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Photosynthetic acclimation to CO2-limiting stress is associated with control of genetic and physiological responses through a signal transduction pathway, followed by integrated monitoring of the environmental changes. Although several CO2-responsive genes have been previously isolated, genome-wide analysis has not been applied to the isolation of CO2-responsive genes that may function as part of a carbon-concentrating mechanism (CCM) in photosynthetic eukaryotes. By comparing expression profiles of cells grown under CO2-rich conditions with those of cells grown under CO2-limiting conditions using a cDNA membrane array containing 10,368 expressed sequence tags, 51 low-CO2 inducible genes and 32 genes repressed by low CO2 whose mRNA levels were changed more than 2.5-fold in Chlamydomonas reinhardtii Dangeard were detected. The fact that the induction of almost all low-CO2 inducible genes was impaired in the ccm1 mutant suggests that CCM1 is a master regulator of CCM through putative low-CO2 signal transduction pathways. Among low-CO2 inducible genes, two novel genes, LciA and LciB, were identified, which may be involved in inorganic carbon transport. Possible functions of low-CO2 inducible and/or CCM1-regulated genes are discussed in relation to the CCM.

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Aquatic photosynthetic organisms, including the green algaChlamydomonas reinhardtii, induce a set of genes for a carbonconcentrating mechanism (CCM) to acclimate to CO 2-limiting conditions. This acclimation is modulated by some mechanisms in the cell to sense CO2 availability. Previously, a high-CO2-requiring mutant C16 defective in an induction of the CCM was isolated from C. reinhardtii by gene tagging. By using this pleiotropic mutant, we isolated a nuclear regulatory gene, Ccm1, encoding a 699-aa hydrophilic protein with a putative zinc-finger motif in its Nterminal region and a Gln repeat characteristic of transcriptional activators. Introduction of Ccm1 into this mutant restored an active carbon transport through the CCM, development of a pyrenoid structure in the chloroplast, and induction of a set of CCM-related genes. That a 5,128-base Ccm1 transcript and also the translation product of 76 kDa were detected in both high-and low-CO2 conditions suggests that CCM1 might be modified posttranslationally. These data indicate that Ccm1 is essential to control the induction of CCM by sensing CO2 availability in Chlamydomonas cells. In addition, complementation assay and identification of the mutation site of another pleiotropic mutant, cia5, revealed that His-54 within the putative zinc-finger motif of the CCM1 is crucial to its regulatory function.zinc-finger motif ͉ carbon transport ͉ signal transduction ͉ photosynthesis ͉ acclimation P hotosynthetic organisms sense environmental changes, e.g., light, temperature, and various nutrient availabilities, to modulate and optimize photosynthetic activities. A number of aquatic photosynthetic organisms are able to concentrate dissolved inorganic carbon (DIC) intracellularly, allowing rapid growth despite low-CO 2 availability externally (1). This carbonconcentrating mechanism (CCM) shows acclimation to external DIC to optimize CO 2 fixation efficiency (2). During acclimation these organisms induce the expression of a set of genes required for various aspects of the CCM. In Chlamydomonas reinhardtii, several genes have been shown to be regulated in response to changes in external CO 2 concentration, including periplasmic carbonic anhydrase (CA; ref.3), mitochondrial CA (4), and a chloroplast envelope protein, LIP-36 (5). A development of pyrenoid structure in the chloroplasts also is modulated by the supply of CO 2 (6). This acclimation to CO 2 -limiting conditions suggests the existence of sensory mechanisms by which cells perceive the shortage of CO 2 and pathways by which the signal is transduced into specific gene regulation. However, the regulation of gene expression during this acclimation in eukaryotic organisms is still poorly understood. One reason is the paucity of mutants impaired in induction processes. We have isolated previously one C. reinhardtii mutant (C16) by gene-tagging mutagenesis, which was high-CO 2 requiring (7), and we showed that C16 exhibited a defect in CCM induction, with a low affinity for DIC and a low level of DIC accumulation under C...

