stages of embryogenesis in plants are also expressed prior to fertilization.     

An Embryo-Defective Mutant of Arabidopsis Disrupted in the Final Step of Biotin Synthesis

Auxotrophic mutants have played an important role in the genetic dissection of biosynthetic pathways in microorganisms. Equivalent mutants have been more difficult to identify in plants. The bio1 auxotroph of Arabidopsis thaliana was shown previously to be defective in the synthesis of the biotin precursor 7,8-diaminopelargonic acid. A second biotin auxotroph of A. thaliana has now been identified. Arrested embryos from this bio2 mutant are defective in the final step of biotin synthesis, the conversion of dethiobiotin to biotin. This enzymatic reaction, catalyzed by the bioB product (biotin synthase) in Escherichia coli, has been studied extensively in plants and bacteria because it involves the unusual addition of sulfur to form a thiophene ring. Three lines of evidence indicate that bio2 is defective in biotin synthase production: mutant embryos are rescued by biotin but not dethiobiotin, the mutant allele maps to the same chromosomal location as the cloned biotin synthase gene, and gel-blot hybridizations and polymerase chain reaction amplifications revealed that homozygous mutant plants contain a deletion spanning the entire BIO2-coding region. Here we describe how the isolation and characterization of this null allele have provided valuable insights into biotin synthesis, auxotrophy, and gene redundancy in plants.     

An embryo-lethal mutant of Arabidopsis thaliana is a biotin auxotroph

Lethal mutants have been used in a variety of animal systems to study the genetic control of morphogenesis and differentiation. Abnormal development has been shown in some cases to be caused by defects in basic cellular processes. We describe in this report an embryo-lethal mutant of Arabidopsis thaliana that can be rescued by the addition of biotin to arrested embryos cultured in vitro and to mutant plants grown in soil. Mutant plants rescued in culture produced phenotypically normal seeds when supplemented with biotin but became chlorotic and failed to produce fertile flowers in the absence of biotin. Arrested embryos were also rescued by desthiobiotin, the immediate precursor of biotin in bacteria. Langridge proposed 30 years ago (1958, Aust. J. Biol. Sci. 11, 58-68) that the scarcity of plant auxotrophs might be caused by lethality prior to germination. The bio1 mutant of Arabidopsis described in this report clearly demonstrates that some auxotrophs in higher plants are eliminated through embryonic lethality. Further analysis of this mutant should provide valuable information on the nature of plant auxotrophs, the biosynthesis and utilization of biotin in plants, and the underlying causes of developmental arrest in lethal mutants of Arabidopsis.     

Complementation of anArabidopsis thaliana biotin auxotroph with anEscherichia coli biotin biosynthetic gene

TheArabidopsis thaliana biotin auxotrophbio1 was rendered prototrophic by transformation with a chimeric transgene containing theEscherichia coli bioA gene driven by a constitutive promoter. ThebioA gene encodes the biotin biosynthetic enzyme 7,8-diaminopelargonic acid aminotransferase. Unlike the untransformed control plants, transgenic plants expressing the bacterial transgene synthesized biotin and grew to maturity without biotin-deficiency symptoms. These findings demonstrate thatbio1/bio1 mutant plants are defective in the gene encoding 7,8-diaminopelargonic acid aminotransferase.     

Transformation of Soybean (Glycine max) by Infecting Germinating Seeds with Agrobacterium tumefaciens

The transfer of genetic material into soybean tissue was accomplished by using an avirulent strain of Agrobacterium tumefaciens which contained the binary vector pGA482. The method used for transformation requires no tissue culture steps as it involves the inoculation of the plumule, cotyledonary node, and adjacent cotyledon tissues of germinating seeds. The identification of neomycin phosphotransferase (NPT) II enzyme activity in the tissues of 16 (R₀) soybean plants indicated that the plant expressible Nos-NPT II gene, contained within the T-DNA region from pGA482, had been transferred at least into somatic tissues. Putative transformed R₀ soybean plants were advanced to produce R₁ plants which were also assayed for the presence of the transferred Nos-NPT II gene. The combined results of these assays indicated that about 0.7% of the surviving inoculated seeds yielded transformed tissues in the R₀ plant, and that about 1/10 of these plants yielded transformed R₁ plants. The presence of the Nos-NPT II gene in DNAs isolated from both R₀ and R₁ plant was demonstrated by using genomic blot hybridization and polymerase chain reaction methods. Integration of this gene into the soybean genome was demonstrated for three R₁ soybean plants.     

