Transcriptome analysis of soybean (Glycine max) root genes differentially expressed in rhizobial, arbuscular mycorrhizal, and dual symbiosis.

Soybean (Glycine max) roots establish associations with nodule-inducing rhizobia and arbuscular mycorrhizal (AM) fungi. Both rhizobia and AM fungi have been shown to affect the activity of and colonization by the other, and their interactions can be detected within host plants. Here, we report the transcription profiles of genes differentially expressed in soybean roots in the presence of rhizobial, AM, or rhizobial-AM dual symbiosis, compared with those in control (uninoculated) roots. Following inoculation, soybean plants were grown in a glasshouse for 6 weeks; thereafter their root transcriptomes were analyzed using an oligo DNA microarray. Among the four treatments, the root nodule number and host plant growth were highest in plants with dual symbiosis. We observed that the expression of 187, 441, and 548 host genes was up-regulated and 119, 1,439, and 1,298 host genes were down-regulated during rhizobial, AM, and dual symbiosis, respectively. The expression of 34 host genes was up-regulated in each of the three symbioses. These 34 genes encoded several membrane transporters, type 1 metallothionein, and transcription factors in the MYB and bHLH families. We identified 56 host genes that were specifically up-regulated during dual symbiosis. These genes encoded several nodulin proteins, phenylpropanoid metabolism-related proteins, and carbonic anhydrase. The nodulin genes up-regulated by the AM fungal colonization probably led to the observed increases in root nodule number and host plant growth. Some other nodulin genes were down-regulated specifically during AM symbiosis. Based on the results above, we suggest that the contribution of AM fungal colonization is crucial to biological N2-fixation and host growth in soybean with rhizobial-AM dual symbiosis.

The conservation of polyol transporter proteins and their involvement in lichenized Ascomycota.

In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. However, the existence of polyol transporter genes and their correlation with lichenization remain unclear. Here, we report candidate polyol transporter genes selected from the genome of the lichen-forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and various ascomycetous fungi suggested that the characterized yeast' polyol transporters form multiple clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless of lichen-forming status. In addition, the phylogenetic clusters suggested that LFFs belonging to Lecanoromycetes have duplicated proteins in each cluster. Consequently, the number of sequences similar to characterized yeast' polyol transporters were evaluated using the genomes of 472 species or strains of Ascomycota. Among these, LFFs belonging to Lecanoromycetes had greater numbers of deduced polyol transporter proteins. Thus, various polyol transporters are conserved in Ascomycota and polyol transporter genes appear to have expanded during the evolution of Lecanoromycetes.

Chiba Tendril-Less locus determines tendril organ identity in melon (Cucumis melo L.) and potentially encodes a tendril-specific TCP homolog.

Tendrils are filamentous plant organs that coil on contact with an object, thereby providing mechanical support for climbing to reach more sunlight. Plant tendrils are considered to be modified structure of leaves, stems, or inflorescence, but the origin of cucurbit tendrils is still argued because of the complexity in the axillary organ patterning. We carried out morphological and genetic analyses of the Chiba Tendril-Less (ctl) melon (Cucumis melo) mutant, and found strong evidence that the melon tendril is a modified organ derived from a stem-leaf complex of a lateral shoot. Heterozygous (CTL/ctl) plants showed traits intermediate between tendril and shoot, and ontogenies of wild-type tendrils and mutant modified shoots coincided. We identified the CTL locus in a 200-kb region in melon linkage group IX. A single base deletion in a melon TCP transcription factor gene (CmTCP1) was detected in the mutant ctl sequence, and the expression of CmTCP1 was specifically high in wild-type tendrils. Phylogenetic analysis demonstrated the novelty of the CmTCP1 protein and the unique molecular evolution of its orthologs in the Cucurbitaceae. Our results move us closer to answering the long-standing question of which organ was modified to become the cucurbit tendril, and suggest a novel function of the TCP transcription factor in plant development.

Denitrification in soil amended with thermophile-fermented compost suppresses nitrate accumulation in plants.

NO (3) (-) is a major nitrogen source for plant nutrition, and plant cells store NO (3) (-) in their vacuoles. Here, we report that a unique compost made from marine animal resources by thermophiles represses NO (3) (-) accumulation in plants. A decrease in the leaf NO (3) (-) content occurred in parallel with a decrease in the soil NO (3) (-) level, and the degree of the soil NO (3) (-) decrease was proportional to the compost concentration in the soil. The compost-induced reduction of the soil NO (3) (-) level was blocked by incubation with chloramphenicol, indicating that the soil NO (3) (-) was reduced by chloramphenicol-sensitive microbes. The compost-induced denitrification activity was assessed by the acetylene block method. To eliminate denitrification by the soil bacterial habitants, soil was sterilized with γ irradiation and then compost was amended. After the 24-h incubation, the N(2)O level in the compost soil with presence of acetylene was approximately fourfold higher than that in the compost soil with absence of acetylene. These results indicate that the low NO (3) (-) levels that are often found in the leaves of organic vegetables can be explained by compost-mediated denitrification in the soil.