To characterize genes whose expression is induced in carbon-stress conditions, 12,969 and 13,450 5'-end expressed sequence tags (ESTs) were generated from cells grown in low-CO2 and high-CO2 conditions of the unicellular green alga, Chlamydomonas reinhardtii. These ESTs were clustered into 4436 and 3566 non-redundant EST groups, respectively. Comparison of their sequences with those of 3433 non-redundant ESTs previously generated from the cells under the standard growth condition indicated that 2665 and 1879 EST groups occurred only in the low-CO2 and high-CO2 populations, respectively. It was also noted that 96.2% and 96.0% of the cDNA species respectively obtained from the low-CO2 and high-CO2 conditions had no similar EST sequence deposited in the public databases. The EST species identified only in the low-CO2 treated cells included genes previously reported to be expressed specifically in low-CO2 acclimatized cells, suggesting that the ESTs generated in this study will be a useful source for analysis of genes related to carbon-stress acclimatization. The sequence information and search results of each clone will appear at the web site: http://www.kazusa.or.jp/en/plant/chlamy/EST/.

Brassica napus (canola) plants were genetically manipulated to increase the amount and composition of carotenoids in seeds by using seven key enzyme genes involved in ketocarotenoid formation, which originated from a soil bacterium Pantoea ananatis (formerly called Erwinia uredovora 20D3), and marine bacteria Brevundimonas sp. strain SD212 and Paracoccus sp. strain N81106 (formerly called Agrobacterium aurantiacum). The seven key gene cassettes, in which each gene was surrounded by an appropriate promoter and terminator, were connected in a tandem manner, and the resulting constructs (17 kb) were inserted into a binary vector and used for transformation of B. napus. Surprisingly, 73-85% of the regenerated plants retained all seven genes, and formed orange- or pinkish orange-coloured seeds (embryos), while untransformed controls had light yellow-coloured seeds with predominant accumulation of lutein. Three of the transgenic lines were analysed further. The total amount of carotenoids in these seeds was 412-657 microg g(-1) fresh weight, which was a 19- to 30-fold increase compared with that of untransformed controls. The total amount of ketocarotenoids was 60-190 microg g(-1) fresh weight. beta-Carotene was the predominant carotenoid, with significant amounts of alpha-carotene, echinenone, phytoene, lutein, and canthaxanthin also detected in the transgenic seeds. The ratio of hydroxylated carotenoids to overall carotenoids was quite small relative to the ratio of ketocarotenoids to overall carotenoids. Interestingly, expression of many endogenous carotenogenic genes was also altered in the transgenic seeds, suggesting that their expression was affected by an increase in carotenoid biosynthesis.

Chlamydomonas reinhardtii adapts to the stress of CO 2 -limiting conditions through the induction of a set of genes including CAH1, which encodes a periplasmic carbonic anhydrase. CAH1 is upregulated under low-CO 2 conditions (air containing 0.04% [v/v] CO 2 ) in the presence of light, whereas it is down-regulated under high-CO 2 conditions (5% [v/v] CO 2 ) or in the dark. In an effort to identify cis-elements involved in the transcriptional regulation of CAH1, a series of 5-nested deletions of the region upstream of CAH1 were fused to a promoterless arylsulfatase reporter gene (ARS). The upstream region from ؊651 to ؉41 relative to the transcription start site was sufficient to regulate the expression of ARS with kinetics similar to those of endogenous CAH1. Deletion of the region between ؊651 and ؊294 resulted in a significant decrease in the level of arylsulfatase activity expressed under low-CO 2 conditions. The 543-bp upstream region from ؊651 to ؊109, without any promoter elements, CAAT-box, or TATA-box, could confer CO 2 and light responsiveness on the ␤ 2 -tubulin minimal promoter. This 543-bp region was divided into two parts: a 358-bp silencer region from ؊651 to ؊294, which represses the minimal promoter activity under high-CO 2 conditions, and a 185-bp enhancer region from ؊293 to ؊109, which activates the promoter under low-CO 2 conditions in the presence of light.