A RING-H2 zinc-finger protein gene RIE1 is essential for seed development in Arabidopsis

RING zinc-finger proteins play important roles in the regulation of development in a variety of organisms. In the plant kingdom, few genes encoding RING zinc-finger proteins have been documented with visible effects on plant growth and development. A novel gene, RIE1, encoding a RING-H2 zinc-finger protein was identified in Arabidopsis thaliana and is characterized in this paper. RIE1 encodes a predicted protein product of 359 amino acids residues with a molecular mass of 40 kDa, with a RING-H2 zinc-finger motif located at the extreme end of the C-terminus. Characterization of a Dissociation (Ds) insertion line (SGT4559) and a T-DNA insertion line (SRIE1) demonstrated that disruption of RIE1 is embryo-lethal. SGT4559 heterozygous plants produced seeds with embryo development arrested from globular to torpedo stages. Some mutant seeds were rescued by embryo culture, and the mutant (rie1) plants seemed to grow normally compared to wild-type plants, except that the mutants produced only abnormal seeds. However, RIE1 was expressed in different tissues throughout the whole plant as revealed by northern blot analysis and gene fusion assay of RIE1 promoter with the beta-glucuronidase (GUS) gene. Our results indicated that RIE1 plays an essential role in seed development.     

Selenium Hyperaccumulator Plants Stanleya pinnata and Astragalus bisulcatus Are Colonized by Se-Resistant, Se-Excluding Wasp and Beetle Seed Herbivores

Selenium (Se) hyperaccumulator plants can concentrate the toxic element Se up to 1% of shoot (DW) which is known to protect hyperaccumulator plants from generalist herbivores. There is evidence for Se-resistant insect herbivores capable of feeding upon hyperaccumulators. In this study, resistance to Se was investigated in seed chalcids and seed beetles found consuming seeds inside pods of Se-hyperaccumulator species Astragalus bisulcatus and Stanleya pinnata. Selenium accumulation, localization and speciation were determined in seeds collected from hyperaccumulators in a seleniferous habitat and in seed herbivores. Astragalus bisulcatus seeds were consumed by seed beetle larvae (Acanthoscelides fraterculus Horn, Coleoptera: Bruchidae) and seed chalcid larvae (Bruchophagus mexicanus, Hymenoptera: Eurytomidae). Stanleya pinnata seeds were consumed by an unidentified seed chalcid larva. Micro X-ray absorption near-edge structure (µXANES) and micro-X-Ray Fluorescence mapping (µXRF) demonstrated Se was mostly organic C-Se-C forms in seeds of both hyperaccumulators, and S. pinnata seeds contained ∼24% elemental Se. Liquid chromatography–mass spectrometry of Se-compounds in S. pinnata seeds detected the C-Se-C compound seleno-cystathionine while previous studies of A. bisulcatus seeds detected the C-Se-C compounds methyl-selenocysteine and γ-glutamyl-methyl-selenocysteine. Micro-XRF and µXANES revealed Se ingested from hyperaccumulator seeds redistributed throughout seed herbivore tissues, and portions of seed C-Se-C were biotransformed into selenocysteine, selenocystine, selenodiglutathione, selenate and selenite. Astragalus bisulcatus seeds contained on average 5,750 µg Se g⁻¹, however adult beetles and adult chalcid wasps emerging from A. bisulcatus seed pods contained 4–6 µg Se g⁻¹. Stanleya pinnata seeds contained 1,329 µg Se g⁻¹ on average; however chalcid wasp larvae and adults emerging from S. pinnata seed pods contained 9 and 47 µg Se g⁻¹. The results suggest Se resistant seed herbivores exclude Se, greatly reducing tissue accumulation; this explains their ability to consume high-Se seeds without suffering toxicity, allowing them to occupy the unique niche offered by Se hyperaccumulator plants.     

Cytochrome b5 Reductase Encoded by CBR1 Is Essential for a Functional Male Gametophyte in Arabidopsis

In all eukaryotes, NADH:cytochrome b5 reductase provides electrons, via cytochrome b5, for a range of biochemical reactions in cellular metabolism, including for fatty acid desaturation in the endoplasmic reticulum. Studies in mammals, yeast, and in vitro plant systems have shown that cytochrome b5 can, at least in some circumstances, also accept electrons from NADPH:cytochrome P450 reductase, potentially allowing for redundancy in reductase function. Here, we report characterization of three T-DNA insertional mutants of the gene encoding cytochrome b5 reductase in Arabidopsis thaliana, CBR1. The progeny of plants heterozygous for the cbr1-2 allele segregated 6% homozygous mutants, while cbr1-3 and cbr1-4 heterozygotes segregated 1:1 heterozygous:wild type, indicating a gametophyte defect. Homozygous cbr1-2 seeds were deformed and required Suc for successful germination and seedling establishment. Vegetative growth of cbr1-2 plants was relatively normal, and they produced abundant flowers, but very few seeds. The pollen produced in cbr1-2 anthers was viable, but when germinated on cbr1-2 or wild-type stigmas, most of the resulting pollen tubes did not extend into the transmitting tract, resulting in a very low frequency of fertilization. These results indicate that cytochrome b5 reductase is not essential during vegetative growth but is required for correct pollen function and seed maturation.     

