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Graminaceous cereal crops as well as all other green plants require iron (Fe), as Fe is a component of heme and [Fe-S] clusters in the photosystems (PSI, PSII) containing green pigments (chlorophyl...

International audience; Roots of the higher plants can assimilate inorganic nitrogen by an enzymatic reduction of the most oxidized form (+6) nitrate to the reduced form (-2) glutamate. For such reactions, the substrates (originated from photosynthates) must be imported to supply energy through the reductant-generating systems within the root cells. Intensive studies over last 70 years (reviewed here) revealed the precise mechanisms of nitrate-to-glutamate transformation in roots with elaborate searches of N-15-tracing, enzymes involved, the reductant-supplying system, and nitrate signaling. In the 1970s, the tracing of N-15-labeled nitrate and ammonia in the roots demonstrated the sequential reduction and assimilation of nitrate to nitrite, ammonia, glutamine amide, and then glutamate. These reactions involve nitrate reductase (NADH-NR, EC 1.7.1.1) in the cytosol, nitrite reductase (ferredoxin [Fd]-NiR, EC 1.7.7.1), glutamine synthetase (GS2, EC 6.3.1.2), and glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in the plastids. NADH for NR is generated by glycolysis in the cytosol, and NADPH for Fd-NIR and Fd-GOGAT are produced by the oxidative pentose phosphate pathway (OPPP). Electrons from NADPH are conveyed to reduce NIR and Fd-GOGAT through Fd-NADP(+) reductase (FNR, EC 1.6.7.1) specifically in the roots. Physiological and molecular analyses showed the parallel inductions of NR, NIR, GS2, Fd-GOGAT, OPPP enzymes, FNR, and Fd in response to a short-term nitrate supply. Recent studies proposed a molecular mechanism of nitrate-induction of these genes and proteins. Roots can also assimilate the reduced form of inorganic ammonia by the combination of cytosolic GS1 and plastidic NADH-GOGAT.

Biological nitrogen fixation (BNF) by plants and its bacterial associations represent an important natural system for capturing atmospheric dinitrogen (N2) and processing it into a reactive form of nitrogen through enzymatic reduction. The study of BNF in non-leguminous plants has been difficult compared to nodule-localized BNF in leguminous plants because of the diverse sites of N2 fixation in non-leguminous plants. Identification of the involved N2-fixing bacteria has also been difficult because the major nitrogen fixers were often lost during isolation attempts. The past 20 years of molecular analyses has led to the identification of N2 fixation sites and active nitrogen fixers in tissues and the rhizosphere of non-leguminous plants. Here, we examined BNF hotspots in six reported non-leguminous plants. Novel rhizobia and methanotrophs were found to be abundantly present in the free-living state at sites where carbon and energy sources were predominantly available. In the carbon-rich apoplasts of plant tissues, rhizobia such as Bradyrhizobium spp. microaerobically fix N2. In paddy rice fields, methane molecules generated under anoxia are oxidized by xylem aerenchyma-transported oxygen with the simultaneous fixation of N2 by methane-oxidizing methanotrophs. We discuss the effective functions of the rhizobia and methanotrophs in non-legumes for the acquisition of fixed nitrogen in addition to research perspectives.

Roots of the higher plants can assimilate inorganic nitrogen by an enzymatic reduction of the most oxidized form (+6) nitrate to the reduced form (-2) glutamate. For such reactions, the substrates (originated from photosynthates) must be imported to supply energy through the reductant-generating systems within the root cells. Intensive studies over last 70 years (reviewed here) revealed the precise mechanisms of nitrate-to-glutamate transformation in roots with elaborate searches of

Zinc (Zn) and iron (Fe) are essential but are sometimes deficient in humans, while cadmium (Cd) is toxic if it accumulates in the liver and kidneys at high levels. All three are contained in the grains of rice, a staple cereal. Zn and Fe concentrations in rice grains harvested under different levels of soil/hydroponic metals are known to change only within a small range, while Cd concentrations show greater changes. To clarify the mechanisms underlying such different metal contents, we synthesized information on the routes of metal transport and accumulation in rice plants by examining metal speciation, metal transporters, and the xylem-to-phloem transport system. At grain-filling, Zn and Cd ascending in xylem sap are transferred to the phloem by the xylem-to-phloem transport system operating at stem nodes. Grain Fe is largely derived from the leaves by remobilization. Zn and Fe concentrations in phloem-sap and grains are regulated within a small range, while Cd concentrations vary depending on xylem supply. Transgenic techniques to increase concentrations of the metal chelators (nicotianamine, 2′-deoxymugineic acid) are useful in increasing grain Zn and Fe concentrations. The elimination of OsNRAMP5 Cd-uptake transporter and the enhancement of root cell vacuolar Cd sequestration reduce uptake and root-to-shoot transport, respectively, resulting in a reduction of grain Cd accumulation.

