Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the~120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

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A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

(H.K., M.T.)Phototropins (phot1 and phot2, formerly designated nph1 and npl1) are blue-light receptors that mediate phototropism, blue light-induced chloroplast relocation, and blue light-induced stomatal opening in Arabidopsis. Phototropins contain two light, oxygen, or voltage (LOV) domains at their N termini (LOV1 and LOV2), each a binding site for the chromophore flavin mononucleotide (FMN). Their C termini contain a serine/threonine protein kinase domain. Here, we examine the kinetic properties of the LOV domains of Arabidopsis phot1 and phot2, rice (Oryza sativa) phot1 and phot2, and Chlamydomonas reinhardtii phot. When expressed in Escherichia coli, purified LOV domains from all phototropins examined bind FMN tightly and undergo a self- The photocycle involves the light-induced formation of a cysteinyl adduct to the C(4a) carbon of the FMN chromophore, which subsequently breaks down in darkness. In each case, the relative quantum efficiencies for the photoreaction and the rate constants for dark recovery of LOV1, LOV2, and peptides containing both LOV domains are presented. Moreover, the data obtained from full-length Arabidopsis phot1 and phot2 expressed in insect cells closely resemble those obtained for the tandem LOV-domain fusion proteins expressed in E. coli. For both Arabidopsis and rice phototropins, the LOV domains of phot1 differ from those of phot2 in their reaction kinetic properties and relative quantum efficiencies. Thus, in addition to differing in amino acid sequence, the phototropins can be distinguished on the basis of the photochemical cycles of their LOV domains. The LOV domains of C. reinhardtii phot also undergo light-activated spectral changes consistent with cysteinyl adduct formation. Thus, the phototropin family extends over a wide evolutionary range from unicellular algae to higher plants.Plants use light not only as an energy source for photosynthesis but also as a signal to indicate the properties of their surrounding environment. UV-A (320-390 nm) and blue (390-500 nm) light regulate a wide variety of responses in higher plants. These responses include phototropism, chloroplast relocation, inhibition of hypocotyl elongation, circadian timing, regulation of gene expression, and stomatal opening (Briggs and Huala, 1999;Lin, 2000;Christie and Briggs, 2001;Briggs et al., 2001b). Four blue-light receptors have been identified from the model higher plant Arabidopsis: cryptochrome 1 (Ahmad et al., 1993), cryptochrome 2 (Hoffman et al., 1996;Lin et al., 1996Lin et al., , 1998, phototropin 1 (phot1; Huala et al., 1997;Christie et al., 1998Christie et al., , 1999, and phototropin 2 (phot2; Jarillo et al., 1998).In Arabidopsis, phot1 and phot2 (formerly known as nph1 and npl1, respectively; see Briggs et al., 2001a) have been shown to serve as photoreceptors mediating phototropism (Huala et al., 1997;Christie et al., 1998), blue light-mediated chloroplast movement Sakai et al., 2001;Jarillo et al., 2001), and blue light-induced stomatal opening (Kinoshita et al., 2002 Article, publicati...

Cyanobacteria possess a CO2-concentating mechanism that involves active CO2 uptake and HCO 3 ؊ transport. For CO2 uptake, we have identified two systems in the cyanobacterium Synechocystis sp. strain PCC 6803, one induced at low CO 2 and one constitutive. The low CO2-induced system showed higher maximal activity and higher affinity for CO2 than the constitutive system. On the basis of speculation that separate NAD(P)H dehydrogenase complexes were essential for each of these systems, we reasoned that inactivation of one system would allow selection of mutants defective in the other. Thus, mutants unable to grow at pH 7.0 in air were recovered after transformation of a ⌬ndhD3 mutant with a transposon-bearing library. Four of them had tags within slr1302 (designated cupB), a homologue of sll1734 (cupA), which is cotranscribed with ndhF3 and ndhD3. The ⌬cupB, ⌬ndhD4, and ⌬ndhF4 mutants showed CO2-uptake characteristics of the low CO2-induced system observed in wild type. In contrast, mutants ⌬cupA, ⌬ndhD3, and ⌬ndhF3 showed characteristics of the constitutive CO2-uptake system. Double mutants impaired in one component of each of the systems were unable to take up CO 2 and required high CO2 for growth. Phylogenetic analysis indicated that the ndhD3͞ ndhD4-, ndhF3͞ndhF4-, and cupA͞cupB-type genes are present only in cyanobacteria. Most of the cyanobacterial strains studied possess the ndhD3͞ndhD4-, ndhF3͞ndhF4-, and cupA͞cupB-type genes in pairs. Thus, the two types of NAD(P)H dehydrogenase complexes essential for low CO2-induced and constitutive CO2-uptake systems associated with the NdhD3͞NdhF3͞CupA-homologues and NdhD4͞NdhF4͞CupB-homologues, respectively, appear to be present in these cyanobacterial strains but not in other organisms.NAD(P)H dehydrogenase ͉ constitutive CO2 uptake ͉ affinity to CO2 ͉ CO2-concentrating mechanism I n cyanobacteria, NAD(P)H dehydrogenase (NDH-1) is essential for both CO 2 uptake (1-3) and photosystem-1 (PSI) cyclic electron transport (4). It has been postulated that uptake of CO 2 is energized by NDH-1-dependent PSI-cyclic electron transport (1). However, observations that mutants defective in ndhD3 display normal cyclic electron transport but are unable to induce high-affinity CO 2 uptake suggest the presence of multiple NDH-1 complexes (5-7). Two types of functionally distinct NDH-1 complexes were recently recognized in Synechocystis sp. strain PCC 6803 with the aid of mutants impaired in one or more subunits of NDH-1 (7). One complex, containing NdhD1 or NdhD2, plays a major role in PSI-cyclic electron f low but is not involved in CO 2 uptake (7). When the second type of NDH-1 complex is inactivated (in the double mutant ⌬ndhD3͞⌬ndhD4), nearly normal PSI-cyclic electron f low is observed, but the mutant does not take up CO 2 and is unable to grow under an air level of CO 2 (7). The single mutants ⌬ndhD3 and ⌬ndhD4, on the other hand, possess CO 2 -uptake activity and can grow under low CO 2 conditions (7). These results raised the possibility of multiple systems for CO 2 uptake. In...

