Pink (P), a new locus responsible for a pink trait in onions (Allium cepa) resulting from natural mutations of anthocyanidin synthase.

A new locus conditioning a pink trait in onions was identified. Unusual pink onions were found in haploid populations induced from an F(1) hybrid between yellow and dark red parents and in F(3) populations originating from the same cross. Segregation ratios of red to pink in F(2), backcross, and F(3) populations indicated that this pink trait is determined by a single recessive locus. RT-PCR was carried out to look for any differential expression of anthocyanin synthesis genes between dark red and pink F(3) lines. The transcript level of anthocyanidin synthase (ANS) was significantly reduced in the pink line. To determine whether this reduced transcription is caused by other regulatory factors or by mutations in the ANS gene itself, ANS gene sequences from both dark red and pink F(3) lines were compared to detect any polymorphisms. Polymorphisms were identified, and subsequently utilized as molecular markers for the selection of ANS alleles. Absolute co-segregation of the pink allele and the ANS allele from the pink line was observed in parents, F(1) and F(3) populations. These results indicate that reduced transcription of the ANS gene caused by mutations in a cis -acting element is likely to result in the pink trait in onions.

Differential immunological cross-reactions with antisera against the V-ATPase of Kalanchoë daigremontiana reveal structural differences of V-ATPase subunits of different plant species.

Two antisera (ATP88 and ATP95) raised against the V-ATPase holoenzyme of Kalanchoë daigremontiana were tested for their cross-reactivity with subunits of V-ATPases from other plant species. V-ATPases from Kalanchoë blossfeldiana, Mesembryanthemum crystallinum, Nicotiana tabacum, Lycopersicon esculentum, Citrus limon, Lemna gibba, Hordeum vulgare and Zea mays were immunoprecipitated with an antiserum against the catalytic V-ATPase subunit A of M. crystallinum. As shown by silver staining and Western blot analysis with ATP88, subunits A, B, C, D and c were present in all immunoprecipitated V-ATPases. In contrast, ATP95 recognized the whole set of subunits only in K. blossfeldiana, M. crystallinum, H. vulgare and Z. mays. This differential cross reactivity of ATP95 indicates the presence of structural differences of certain V-ATPase subunits. Based on the Bafilomycin A1-sensitive ATPase activity of tonoplast enriched vesicles, and on the amount of V-ATPase solubilized and immunoprecipitated, the specific ATP-hydrolysis activity of the V-ATPases under test was determined. The structural differences correlate with the ability of V-ATPases from different species to hydrolyze ATP at one given assay condition for ATP-hydrolysis measurements. Interestingly V-ATPases showing cross-reactivity of subunits A, B, C, D and c with ATP95 showed higher rates of specific ATP hydrolysis compared to V-ATPases containing subunits which were not labeled by ATP95. Thus, V-ATPases with high turnover rates in our assay conditions may show common structural characteristics which separate them from ATPases with low turnover rates.

NaCI Reduces Indole-3-Acetic Acid Levels in the Roots of Tomato Plants Independent of Stress-Induced Abscisic Acid.

Indole-3-acetic acid (IAA) was measured in leaves and roots of tomato (Lycopersicon esculentum) genotypes subjected to salt stress. An abscisic acid (ABA)-deficient mutant of tomato (sitiens), the genetic parent (Rheinlands Ruhm, RR), and a commercial variety (Large Cherry Red, LCR) of tomato were treated with 50 to 300 mM NaCl in nutrient culture. Both LCR and RR had significantly higher levels of IAA in the roots compared with that in sitiens prior to treatment. The initial levels of IAA in the roots of LCR and RR declined by nearly 75% after exposure to NaCl, whereas those in roots from the sitiens mutant remained unchanged. IAA levels in the leaves of all genotypes remained unchanged or increased slightly in response to NaCl. ABA was highest in leaves from the normal genotypes after exposure to NaCl. ABA levels in the roots of sitiens were similar to the levels in the normal genotypes, whereas levels in the leaves were only 10% of the levels found in normal genotypes regardless of the salt treatment. Treatment of LCR and sitiens with exogenous ABA increased the ABA levels in leaves and roots, but there were no measurable changes in endogenous IAA. Therefore, the reduction in IAA appears to result from an ABA-independent effect of NaCl on IAA metabolism in the roots of stressed plants.

Induction of direct somatic embryogenesis and plant regeneration in pepper (Capsicum annuum L.).

Pepper (cv. New Mexico - 6 and Rajur Hirapur) plants were regenerated from immature zygotic embryos via direct somatic embryogenesis. Somatic embryos were formed directly, without any intervening callus, on the zygotic embryo apex, embryo axis and cotyledons on Murashige and Skoog's (MS) medium containing 2,4-D (418 µM), thidiazuron (10 µM) and a high concentration of sucrose (6-10%). The best response was observed on MS medium containing 2,4-D (9 µM), coconut water (10%) and high sucrose (8%). The entire process of induction and maturation of the embryos was completed on the same medium. Histological examination indicated that secondary embryogenesis also occurred directly from the primary somatic embryos. Differentiation of embryos was nonsynchronous, and some embryos were swollen and distorted with fasciation. More than 70% of the mature normal somatic embryos germinated readily on MS medium containing GA3 or TDZ, alone and in combination, and following transfer to pots developed into normal plants.

Induction of a Putative Ca-ATPase mRNA in NaCl-Adapted Cells.