Soil fertility has been an important factor in sustainable plant production in native and agricultural fields in temperate climates such as that in Japan. Soil fertility is assessed based on the availability of nutrients, in particular inorganic nitrogen (N) and phosphorus (P), from soil-accumulated organic matter (SOM) via microbial immobilization and mineralization. However, the pool sizes of SOM in humid tropics such as those in Thailand are small and they are turned over rapidly; under such circumstances, the tropical soil fertility would soon be depleted. To meet the urgent requirement of plant nutrients for high plant productivity, we define a direct supply of plant nutrients (i.e., residue fertility) from raw plant and microbial residues. The residue fertility may be driven by the activities of soil fauna (e.g., earthworms, collembolans, termites) and micro-organisms (e.g., saprophytic fungi, protozoa, bacteria), and the released nutrients may be collected and absorbed directly by plant roo...

We examined the concentrations of metals (Cd, Zn, Cu, Fe and Mn) and potential metal-binding compounds [nicotianamine (NA), thiol compounds and citrate] in xylem and phloem saps from 4-week-old castor bean plants (Ricinus communis) treated with 0 (control), 0.1, 1.0, and 10 μM Cd for 3 weeks. Treatment with 0.1 and 1 μM Cd produced no visible damage, while 10 μM Cd retarded growth. Cadmium concentrations in both saps were higher than those in the culture solution at 0.1 μM, similar at 1.0 μM and lower at 10 μM. Cd at 10 μM reduced Cu and Fe concentrations in both saps. NA concentrations measured by capillary electrophoresis-mass spectrometry (MS) in xylem sap (20 μM) were higher than the Cu concentrations, and those in phloem sap (150 μM) were higher than those of Zn, Fe and Cu combined. Reduced glutathione concentrations differed in xylem and phloem saps (1-2 and 30-150 μM, respectively), but oxidized glutathione concentrations were similar. Phloem sap phytochelatin 2 concentration increased from 0.8 μM in controls to 8 μM in 10 μM Cd. Free citrate was 2-4 μM in xylem sap and 70-100 μM in phloem sap. Total bound forms of Cd in phloem and xylem saps from 1 μM Cd-treated plants were 54 and 8%, respectively. Treatment of phloem sap with proteinaseK reduced high-molecular compounds while increasing fractions of low-molecular Cd-thiol complexes. Zinc-NA, Fe-NA and Cu-NA were identified in the phloem sap fraction of control plants by electrospray ionization time-of-flight MS, and the xylem sap contained Cu-NA.

Despite a general view that asparagine synthetase generates asparagine as an amino acid for long-distance transport of nitrogen to sink organs, its role in nitrogen metabolic pathways in floral organs during seed nitrogen filling has remained undefined. We demonstrate that the onset of pollination in Arabidopsis induces selected genes for asparagine metabolism, namely ASN1 (At3g47340), GLN2 (At5g35630), GLU1 (At5g04140), AapAT2 (At5g19950), ASPGA1 (At5g08100) and ASPGB1 (At3g16150), particularly at the ovule stage (stage 0), accompanied by enhanced asparagine synthetase protein, asparagine and total amino acids. Immunolocalization confined asparagine synthetase to the vascular cells of the silique cell wall and septum, but also to the outer and inner seed integuments, demonstrating the post-phloem transport of asparagine in these cells to developing embryos. In the asn1 mutant, aberrant embryo cell divisions in upper suspensor cell layers from globular to heart stages assign a role for nitrogen in differentiating embryos within the ovary. Induction of asparagine metabolic genes by light/dark and nitrate supports fine shifts of nitrogen metabolic pathways. In transgenic Arabidopsis expressing promoter CaMV35S::ASN1 fusion, marked metabolomics changes at stage 0, including a several-fold increase in free asparagine, are correlated to enhanced seed nitrogen. However, specific promoter Napin2S::ASN1 expression during seed formation and a six-fold increase in asparagine toward the desiccation stage result in wild-type seed nitrogen, underlining that delayed accumulation of asparagine impairs the timing of its use by releasing amide and amino nitrogen. Transcript and metabolite profiles in floral organs match the carbon and nitrogen partitioning to generate energy via the tricarboxylic acid cycle, GABA shunt and phosphorylated serine synthetic pathway.

