NADPH-protochlorophyllide oxidoreductase (POR; EC 1.6.99.1) catalyzes the only known light-dependent step in chlorophyll synthesis of higher plants, the reduction of protochlorophyllide (Pchlide) to chlorophyllide. In barley, two distinct immunoreactive POR proteins were identified. In contrast to the light-sensitive POR enzyme studied thus far (POR-A), levels of the second POR protein remained constant in seedlings during the transition from dark growth to the light and in green plants. The existence of a second POR-related protein was verified by isolating and sequencing cDNAs that encode a second POR polypeptide (POR-B) with an amino acid sequence identity of 75% to the POR-A. In the presence of NADPH and Pchlide, the in vitro-synthesized POR-A and POR-B proteins could be reconstituted to ternary enzymatically active complexes that reduced Pchlide to chlorophyllide only after illumination. Even though the in vitro activities of the two enzymes were similar, the expression of their genes during the light-induced transformation of etiolated to green seedlings was distinct. While the POR-A mRNA rapidly declined during illumination of dark-grown seedlings and soon disappeared, POR-B mRNA remained at an approximately constant level in dark-grown and green seedlings. Thus these results suggest that chlorophyll synthesis is controlled by two light-dependent POR enzymes, one that is active only transiently in etiolated seedlings at the beginning of illumination and the other that also operates in green plants.

SummaryThe moss Physcomitrella patens contains high proportions of polyunsaturated very-long-chain fatty acids with up to 20 carbon atoms. Starting from preformed C 18 polyunsaturated fatty acids, their biosynthesis involves a sequence of D6-desaturation, D6-elongation and D5-desaturation. In this report we describe for the ®rst time the characterisation of a cDNA (PSE1) of plant origin with homology to the ELO-genes from Saccharomyces cerevisiae, encoding a component of the D6-elongase. Functional expression of PSE1 in S. cerevisiae led to the elongation of exogenously supplied D6-polyunsaturated fatty acids. By feeding experiments with different trienoic fatty acids of natural and synthetic origin, both substrate speci®city and substrate selectivity of the enzyme were investigated. The activity of Pse1, when expressed in yeast, was not sensitive to the antibiotic cerulenin, which is an effective inhibitor of fatty acid synthesis and elongation. Furthermore, the PSE1 gene was disrupted in the moss by homologous recombination. This led to a complete loss of all C 20 polyunsaturated fatty acids providing additional evidence for the function of the cDNA as coding for a component of the D6-elongase. The elimination of the elongase was not accompanied by a visible alteration in the phenotype, indicating that C 20 -PUFAs are not essential for viability of the moss under phytotron conditions.

Transient cytosolic Ca 2+ ([Ca 2+ ] cyt ) elevations are early events in plant signaling pathways including those related to abiotic stress. The restoration of [Ca 2+ ] cyt to prestimulus levels involves ATP-driven Ca 2+ pumps, but direct evidence for an essential role of a plant Ca 2+ -ATPase in abiotic stress adaptation is missing. Here, we report on a stress-responsive Ca 2+ -ATPase gene ( PCA1 ) from the moss Physcomitrella patens. Functional analysis of PCA1 in a Ca 2+ transport-deficient yeast mutant suggests that PCA1 encodes a P IIB -type Ca 2+ -ATPase harboring an N-terminal autoinhibitory domain. In vivo localizations identified membranes of small vacuoles as the integration site for a PCA1:GFP fusion protein. PCA1 mRNA levels are up-regulated by dehydration, NaCl, and abscisic acid, and PCA1 loss-of-function mutants (Δ PCA1 ) exhibit an enhanced susceptibility to salt stress. The Δ PCA1 lines show sustained elevated [Ca 2+ ] cyt in response to salt treatment in contrast to WT that shows transient Ca 2+ elevations, indicating a direct role for PCA1 in the restoration of prestimulus [Ca 2+ ] cyt . The altered Ca 2+ response of the Δ PCA1 mutant lines correlates with altered expression levels of stress-induced genes, suggesting disturbance of a stress-associated signaling pathway. We propose that PCA1 is an essential component for abiotic stress adaptation in Physcomitrella involved in the generation of a specific salt-induced Ca 2+ signature.

Chlorophyll synthesis in barley is controlled by two different light-dependent NADPH:protochlorophyllide oxidoreductases, termed PORA and PORB. PORA is present abundantly in etioplasts but selectively disappears soon after the beginning of illumination. This negative light effect is mediated simultaneously at three different levels. First, the concentration of porA mRNA declines drastically during illumination of dark-grown seedlings. Second, the plastids' ability to import the precursor of PORA (pPORA) is reduced during the transition from etioplasts to chloroplasts. This effect is due to a rapid decline in the plastidic level of protochlorophyllide (Pchlide), which is required for the translocation of the pPORA. Third, PORA becomes selectively destabilized in illuminated seedlings. When illuminated, PORA-Pchlide-NADPH complexes formed in the dark photoreduce their Pchlide to Chlide and become simultaneously susceptible to attack by plastid proteases. The PORA-degrading protease activity is not detectable in etioplasts but is induced during illumination. In contrast to PORA, the second Pchlide-reducing enzyme, PORB, remains operative in both illuminated and green plants. Its translocation into plastids does not depend on its substrate, Pchlide.