Legume plants host nitrogen-fixing endosymbiotic Rhizobium bacteria in root nodules. In Medicago truncatula, the bacteria undergo an irreversible (terminal) differentiation mediated by hitherto unidentified plant factors. We demonstrated that these factors are nodule-specific cysteine-rich (NCR) peptides that are targeted to the bacteria and enter the bacterial membrane and cytosol. Obstruction of NCR transport in the dnf1-1 signal peptidase mutant correlated with the absence of terminal bacterial differentiation. On the contrary, ectopic expression of NCRs in legumes devoid of NCRs or challenge of cultured rhizobia with peptides provoked symptoms of terminal differentiation. Because NCRs resemble antimicrobial peptides, our findings reveal a previously unknown innovation of the host plant, which adopts effectors of the innate immune system for symbiosis to manipulate the cell fate of endosymbiotic bacteria.

Cryptochrome flavoproteins, which share sequence homology with light-dependent DNA repair photolyases, function as photoreceptors in plants and circadian clock components in animals. Here, we coupled sequencing of an Arabidopsis cryptochrome gene with phylogenetic, structural, and functional analyses to identify a new cryptochrome class (cryptochrome DASH) in bacteria and plants, suggesting that cryptochromes evolved before the divergence of eukaryotes and prokaryotes. The cryptochrome crystallographic structure, reported here for Synechocystis cryptochrome DASH, reveals commonalities with photolyases in DNA binding and redox-dependent function, despite distinct active-site and interaction surface features. Whole genome transcriptional profiling together with experimental confirmation of DNA binding indicated that Synechocystis cryptochrome DASH functions as a transcriptional repressor.

Cyanobacteria are the only bacterial species found to have a circadian clock. We used DNA microarrays to examine circadian expression patterns in the cyanobacterium Synechocystis sp. strain PCC 6803. Our analysis identified 54 (2%) and 237 (9%) genes that exhibited circadian rhythms under stringent and relaxed filtering conditions, respectively. The expression of most cycling genes peaked around the time of transition from subjective day to night, suggesting that the main role of the circadian clock in Synechocystis is to adjust the physiological state of the cell to the upcoming night environment. There were several chromosomal regions where neighboring genes were expressed with similar circadian patterns. The physiological functions of the cycling genes were diverse and included a wide variety of metabolic pathways, membrane transport, and signal transduction. Genes involved in respiration and poly(3-hydroxyalkanoate) synthesis showed coordinated circadian expression, suggesting that the regulation is important for the supply of energy and carbon source in the night. Genes involved in transcription and translation also followed circadian cycling patterns. These genes may be important for output of the rhythmic information generated by the circadian clock. Our findings provided critical insights into the importance of the circadian clock on cellular physiology and the mechanism of clock-controlled gene regulation.Circadian rhythm is a self-sustaining oscillation whose period length coincides with the 24-h day-night cycle. Many biological activities show circadian patterns, allowing organisms to adapt to daily fluctuations in the environment. Circadian rhythms are widespread and involve functions as diverse as human sleep-wake cycles and cyanobacterial nitrogen fixation. The molecular basis of circadian rhythms involves negative and positive feedback regulation of clock genes (16).Cyanobacteria are the only bacterial species found to have a circadian clock. Three clock genes, kaiA, kaiB, and kaiC, have been identified in Synechococcus sp. strain PCC 7942 (25). kaiB and kaiC form an operon and are coordinately transcribed, while kaiA is transcribed independently. All of the kai genes show circadian rhythms of expression. Continuous overexpression of kaiC represses expression of kaiBC, but overexpression of kaiA enhances expression of kaiBC. This suggests that kaiC is regulated by a negative feedback autoregulatory loop and that KaiA activates kaiBC expression, thus sustaining the cyclical expression of kaiBC (25).In cyanobacteria, activities as diverse as cell division, amino acid uptake, nitrogen fixation, respiration, and carbohydrate synthesis are under circadian control (18), but a clear mechanistic link between physiological rhythms and the regulation of output genes is still lacking. Promoter trap analyses were performed with two cyanobacterial species, Synechococcus sp. strain PCC 7942 (33) and Synechocystis sp. strain PCC 6803 (4). The percentage of rhythmic clones was lower in Synechocystis organisms (77...