Isolation and characterization of six embryo-lethal mutants of Arabidopsis thaliana

Mature seeds of Arabidopsis thaliana strain “Columbia” were soaked for 7.5 hr in an aqueous solution of the chemical mutagen ethyl methanesulfonate (0.05, 0.10, or 0.50%, v/v). Embryo-lethal mutants were identified in the resulting M-1 chimeral plants by screening the first five siliques of each plant and noting the frequency of aborted seeds. Three hundred sixty seeds were treated at each mutagen dose; the frequency of embryo-lethal mutants ranged from 1–3% of the M-1 plants grown from seeds exposed to 0.05% EMS, to 20–30% of the M-1 plants at the highest mutagen dose. Six embryo-lethal mutants identified through screening of M-1 plants were chosen for detailed studies in subsequent generations. All six mutants segregate as nonallelic, Mendelian recessive lethals, and are maintained as heterozygotes since homozygotes die as embryos. Fruits of heterozygous plants contain 25% aborted seeds and 75% phenotypically normal seeds ($\text{2}\text{3}$ heterozygotes and $\text{1}\text{3}$ wild type). Segregation ratios are not temperature sensitive; the same frequency of aborted seeds is found in plants grown at 18, 25, and 32°C. Embryo arrest and eventual lethality in each mutant occur at a characteristic stage of early embryo development: globular-heart, globular, early globular, or preglobular. Arrested embryos from five of the six mutants resemble normal embryos at early stages of development. Developmental arrest of the embryo proper in the remaining mutant is followed by abnormal growth of the suspensor, an embryonic structure that attaches the embryo proper to the maternal tissue.     

Chimeras for mutations induced in dormant tomato seeds

Genetic chimeras of the VFNT cherry tomato line (Lycopersicon esculentum) were produced by mutagenizing seeds with ethyl methane sulfonate (EMS). The chimeras thereby produced were evaluated by progeny-testing the fruits of the genetically mosaic tissue. A total of 2011 M₁ plants was grown from treated seeds and evaluated by screening their 95175 (M₂) progeny for mutations affecting seedling phenotype. Three vigorous and fertile M₁ plants bearing mutant progeny with definitive phenotypes were selected for systematic harvesting and analysis. The specific location of each fruit was noted at harvest time, enabling the mutated sporogenous tissue of the mosaic M₁ plants to be traced. Sectoring appeared in both branch and floral tissues. In several cases, mutant progenies were restricted to individual branches or parts thereof. True-breeding recessive mutants whose monogenic mode of inheritance was later established occasionally segregated within M₁ fruit progenies at frequencies that indicate a non-homogeneous floral meristem origin. The data emphasize the necessity of making a well-distributed harvest of mosaic plants in order to detect as many variants as possible, as mosaic sectors may or may not recur late in ontogeny and may not contribute to sporogenous tissue early in development.     

Selection and characterization of a rice mutant resistant to 5-methyltryptophan

Summary A rice plant resistant to 5-methyltryptophan (5MT) was selected from mutagenized M3 seeds (Oryza sativa L. var. Sasanishiki) originating from panicles treated with ethylene imine (0.2%) 2 h after flowering. When germinated on 5MT-containing medium, the seeds (M4) from selfed plants segregated with a 3 resistant:1 sensitive ratio, indicating that the plant was heterozygous for a resistance gene and that the resistance was dominant. The resistance was also expressed in callus derived from seeds. Analysis of the free amino acids in seeds, seedlings, and calli showed that homozygous resistant plants (TR1) contained higher levels of total free amino acids than sensitive plants. In particular the levels of tryptophan, phenylalanine, and histidine were, respectively, 8.5, 5.4, and 4.9 times higher than those in the sensitive plants.     

Role of Endogenous Plant Growth Regulators in Seed Dormancy of Avena fatua: II. Gibberellins

Gibberellin A₁ (GA₁) was identified by combined gas chromatographymass spectrometry as the major biologically active gibberellin (GA) in seeds of wild oat (Avena fatua L.) regardless of the depth of dormany or stage of imbibition. Both unimbibed dormant and nondromant seeds contained similar amounts of GA₁ as estimated by the d5-maize bioassay. During imbibition, the level of GA₁ declined in both dormant and non-dormant seeds, although the decline was more rapid in dormant seeds. Only in imbibing nondormant seeds did the GA biosynthesis inhibitor, 2-chloroethyltrimethyl ammonium chloride (CCC), cause a reduction in the level of GA₁ from that observed in control seeds. These results are interpreted as an indication that while afterripening does not cause a direct change in the levels of GAs during dry storage, it does induce a greater capacity for GA biosynthesis during imbibition.

