Thylakoid dismantling of damaged unfunctional chloroplasts modulates the Cab and RbcS gene expression in wheat leaves.

Thylakoid membrane dismantling and Lhcb and RbcS nuclear gene expression have been analysed in leaves of wheat plants grown in high fluence rate light and deprived of photoprotective carotenoids by treatments with the two bleaching herbicides, either norflurazon or amitrole. The Lhcb transcript was not detectable in cells of norflurazon-supplied leaves, having chloroplasts totally devoid of both inner membranes and pigments. In contrast, a substantial amount of Lhcb mRNA could be found in cells of amitrole-treated leaves, whose severely damaged organelles still contained few strikingly altered and photosynthetically unfunctional thylakoids, as well as chlorophyll traces. A possible relationship between chlorophyll synthesis and Lhcb expression, with the transcript level depending on the rate of pigment production in photodamaged chloroplasts is discussed. Also the RbcS expression was linked to the chloroplast membrane photodamage. However, a detectable level of transcript was still produced in norflurazon-treated cells, despite complete thylakoid demolition. Thus, the wheat cell behaviour had to be placed between that of species, such as maize, in which the RbcS expression is broken off in these conditions, and that of species, such as pea, in which it is slightly lowered. Interestingly, the dramatically photodamaged chloroplasts still maintained the ability to synthesize proteins and this allowed SSU and LSU Rubisco subunits to be found in the organelles of both norflurazon- and amitrole-treated plants.

A deficiency at the gene coding for zeta-carotene desaturase characterizes the sunflower non dormant-1 mutant.

The non dormant-1 (nd-1) mutant of sunflower (Helianthus annuus L.) is characterized by an albino and viviparous phenotype. Pigment analysis by spectrophotometer and HPLC demonstrated in nd-1 cotyledons the absence of beta-carotene, lutein and violaxanthin. Additionally, we found a strong accumulation of zeta-carotene and, to a lesser extent, of phytofluene and cis-phytoene in nd-1 seedlings grown in very dim light (1 micro mol m(-2) s(-1)). These results suggested that zeta-carotene desaturation was impaired in the mutant plants. To understand the molecular basis of the nd-1 mutation, we cloned and characterized the zeta-carotene desaturase (Zds) gene from sunflower. A reconstructed full-length sequence (1,916 bp) of the Zds cDNA was obtained from homozygous Nd-1/Nd-1 wild-type plants. It contains a 1,761-bp CDS, 62 nucleotides of 5'-untranslated region (UTR), and 77 nucleotides of 3'-UTR. The predicted protein (64.9 kDa) consists of 587 amino acid residues with a putative transit sequence for plastid targeting in the N-terminal region and a typical amino oxidase domain that includes the flavin adenosine dinucleotide (FAD) binding motif. The phylogenetic analysis demonstrated that the sunflower Zds was clustered to marigold (Tagetes) Zds gene, for which it showed an overall aminoacidic identity of 96.6% and resulted strictly correlated with other Zds sequences of higher plants. Interestingly, RT-PCR analyses showed that nd-1 plants were unable to accumulate Zds transcripts. Sequence information from the Zds cDNA was used to design specific primers and to isolate the full-length exons/introns region of the gene. The sunflower Zds gene (HaZds) comprises 14 exons and 13 introns scattered in a ca. 5.0-kb region. Also, HaZds showed a high conservation of the distribution and size of the exons with rice Zds gene. Based on genomic Southern analysis, the nd-1 genome disclosed a large deficiency at the Zds locus.

Room temperature microspectrofluorimetry as a useful tool for studying the assembly of the PSII chlorophyll-protein complexes in single living cells of etiolated Euglena gracilis Klebs during the greening process.

The assembly kinetics of the PSII chlorophyll-protein complexes was followed during the greening of Euglena gracilis by microspectrofluorimetry in vivo, at room temperature, on single living cells. The study was correlated to micro- and submicroscopic events accompanying the proplastid to chloroplast transformation and with the immunolocalization of the LHCPII. Etiolated cells of Euglena gracilis were grown in darkness in Mego's heterotrophic liquid medium under shaking at 25+/-1 degrees C. At the stationary phase of growth, they were exposed to continuous light (330 micromol m(-2) s(-1)) for 72 h. The analyses were carried out on samples collected at different times of illumination. Microspectrofluorimetric data were recorded in the 620-780 nm range (excitation at 436 nm) and were resolved into Gaussian components corresponding to the reaction centres (RCII) and the inner antennae (CP(43-47)) of the PSII and LHCPII. From the RCII/CP(43-47) and LHCPII/PSII ratios, it was inferred that (1) a disconnection between RCII and CP(43-47) syntheses occurs during the lag phase of chloroplast differentiation, RCII being synthesized before the inner antennae. This results in the accumulation of uncoupled PSII Chl-protein complexes; (2) after lag phase, the RCII and CP(43-47) syntheses are connected one to another; (3) the freshly synthesized LHCPII complexes are immediately assembled with the PSII, suggesting that the outer antennae always maintain the form bound to PSII. Micro- and submicroscopical observations and LHCPII immunolocalization were in agreement. These data suggest that microspectrofluorimetry may constitute a useful non-destructive tool for studying the assembly kinetics of PSII, under fully physiological life conditions.