In higher plants, the Dof transcription factors that harbour a conserved plant-specific DNA-binding domain function in the regulation of diverse biological processes that are unique to plants. Although these factors are present in both higher and lower plants, they have not yet been characterized in lower plants. Here six genes encoding Dof transcription factors in the moss Physcomitrella patens are characterized and two of these genes, PpDof1 and PpDof2, are functionally analysed. The targeted disruption of PpDof1 caused delayed or reduced gametophore formation, accompanied by an effect on development of the caulonema from the chloronema. Furthermore, the ppdof1 disruptants were found to form smaller colonies with a reduced frequency of branching of protonemal filaments, depending on the nutrients in the media. Most of these phenotypes were not apparent in the ppdof2 disruptant, although the ppdof2 disruptants also formed smaller colonies on a particular medium. Transcriptional repressor activity of PpDof1 and PpDof2 and modified expression of a number of genes in the ppdof disruptant lines were also shown. These results thus suggest that the PpDof1 transcriptional repressor has a role in controlling nutrient-dependent filament growth.

In rice (Oryza sativa L.), cadmium (Cd), a toxic heavy metal, is found in phloem sap and eventually accumulates in the grains. To further characterize phloem-transported Cd, the chemical forms of Cd and other metals and varietal differences in phloem sap Cd concentrations were investigated in young rice plants. The size-exclusion chromatography elution times for Cd-bound compounds indicated that phloem Cd in cv. Nipponbare exists mainly as an approximately 13 kDa complex. Protease digestion of rice phloem sap reduced the bound Cd content from 92 to 19%. The remaining Cd may bind to low-molecule SH compounds. An experiment examining in vitro addition of Cd2+ to phloem sap from non-Cd-treated plants revealed that rice phloem sap constitutively contains Cd chelators. The major Cd peak is distinguishable from those of Fe, Zn, Cu, Mn, Ni and Co, which probably bind to nicotianamine, 2′-deoxymugineic acid, citrate and histidine. The Cd concentrations of the phloem saps in three varieties (Milyang 23, L...

To explore the presence and expression of the nifH gene of diazotrophic endophytes in sugarcane (Saccharum spp. hybrids) cv. NiF8, we conducted pot experiments for two seasons in a glasshouse from 10 June to 17 September (high temperature and long days) in 2005 and 1 September to 9 December (low temperature and short days) in 2006. The expression of nifH genes in the stems and roots of sugarcane plants at 50 (or 59) and 100 days after transplanting was investigated by reverse transcription (RT)–PCR and by examining the nifH nucleotide sequence diversity. N2 fixation was assessed by the 15N-dilution method. The nifH RNA sequences in the stems and roots of the first experiment were similar to those of Bradyrhizobium sp. and Azorhizobium caulinodans. In the second experiment, nifH expression from these bacteria was not detected in the stems. nifH gene expression in the stems was in accordance with 18–24% N derived from air in the high-temperature season and negligible in the low-temperature season. Both the proliferation of N2 fixers and expression of the nifH gene indicated that bacteria similar to rhizobia may contribute to N2 fixation in sugarcane under our experimental conditions.

Eukaryotic translation initiation factor 6 (eIF6) is an essential component of ribosome biogenesis. In our present study, we characterize plant eIF6 genes for the first time. Although a single gene encodes eIF6 in yeast and animals, two genes were found to encode proteins homologous to animal and yeast eIF6 in Arabidopsis and rice, denoted At-eIF6;1 and At-eIF6;2, and Os-eIF6;1 and Os-eIF6;2, respectively. Analysis of the yeast eif6 (tif6) mutant suggested that plant eIF6, at least in the case of At-eIF6;1, can complement the essential function of eIF6 in yeast. Evidence for the essential role of eIF6 in plants was also provided by the embryonic-lethal phenotype of the at-eif6;1 mutant. In contrast, At-eIF6;2 appears not to be essential due to its very low expression level and the normal growth phenotype of the eif6;2 mutants. Consistent with the putative role of plant eIF6 in ribosome biogenesis, At-eIF6;1 is predominately expressed in tissues where cell division actively proceeds under the control of intronic cis-regulatory elements. On the other hand, both Os-eIF6;1 and Os-eIF6;2 are probably active genes because they are expressed at significant expression levels. Interestingly, the supply of ammonium nitrate as a plant nutrient was found to induce specifically the expression of Os-eIF6;2. Our present findings indicate that the eIF6 genes have differently evolved in plant and animal kingdoms and also in distinct plant species.