Nondormant seeds imbibed in the presence of 50 millimolar CCC germinated equally as well as untreated seeds. When wild oat plants were fed CCC throughout the entire life cycle, viable seeds were produced that lacked detectable GA-like substances. These seeds afterripened at a slightly slower rate than the controls. Moreover, completely afterripened (nondormant) seeds from plants fed CCC continuously contained no detectable GA-like substances, and when these seeds germinated, dwarf seedlings were produced, indicating GA biosynthesis was inhibited during and after germination. In total, these results suggest that the increased capacity for GA biosynthesis observed in imbibing nondormant seeds is not a necessary prerequisite for germination. It is therefore possible that GA biosynthesis in imbibing nondormant seeds is one of many coordinated biochemical events that occur during germination rather than an initiator of the processes leading to germination.

     

Seed set in Guayule (Parthenium argentatum,asteraceae) in relation to insect pollination

Guayule (Tarthenium argentatum, Asteraceae) is one of two major plant species grown for natural rubber. Studies were conducted to determine the effect of honey bee (Apis mellifera) pollination and season on seed set and total seed yield/ha. The experiments involved four pollination treatments: plants caged with bees; plants caged without bees; plants open pollinated (uncovered); and plants individually covered. Seeds were harvested monthly July–September 1984, and May–September 1985. Plots with bees produced at least 150% more seeds than plots without bees, and there were no qualitative differences in the seed weights among treatments. Highest seed yield was in May and September. Results indicate that (1) insect pollination in guayule increases seed yield and (2) fewer seeds are produced in the warmest months.     

Effects of germination time on seed morph ratio in a seed-dimorphic species and possible ecological significance

Background and Aims Diaspores of heteromorphic species may germinate at different times due to distinct dormancy-breaking and germination requirements, and this difference can influence life history traits. The primary aim of this study was to determine the effect of germination time of the two seed morphs of Suaeda corniculata subsp. mongolica on life history traits of the offspring.Methods Germinated brown and black seeds were sown on the 20th of each month from April to September in a simulated but near-natural habitat of the species. Phenological and vegetative traits of the maternal plants, and number, size and germination percentage of the offspring were determined.Key Results Germinated seeds sown late in the year produced smaller plants that had a higher proportion of non-dormant brown than dormant black seeds, and these brown seeds were larger than those produced by germinated seeds sown early in the year. The length of the seedling stage for brown seeds was shorter than that for black seeds, and the root/shoot ratio and reproductive allocation of plants from brown seeds were more variable than they were for plants from black seeds. Late-germinating brown seeds produced larger plants than late-germinating black seeds.Conclusions Altering the proportion of the two seed types in response to germination timing can help alleviate the adverse effects of delayed germination. The flexible strategy of a species, such as S. corniculata, that produces different proportions of dimorphic seeds in response to variation in germination timing may favour the maintenance and regeneration of the population in its unpredictable environment.     

The stromal processing peptidase of chloroplasts is essential in Arabidopsis, with knockout mutations causing embryo arrest after the 16-cell stage

Stromal processing peptidase (SPP) is a metalloendopeptidase located in the stroma of chloroplasts, and it is responsible for the cleavage of transit peptides from preproteins upon their import into the organelle. Two independent mutant Arabidopsis lines with T-DNA insertions in the SPP gene were analysed (spp-1 and spp-2). For both lines, no homozygous mutant plants could be detected, and the segregating progeny of spp heterozygotes contained heterozygous and wild-type plants in a ratio of 2∶1. The siliques of heterozygous spp-1 and spp-2 plants contained many aborted seeds, at a frequency of ～25%, suggesting embryo lethality. By contrast, transmission of the spp mutations through the male and female gametes was found to be normal, and so gametophytic effects could be ruled out. To further elucidate the timing of the developmental arrest, mutant and wild-type seeds were cleared and analysed by Nomarski microscopy. A significant proportion (～25%) of the seeds in mutant siliques exhibited delayed embryogenesis compared to those in wild type. Moreover, the mutant embryos never progressed normally beyond the 16-cell stage, with cell divisions not completing properly thereafter. Heterozygous spp mutant plants were phenotypically indistinguishable from the wild type, indicating that the spp knockout mutations are completely recessive and suggesting that one copy of the SPP gene is able to produce sufficient SPP protein for normal development under standard growth conditions.