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...

The actinobacteria Frankia spp. are able to induce the formation of nodules on the roots of a large spectrum of actinorhizal plants, where they convert dinitrogen to ammonia in exchange for plant photosynthates. In the present study, transcriptional analyses were performed on nitrogen-replete free-living Frankia alni cells and on Alnus glutinosa nodule bacteria, using whole-genome microarrays. Distribution of nodule-induced genes on the genome was found to be mostly over regions with high synteny between three Frankia spp. genomes, while nodule-repressed genes, which were mostly hypothetical and not conserved, were spread around the genome. Genes known to be related to nitrogen fixation were highly induced, nif (nitrogenase), hup2 (hydrogenase uptake), suf (sulfur-iron cluster), and shc (hopanoids synthesis). The expression of genes involved in ammonium assimilation and transport was strongly modified, suggesting that bacteria ammonium assimilation was limited. Genes involved in particular in transcriptional regulation, signaling processes, protein drug export, protein secretion, lipopolysaccharide, and peptidoglycan biosynthesis that may play a role in symbiosis were also identified. We also showed that this Frankia symbiotic transcriptome was highly similar among phylogenetically distant plant families Betulaceae and Myricaceae. Finally, comparison with rhizobia transcriptome suggested that F. alni is metabolically more active in symbiosis than rhizobia.

SummaryPlant hemoglobins (Hbs) have been divided into three groups: class 1, class 2, and truncated Hbs. The various physiological functions of class 1 Hb include its role as a modulator of nitric oxide (NO) levels in plants. To gain more insight into the functions of class 1 Hbs, we investigated the physical properties of LjHb1 and AfHb1, class 1 Hbs of a model legume Lotus japonicus and an actinorhizal plant Alnus firma, respectively. Spectrophotometric analysis showed that the recombinant form of the LjHb1 and AfHb1 proteins reacted with NO. The localization of LjHb1 expression was correlated with the site of NO production. Overexpression of LjHb1 and AfHb1 by transformed hairy roots caused changes in symbiosis with rhizobia. The number of nodules formed on hairy roots overexpressing LjHb1 or AfHb1 increased compared with that on untransformed hairy roots. Furthermore, nitrogenase activity as acetylene-reduction activity (ARA) of LjHb1-or AfHb1-overexpressing nodules was higher than that of the vector control nodules. Microscopic observation with a NO-specific fluorescent dye suggested that the NO level in LjHb1-and AfHb1-overexpressing nodules was lower than that of control nodules. Exogenous application of a NO scavenger enhanced ARA in L. japonicus nodules, whereas a NO donor inhibited ARA. These results suggest that the basal level of NO in nodules inhibits nitrogen fixation, and overexpression of class 1 Hbs enhances symbiotic nitrogen fixation activity by removing NO as an inhibitor of nitrogenase.

Symbiotic nitrogen fixation by the collaboration between leguminous plants and rhizobia is an important system in the global nitrogen cycle, and some molecular aspects during the early stage of host-symbiont recognition have been revealed. To understand the responses of a host plant against various bacteria, we examined expression of hemoglobin (Hb) genes and production of nitric oxide (NO) in Lotus japonicus after inoculation with rhizobia or plant pathogens. When the symbiotic rhizobium Mesorhizobium loti was inoculated, expression of LjHb1 and NO production were induced transiently in the roots at 4 h after inoculation. In contrast, inoculation with the nonsymbiotic rhizobia Sinorhizobium meliloti and Bradyrhizobium japonicum induced neither expression of LjHb1 nor NO production. When L. japonicus was inoculated with plant pathogens (Ralstonia solanacearum or Pseudomonas syringae), continuous NO production was observed in roots but induction of LjHb1 did not occur. These results suggest that modulation of NO levels and expression of class 1 Hb are involved in the establishment of the symbiosis.

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/.