Xylem and phloem transport of cadmium (Cd), a toxic element, into rice grains was compared with transport of the nutritional elements zinc (Zn) and iron (Fe) in rice plants (Oryza sativa L.) grown under continuously flooded soil conditions. To explore their transport, the concentrations of Cd, Zn and Fe in xylem and phloem saps and in different tissues were determined at four (10th-leaf, 14th-leaf, early grain-filling, mature) stages using semi-dwarf rice plants (cv. Kantou) grown on soil contaminated with Cd fourfold higher than average Cd contamination in Japan. Phloem saps were collected from mature leaf sheathes at the first two stages and from the uppermost internodes at early grain filling. Xylem saps were collected from cut stems. The Cd concentrations in the xylem and phloem saps collected at the 14th-leaf and early grain-filling stages were lower than those at the 10th-leaf stage, and the Cd concentration in dehusked grain was low (0.05 mg kg−1 dry weight [DW]). In contrast, Zn and Fe co...

The presence of glutamine synthetase (GS) in the rice sieve tube was examined. Proteins in the rice phloem sap from leaf sheaths were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidine difluoride membranes and immunoblotted with anti-GS1 antibody. A cross-reacting band, thought to be GS1, was detected in the phloem sap. Moreover, the phloem sap contained a significant amount of GS transferase activity. Previous studies have shown that the concentrations of substrates and cofactors in the rice phloem sap are sufficient for cytosolic GS reaction. These data suggest that physiologically active GS1 is present in rice phloem sap, which might convert glutamate to glutamine in vivo.

We investigated the role of glutamine synthetases (cytosolic GS1 and chloroplast GS2) and glutamate synthases (ferredoxin-GOGAT and NADH-GOGAT) in the inorganic nitrogen assimilation and reassimilation into amino acids between bundle sheath cells and mesophyll cells for the remobilization of amino acids during the early phase of grain filling in Zea mays L. The plants responded to a light/dark cycle at the level of nitrate, ammonium and amino acids in the second leaf, upward from the primary ear, which acted as the source organ. The assimilation of ammonium issued from distinct pathways and amino acid synthesis were evaluated from the diurnal rhythms of the transcripts and the encoded enzyme activities of nitrate reductase, nitrite reductase, GS1, GS2, ferredoxin-GOGAT, NADH-GOGAT, NADH-glutamate dehydrogenase and asparagine synthetase. We discerned the specific role of the isoproteins of ferredoxin and ferredoxin:NADP(+) oxidoreductase in providing ferredoxin-GOGAT with photoreduced or enzymatically reduced ferredoxin as the electron donor. The spatial distribution of ferredoxin-GOGAT supported its role in the nitrogen (re)assimilation and reallocation in bundle sheath cells and mesophyll cells of the source leaf. The diurnal nitrogen recycling within the plants took place via the specific amino acids in the phloem and xylem exudates. Taken together, we conclude that the GS1/ferredoxin-GOGAT cycle is the main pathway of inorganic nitrogen assimilation and recycling into glutamine and glutamate, and preconditions amino acid interconversion and remobilization.

International audience; The aim of this study was to investigate the role of ASN3-encoded asparagine synthetase (AS, EC 6.3.5.4) during vegetative growth, seed development and germination of Arabidopsis thaliana. Phenotypic analysis of knockout (asn3-1) and knockdown (asn3-2) T-DNA insertion mutants for the ASN3 gene (At5g10240) demonstrated wild-type contents of asparagine synthetase protein, chlorophyll and ammonium in green leaves at 35 days after sowing. In situ hybridization localized ASN3 mRNA to phloem companion cells of vasculature. Young siliques of the asn3-1 knockout line showed a decrease in asparagine but an increase in glutamate. The seeds of asn3-1 and asn3-2 displayed a wild-type nitrogen status expressed as total nitrogen content, indicating that the repression of ASN3 expression had only a limited effect on mature seeds. An analysis of amino acid labeling of seeds imbibed with (N-15) ammonium for 24 h revealed that asn3-1 seeds contained 20% less total asparagine while N-15-labeled asparagine ((2-N-15)asparagine, (4-N-15)asparagine and (2,4-N-15)asparagine) increased by 12% compared to wild-type seeds. The data indicate a fine regulation of asparagine synthesis and hydrolysis in Arabidopsis seeds.