Nitric oxide (NO) as an intermediate in the cryptogein-induced hypersensitive response--a critical re-evaluation.

A hypersensitive response (HR) was induced in tobacco leaves and cell suspensions by the fungal elicitor cryptogein, and NO production was followed by chemiluminescence and occasionally by diaminofluorescein (DAF)-fluorescence. Results from both methods were at least partly consistent, but kinetics was different. NO emission was not induced by cryptogein in leaves, whereas in cell suspensions some weak NO emission was observed, which was nitrate reductase (NR)-dependent, but not required for cell death. Nitric oxide synthase (NOS) inhibitors did not prevent cell death, but PR-1 expression was weakened. In conclusion, neither NR nor NOS appear obligatory for the cryptogein-induced HR. However, a role for NO was still suggested by the fact that the NO scavenger cPTIO prevented the HR. Unexpectedly, cPTI, the reaction product of cPTIO and NO, also impaired the HR but without scavenging NO. Thus, prevention of the HR by cPTIO is not necessarily indicative for a role of NO. Further, even a 100-fold NO overproduction (over wild type) by a nitrite reductase-deficient mutant did not interfere with the cryptogein-induced HR. Accordingly, the role of NO in the HR should be reconsidered.

Nitric oxide emission from tobacco leaves and cell suspensions: rate limiting factors and evidence for the involvement of mitochondrial electron transport.

Quantitative data on nitric oxide (NO) production by plants, and knowledge of participating reactions and rate limiting factors are still rare. We quantified NO emission from tobacco (Nicotiana tabacum) wild-type leaves, from nitrate reductase (NR)- or nitrite reductase (NiR)-deficient leaves, from WT- or from NR-deficient cell suspensions and from mitochondria purified from leaves or cells, by following NO emission through chemiluminescence detection. In all systems, NO emission was exclusively due to the reduction of nitrite to NO, and the nitrite concentration was an important rate limiting factor. Using inhibitors and purified mitochondria, mitochondrial electron transport was identified as a major source for reduction of nitrite to NO, in addition to NR. NiR and xanthine dehydrogenase appeared to be not involved. At equal respiratory activity, mitochondria from suspension cells had a much higher capacity to produce NO than leaf mitochondria. NO emission in vivo by NiR-mutant leaves (which was not nitrite limited) was proportional to photosynthesis (high in light +CO(2), low in light -CO(2), or in the dark). With most systems including mitochondrial preparations, NO emission was low in air (and darkness for leaves), but high under anoxia (nitrogen). In contrast, NO emission by purified NR was not much different in air and nitrogen. The low aerobic NO emission of darkened leaves and cell suspensions was not due to low cytosolic NADH, and appeared only partly affected by oxygen-dependent NO scavenging. The relative contribution of NR and mitochondria to nitrite-dependent NO production is estimated.

Arabidopsis nonsymbiotic hemoglobin AHb1 modulates nitric oxide bioactivity.

Nitric oxide (NO) is a widespread signaling molecule, and numerous targets of its action exist in plants. Whereas the activity of NO in erythrocytes, microorganisms, and invertebrates has been shown to be regulated by several hemoglobins, the function of plant hemoglobins in NO detoxification has not yet been elucidated. Here, we show that Arabidopsis thaliana nonsymbiotic hemoglobin AHb1 scavenges NO through production of S-nitrosohemoglobin and reduces NO emission under hypoxic stress, indicating its role in NO detoxification. However, AHb1 does not affect NO-mediated hypersensitive cell death in response to avirulent Pseudomonas syringae, suggesting that it is not involved in the removal of NO bursts originated from acute responses when NO mediates crucial defense signaling functions.

Genetic elucidation of nitric oxide signaling in incompatible plant-pathogen interactions.

