Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress.

Proteins homologous to fibrillin, a pepper plastid lipid-associated protein involved in carotenoid storage in fruit chromoplasts, have been recently identified in leaf chloroplasts from several species and shown to be induced upon environmental stress. To further investigate the role of the protein, transgenic Nicotiana tabacum plants over-expressing fibrillin using a constitutive promoter were generated. Transgenics grown under standard light intensities (300 micromol photons m-2 sec-1) were found to contain substantial amounts of fibrillin in flowers and leaves. In leaves, the protein was immunolocalized within chloroplasts in both stromal and thylakoid subfractions. No change was noticed in thylakoid structures from transgenics, but chloroplasts contained an increased number of plastoglobules organized in clusters. In petals, leucoplasts were also found to contain more agglutinated plastoglobules. The effects of environmental factors on fibrillin gene expression and protein localization were studied in tobacco leaves. Less fibrillin was present in plants grown under low light intensities, which can be explained by the involvement of a light-dependent splicing step in the control of fibrillin gene expression in leaves. Analysis of protein subfractions from plants subjected to drought or high light showed that both stresses resulted in fibrillin association with thylakoids. Whereas no growth difference between wild-type (WT) and transgenic plants was noticed under low light conditions, transgenics exhibit a longer main stem, enhanced development of lateral stems and accelerated floral development under higher light intensities. These data suggest that fibrillin-related proteins fulfil an important function in plant development in relation to environmental constraints.

A novel thioredoxin-like protein located in the chloroplast is induced by water deficit in Solanum tuberosum L. plants.

By analysing two-dimensional patterns of chloroplastic proteins from Solanum tuberosum, the authors observed the accumulation of a 32-kDa polypeptide in the stroma of plants subjected to water deficit. N-terminus and internal peptides of the protein, named CDSP 32 for chloroplastic drought-induced stress protein, showed no obvious homology with known sequences. Using a serum raised against the protein N-terminus, a cDNA encoding CDSP 32 was cloned by screening an expression library. The deduced mature CDSP 32 protein is 243 amino acids long and displays typical features of thioredoxins in the C-terminal region (122 residues). In particular, CDSP 32 contains a CGPC motif corresponding to a thioredoxin active site and a number of amino acids conferring thioredoxin-type structure. The CDSP 32 C-terminal region was expressed as a fusion protein in Escherichia coli and was shown to possess thioredoxin activity based on reduction assay of insulin disulfide bridges. RNA blot analysis showed that CDSP 32 transcript does not accumulate upon mild water deficit conditions corresponding to leaf relative water contents (RWC) around 85%, but high levels of CDSP 32 transcripts were observed for more severe stress conditions (RWC around 70%). In vivo labelling and immunoprecipitation revealed a substantial increase in CDSP 32 synthesis upon similar stress conditions. Rewatering of wilted plants caused decreases in both transcript and protein abundances. In tomato wild-type plants and ABA-deficient mutants, a similar accumulation of a CDSP 32-related transcript was observed upon water deficit, most likely indicating no requirement for ABA in the regulation of CDSP 32 synthesis. Based on these results, it is proposed that CDSP 32 plays a role in preservation of the thiol: disulfide redox potential of chloroplastic proteins during water deficit.

Effects of CO(2)-Enrichment and of Aminoacetonitrile on Growth and Photosynthesis of Photoautotrophic Calli of Nicotiana plumbaginifolia.

Photoautotrophic calli of Nicotiana plumbaginifolia were grown for 3 weeks under two CO(2) concentrations (500 and 20,000 microliters of CO(2) per liter). Calli cultured at high CO(2) exhibited a two-fold higher rate of growth. At CO(2) test levels, these calli were characterized by a lower net photosynthetic capacity than calli cultured at low CO(2). This diminution due to CO(2) adaptation could be ascribed to a 170% stimulation of dark respiration, a 40% decrease in total ribulose-1,5-bisphosphate carboxylase (Rubisco) activity, and also to a feedback inhibition of photosynthesis: high CO(2) grown calli contained about 5.5-fold more sucrose and three-fold less orthophosphate (Pi) than low CO(2) grown calli. Whether the decrease in Rubisco activity is related to the accumulation of sucrose and to the Pi limitation is discussed. Both calli exhibited a Warburg-effect showing the existence of active photorespiration at low CO(2). In calli grown at low CO(2) with 5 millimolar aminoacetonitrile (AAN), an inhibitor of the glycolate pathway, fresh weight decreased by 25% and chlorophyll content by 40%, dark respiration increased by 50% and net CO(2) uptake decreased by about 60% at 340 microliters of CO(2) per liter and 35% at 10,000 microliters of CO(2) per liter. In these calli, glutamine and glutamate contents were half of control calli. In contrast, AAN did not provoke any noticeable effect in calli grown at high CO(2). In photoautotrophic calli, the inhibition of the glycolate pathway by AAN results in severe perturbations in glutamate metabolism and in chlorophyll biosynthesis.

Atrazine and diuron resistant plants from photoautotrophic protoplast-derived cultures of Nicotiana plumbaginifolia.

Atrazine and diuron resistant clones were isolated from diploid photoautotrophic protoplastderived colonies of Nicotiana plumbaginifolia. Protoplasts were mutagenised with 0.1 mM N-ethyl-N-nitrosourea and colonies were screened for resistance after plating. Selection of calli was carried out on their ability to grow and green on a selective medium containing either atrazine or diuron. Plants were regenerated from most tolerant calli. Herbicide spray showed that plants of 6 and 4 clones were resistant to atrazine and diuron, respectively.

Establishment and characterization of photoautotrophic protoplast-derived cultures ofNicotiana plumbaginifolia.

A procedure is described for the rapid establishment of photoautotrophic protoplast-derived cultures ofNicotiana plumbaginifolia. Photoautotrophic growth was induced by lowering the glucose concentration to 2.5 g.l(-1) in the protoplast culture medium and by omitting glucose from the subsequent dilution medium. Four week-old highly viable suspensions were plated on an agar-medium without glucose in unsealed Petri dishes and kept in illuminated chambers flushed with 0.05 % or 2 % CO2. Air-grown calli had net photosynthesis rates of 1.8 and 17 µmoles CO2.g(-1) fresh wt.h(-1) in air at 0.034 % CO2 and in air enriched with 1 % CO2, respectively. Calli grown in 2 % CO2 exhibited lower rates of net photosynthesis at the two CO2 concentrations tested (0 and 7.5 µmoles CO2.g(-1) fresh wt.h(-1), respectively). The contribution of photosynthesis to growth was estimated to be 80 % in air-grown calli and more than 90 % in calli grown in 2 % CO2. The suitability of this photoautotrophic culture procedure is discussed with regard to the screening of photosynthetic mutants or transformants from protoplasts.