To investigate short term interaction between recently fixed carbon and assimilated nitrogen, the fates of pulse-fed 13 C and continuously-applied 15 N in sugar, nitrate, organic acid, amino acid and insoluble compound fractions from detached leaves of 5-weeks old spinach ( Spinacia oleracea L. cv. Lead) were followed under four light/NO 3 − combined treatments. At the termination of a 75 min pulse 13 CO 2 feeding (defined as 0 h), the total amounts of the 13 C recently fixed by detached leaves in the light were slightly higher for N-free and 1 mM NO 3 − medium than for 4 mM NO 3 − . In shaded leaves, 13 C fixation and 15 NO 3 − uptake, respectively, decreased to 11.2% and 71.3% of the fully illuminated leaves exposed to the same 4 mM NO 3 − concentration. In the light at 0 h, the incorporation of 13 C was mostly into sugars, followed by undefined 13 C (total 13 C – sum of analyzed sub-fractions). In shaded leaves, the largest incorporation was found in the undefined C, followed by insoluble C and sugars. The uptake of 15 NO 3 − from 0 to 3 h was 128, 665 and 620 μg g −1 DW and the percentages of 15 N present as reduced forms were 80, 77 and 41, respectively, in the leaves exposed to, in mM, 1 NO 3 − , 4 NO 3 − in the light and 4 NO 3 − in the shaded condition; thus, shading did a small decrease in uptake but a great decrease in reduction of 15 NO 3 − . Under light treatment, the synthesis of reduced 15 N was negatively related to the reduction of 13 C sugars from 0 to 3 h, but positively related to amounts of organic acid 13 C at 3 h.

The contribution of the phloem in translocation of cadmium (Cd) to grains of rice plants (Oryza sativa L. cv. Kantou) was estimated. We measured Cd concentrations in phloem sap collected from the uppermost internode of rice plants at the grain-filling stage. Cadmium concentration was 17.8 ± 4.5 µmol L−1 in plants treated with a nutrient solution containing 10 µmol L−1 Cd for 2 days. Subsequently, distribution ratios of 109Cd between the grains and the glumes were determined after feeding of 109Cd to the cut ends of the uppermost internodes or onto the surface of the flag leaves or to the roots. The results suggested that 91–100% of the Cd in the grains was deposited from the phloem. To our knowledge, this is the first determination of Cd concentrations in phloem sap transported to grains, and the first estimation of the contribution of the phloem in Cd transport to rice grains.

We found 19 putative genes for plant-specific Dof transcription factors in the moss Physcomitrella patens and one Dof gene in the green alga Chlamydomonas reinhardtii, but no identifiable Dof gene in the red alga Cyanidioschyzon merolae and the diatom Thalassiosira pseudonana, suggesting that the origin of the Dof transcription factors pre-dates the divergence of the green algae and the ancestors of terrestrial plants. The phylogenetic analyses contended that the Dof family in angiosperms formed through a series of evolutionary processes, including intensive duplications of a specific ancestral gene after the divergence of the moss and the angiosperm lineages.

The Arabidopsis thaliana mutants altered sulfur response 1 -1 (asrl-1) and asr1-2 were isolated using the green fluorescent protein gene (GFP), as a marker, driven by a sulfur deficiency-responsive promoter containing the BSR fragment, which is s responsible for the induction of gene expression under sulfur deficiency. In the asr1 mutants, the expression of three sulfur deficiency-responsive genes BSR-driven GFP, sulfate transporter 2;2 (SULTR2;2) and adenosine-5'-phosphosulfate reductase 1 (APR1) were induced in medium containing a normal sulfate concentration. The ASR1 locus was mapped to a 53-kb region on the upper arm of chromosome III; this is also the region of the BIG gene, which encodes a calossin-l ike protein necessary for the polar transport of auxin. The morphology of the asr1 mutants, i.e. reduced leaf size and inflorescence elongation, resembled that of bigmutants. Using nucleotide sequence analysis of the BIG gene, we identified independent nonsense mutations in asrl-1 I and asr1 -2. To confirm that ASR1 was BIG, we established lines of transgenic A. thaliana carrying a transfer DNA (T-DNA) insertion in the BIG gene. In these T-DNA insertion mutants, mRNA levels of BsR-driven GFP and APR1 were upregulated in normal sulfate medium. The F1 plants from crosses between asrl-1 and T-DNA insertion lines exhibited reduced leaf size and inflorescence length, indicating that ASR1 was indeed BIG. Taken together, the present results established that BIG is involved in n the regulation of BsR-driven GFPand APR1 mRNA level gene expression. Indole-3-acetic acid also upregulated BsR-driven GFP and APR1 together with SULTR2;2 mRNA level, suggesting that the big effect on BsR-driven GFP and APR1 is a pleiotropic aspect of the BIG gene.