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.

The cyanobacterium Synechocystis sp. strain PCC 6803 possesses two CO 2 uptake systems and two HCO 3 ؊ transporters. We transformed a mutant impaired in CO 2 uptake and in cmpA-D encoding a HCO 3 ؊ transporter with a transposon inactivation library, and we recovered mutants unable to take up HCO 3 ؊ and grow in low CO 2 at pH 9.0. They are all tagged within slr1512 (designated sbtA). We show that SbtA-mediated transport is induced by low CO 2 , requires Na ؉ , and plays the major role in HCO 3 ؊ uptake in Synechocystis. Inactivation of slr1509 (homologous to ntpJ encoding a Na ؉ /K ؉ -translocating protein) abolished the ability of cells to grow at [Na ؉ ] higher than 100 mM and severely depressed the activity of the SbtA-mediated HCO 3 ؊ transport. We propose that the SbtA-mediated HCO 3 ؊ transport is driven by ⌬Na ؉ across the plasma membrane, which is disrupted by inactivating ntpJ. Phylogenetic analyses indicated that two types of sbtA exist in various cyanobacterial strains, all of which possess ntpJ. The sbtA gene is the first one identified as essential to Na ؉ -dependent HCO 3 ؊ transport in photosynthetic organisms and may play a crucial role in carbon acquisition when CO 2 supply is limited, or in Prochlorococcus strains that do not possess CO 2 uptake systems or Cmp-dependent HCO 3 ؊ transport.Growth of many photosynthetic microorganisms depends on the activity of a CO 2 -concentrating mechanism (CCM), 1 which raises the [CO 2 ] in close proximity to ribulose-1,5-bisphosphate carboxylase/oxygenase and thereby enables efficient CO 2 fixation despite the low affinity of the enzyme for CO 2 (1, 2). In the cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803), the CCM involves active CO 2 uptake and HCO 3 Ϫ transport. We have recently identified two systems for CO 2 uptake in Synechocystis 6803, one constitutive and the other inducible by low CO 2 (3). As deduced from phylogenetic analysis of proteins encoded by the genes involved, these CO 2 uptake systems are present in various cyanobacteria with the exception of Prochlorococcus marinus (3). The inducible system that depends on NdhD3/ NdhF3/CupA shows higher maximal activity and higher affinity for CO 2 than the constitutive, NdhD4/NdhF4/CupB-dependent system. Inactivation of two different genes, one encoding a component of the constitutive system and the other a constituent of the inducible system, abolished CO 2 uptake activity. The double mutants were unable to grow at pH 7.0 under air level of CO 2 (3, 4). In contrast, because the mutants possessed HCO 3 Ϫ transport capability, they could grow like the wild type (WT) at pH 9.0 in air.An ABC-type HCO 3 Ϫ transporter encoded by cmpABCD has been identified in Synechococcus sp. strain PCC 7942 (thereafter Synechococcus 7942) (5). Inactivation of cmp genes in Synechocystis 6803, however, had little effect on the HCO 3 Ϫ transport activity. This indicated that another HCO 3 Ϫ transporter, as yet unidentified, plays a central role in HCO 3 Ϫ uptake. Sodium ions are essential for ...