A cDNA clone was isolated that encodes the partial sequence of a putative endoplasmic reticulum Ca(2+)-ATPase of tobacco. The 1.497-kb insert had an open reading frame of 1.149 kb. The deduced peptide had the greatest homology to the endoplasmic reticulum Ca(2+)-ATPases of Drosophila and Artemia, followed by the mammalian and avian enzymes (SERCA2 and 3). The cDNA insert hybridized to a single mRNA of 4.4 kb from tobacco cultured cells or plant tissues. The level of this transcript was induced about 2-fold by NaCl shock in 428 mm NaCl-deadapted tobacco cells that were maintained in medium without salt, but not in unadapted cells. The level of this transcript was 3- to 4-fold higher in 428 mm NaCl-adapted cells growing in salt than in unadapted cells growing without salt.

NaCl Regulation of Tonoplast ATPase 70-Kilodalton Subunit mRNA in Tobacco Cells.

A cDNA clone encoding the 70-kilodalton subunit of the tobacco (Nicotiana tabacum var Wisconsin 38) tonoplast ATPase has been isolated. The 1.656 kilobase insert contains only open reading frame that represents more than 80% of the carrot cDNA coding region. The deduced amino acid sequence has greater than 95% sequence identity with the homologous carrot sequence. A transcript of approximately 2.7 kilobase was detected on Northern blots of tobacco poly(A)(+) selected or total RNA using labeled probe produced from this clone. The gene was expressed throughout the growth cycle in unadapted and 428 millimolar NaCl adapted cells. Transcription of the 70-kilodalton subunit gene or mRNA stability was induced by short-term NaCl treatment in NaCl adapted cells or by abscisic acid treatment in both adapted and unadapted cells. Southern analysis indicated the presence of up to four genes encoding the 70-kilodalton subunit.

Intracellular compartmentation of ions in salt adapted tobacco cells.

Na(+) and Cl(-) are the principal solutes utilized for osmotic adjustment in cells of Nicotiana tabacum L. var Wisconsin 38 (tobacco) adapted to NaCl, accumulating to levels of 472 and 386 millimolar, respectively, in cells adapted to 428 millimolar NaCl. X-ray microanalysis of unetched frozen-hydrated cells adapted to salt indicated that Na(+) and Cl(-) were compartmentalized in the vacuole, at concentrations of 780 and 624 millimolar, respectively, while cytoplasmic concentrations of the ions were maintained at 96 millimolar. The morphometric differences which existed between unadapted and salt adapted cells, (cytoplasmic volume of 22 and 45% of the cell, respectively), facilitated containment of the excited volume of the x-ray signal in the cytoplasm of the adapted cells. Confirmation of ion compartmentation in salt adapted cells was obtained based on kinetic analyses of (22)Na(+) and (36)Cl(-) efflux from cells in steady state. These data provide evidence that ion compartmentation is a component of salt adaptation of glycophyte cells.

Solute Accumulation in Tobacco Cells Adapted to NaCl.

Cells of Nicotiana tabacum L. var Wisconsin 38 adapted to NaCl (up to 428 millimolar) which have undergone extensive osmotic adjustment accumulated Na(+) and Cl(-) as principal solutes for this adjustment. Although the intracellular concentrations of Na(+) and Cl(-) correlated well with the level of adaptation, these ions apparently did not contribute to the osmotic adjustment which occurred during a culture growth cycle, because the concentrations of Na(+) and Cl(-) did not increase during the period of most active osmotic adjustment. The average intracellular concentrations of soluble sugars and total free amino acids increased as a function of the level of adaptation; however, the levels of these solutes did not approach those observed for Na(+) and Cl(-). The concentration of proline was positively correlated with cell osmotic potential, accumulating to an average concentration of 129 millimolar in cells adapted to 428 millimolar NaCl and representing about 80% of the total free amino acid pool as compared to an average of 0.29 millimolar and about 4% of the pool in unadapted cells. These results indicate that although Na(+) and Cl(-) are principal components of osmotic adjustment, organic solutes also may make significant contributions.

Adaptation of Tobacco Cells to NaCl.

Cell lines of tobacco (Nicotiana tabacum L. var Wisconsin 38) were obtained which are adapted to grow in media with varying concentrations of NaCl, up to 35 grams per liter (599 millimolar). Salt-adapted cells exhibited enhanced abilities to gain both fresh and dry weight in the presence of NaCl compared to cells which were growing in medium without NaCl (unadapted cells). Tolerance of unadapted cells and cells adapted to 10 grams per liter NaCl was influenced by the stage of growth, with the highest degree of tolerance exhibited by cells in the exponential phase. Cell osmotic potential and turgor varied through the growth cycle of unadapted cells and cells at all levels of adaptation, with maximum turgor occurring at approximately the onset of exponential fresh weight accumulation.Adaptation to NaCl led to reduced cell expansion and fresh weight gain, while dry weight gain remained unaffected. This reduction in cell expansion was not due to failure of the cells to maintain turgor since cells adapted to NaCl underwent osmotic adjustment in excess of the change in water potential caused by the addition of NaCl to the medium. Tolerance of the adapted cells, as indicated by fresh or dry weight gain, did not increase proportionately with the increase in turgor. Adaptation of these glycophytic cells to NaCl appears to involve mechanisms which result in an altered relationship between turgor and cell expansion.