Recent experiments indicate that nitric oxide (NO) plays a pivotal role in disease resistance and several other physiological processes in plants. However, most of the current information about the function of NO in plants is based on pharmacological studies, and additional approaches are therefore required to ascertain the role of NO as an important signaling molecule in plants. We have expressed a bacterial nitric oxide dioxygenase (NOD) in Arabidopsis plants and/or avirulent Pseudomonas syringae pv tomato to study incompatible plant-pathogen interactions impaired in NO signaling. NOD expression in transgenic Arabidopsis resulted in decreased NO levels in planta and attenuated a pathogen-induced NO burst. Moreover, NOD expression in plant cells had very similar effects on plant defenses compared to NOD expression in avirulent Pseudomonas. The defense responses most affected by NO reduction during the incompatible interaction were decreased H(2)O(2) levels during the oxidative burst and a blockage of Phe ammonia lyase expression, the key enzyme in the general phenylpropanoid pathway. Expression of the NOD furthermore blocked UV light-induced Phe ammonia lyase and chalcone synthase gene expression, indicating a general signaling function of NO in the activation of the phenylpropanoid pathway. NO possibly functions in incompatible plant-pathogen interactions by inhibiting the plant antioxidative machinery, and thereby ensuring locally prolonged H(2)O(2) levels. Additionally, albeit to a lesser extent, we observed decreases in salicylic acid production, a diminished development of hypersensitive cell death, and a delay in pathogenesis-related protein 1 expression during these NO-deficient plant-pathogen interactions. Therefore, this genetic approach confirms that NO is an important regulatory component in the signaling network of plant defense responses.

Cloning of a nitrate reductase inactivator (NRI) cDNA from Spinacia oleracea L. and expression of mRNA and protein of NRI in cultured spinach cells.

The spinach ( Spinacia oleracea L. (cv. Hoyo) nitrate reductase inactivator (NRI) is a novel protein that irreversibly inactivates NR. Using degenerate primers based on an N-terminal amino acid sequence of NRI purified from spinach leaves and a cDNA library, we isolated a full-length NRI cDNA from spinach that contains an open reading frame encoding 479 amino acid residues. This protein shares 67.4% and 51.1-68.3% amino acid sequence similarities with a nucleotide pyrophosphatase (EC 3.6.1.9) from rice and three types of the nucleotide pyrophosphatase-like protein from Arabidopsis thaliana, respectively. Immunoblot analysis revealed that NRI was constitutively expressed in suspension-cultured spinach cells; however, its expression level is quite low in 1-day-subcultured cells. Moreover, northern blot analysis indicated that this expression was regulated at the mRNA level. These results suggest that NRI functions in mature cells.

Genes essential to sodium-dependent bicarbonate transport in cyanobacteria: function and phylogenetic analysis.

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 DeltamuNa(+) 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.

Modulation of nitrate reductase: some new insights, an unusual case and a potentially important side reaction.

The mechanism of the post-translational modulation of nitrate reductase activity (NR, EC 1.6.6.1) is briefly summarized, and it is shown that by this mechanism nitric oxide production through NR is also rapidly modulated. New and partly unexpected details on the modulation mechanism have been obtained by using immunological techniques. The phosphorylation state of NR has been assessed with peptide antibodies raised against the serine phosphorylation motive of spinach NR. By co-immunoprecipitation experiments, 14-3-3 binding to phospho-NR and the function of Mg(2+) in that process has been elucidated. Conflicting data on the role of NR phosphorylation and 14-3-3 binding in controlling NR proteolysis are discussed. A possible role of other NR inactivating proteins is also briefly considered and the regulation of NR of Ricinus communis is described as an interesting special case that differs from the 'normal' mechanism in several important aspects.

Functionally distinct NAD(P)H dehydrogenases and their membrane localization in Synechocystis sp. PCC6803.

The type I NAD(P)H dehydrogenase complex (NDH-1) in cyanobacteria is involved in both respiratory and photosynthetic electron transport processes. NDH-1 is also essential for inorganic carbon transport. It has been postulated that NDH-1-dependent cyclic electron flow around PSI energizes CO2 uptake. The genome information of Synechocystis sp. PCC6803 has enabled us to provide an integrative view of the CO2 concentrating mechanism in this organism. In an attempt to dissect the role of the NDH-1 complex, we have constructed single and double mutants of Synechocystis 6803 by disrupting highly homologous ndhD genes in pairs, and have analysed the growth, CO2 uptake activities, and redox levels of P700 and the plastoquinone pool in these mutants under various conditions. We have also determined the membrane localization of this membrane protein. Our studies have revealed that: (i) mutations in ndh genes lead to inhibition of CO2 uptake, rather than HCO3- uptake; (ii) NDH-1 complexes are localized only in the thylakoid membrane; (iii) there are functionally distinct NDH-1 complexes in Synechocystis #6803. Based on these data, we propose a schematic view of the roles of different NDH-1 complexes in cyanobacteria.