The role of the Arabidopsis thaliana genes SNRK2s, which are similar to the sulfur-regulatory gene SAC3 of Chlamydomonas reinhardtii, in the response of plants to sulfur was studied. The Arabidopsis genome contains 10 genes (SNRK2.1 to SNRK2.10) similar to SAC3. Among these genes, transcript accumulation of several genes including SNRK2.3 was induced in response to sulfur starvation. Independently isolated transgenic A. thaliana lines carrying T-DNA insertions in SNRK2.3 exhibited reduced sulfur-starvation induction of the transcript of the sulfate transporter SULTR2;2 gene, whereas the accumulation of O-acetyl-l-serine under sulfur deficiency conditions increased. These results suggest that SNRK2.3 plays a beneficial role in the regulation of gene expression and metabolism in response to sulfur starvation.

In legumes, the number of root nodules is controlled by a mechanism called autoregulation. Recently, we found that the foliar brassinosteroid (BR), a plant growth-regulating hormone, systemically regulates the nodule number in soybean plants. In the present study we report that such down-regulation of root nodule formation by a BR may occur through a change of the polyamine contents, with the experimental evidence as follows. The foliar contents of both spermidine (Spd) and spermine (Spm) in the super-nodulating soybean mutant, En6500, were always lower than those in its parent line, Enrei. This lower Spd and Spm content accompanied a striking accumulation of putrescine (Put) in the former plant. This finding indicates that Spd and Spm biosynthesis from their precursor Put is repressed in En6500. The foliar treatments with Spd or Spm of En6500 led to a reduction of both nodule number and root growth. On the other hand, foliar treatment with MDL74038, a specific inhibitor of Spd biosynthesis, apparently increased the root nodule number in Enrei. Foliar application of brassinolide (BL) of En6500 increased the leaf Spd level and reduced the nodule number. These results suggested that BL-induced Spd synthesis in shoots might suppress the root nodule formation.

The carbon (C) content and natural 13C abundance (δ13C) of soil organic matter were analyzed in soils collected from forests and agricultural fields in Thailand to determine the origin and turnover of soil organic carbon in the Asian tropics. The δ13C values of the soil horizons in two forests were in the range of C3 plant values. Soil δ13C values in fields with continuous cultivation of lowland rice (C3 plant) and sugarcane (C4 plant) were less negative than those in the forests. The organic carbon contents and δ13C values in 226 top-soils from agricultural fields varied in four regions of Thailand and were affected by the cultivation of preceding crops and weeds. The δ13C values of two series of top-soils from forests and sugarcane fields were investigated in the Northeastern and Central Plain Regions. Conversion of forest land to fields for sugarcane cultivation led to a rapid decrease in the content of soil organic carbon and an increase in the soil δ13C values. The calculated forest-derived ...

Leguminous plants form nitrogen-fixing root nodules and the number of nodules is controlled by a self-regulating mechanism called autoregulation. However, signaling substances involved in nodule regulation have not been identified. In the present study, we used brassinolide, a most effective molecular species of plant hormone brassinosteroids, and brassinazole, an effective inhibitor of brassinosteroid biosynthesis to determine whether brassinolide played a role in systemic regulation of noduel formation in wild type soybean and its super-nodulating mutant. Foliar application or direct injection of brassinolide into the root base inhibited nodule formation and root development in the super-nodulating mutant (En6500), but not in the parent line (cv. Enrei). The internodes in the plants subjected to foliar application were significantly longer than those in the untreated plants. In contrast, the application of brassinazole on mature leaves or into the culture media resulted in the increase of the nodule num...

Cold-induced soluble sugar accumulation enhances the degree of freezing tolerance in various cold-hardy plants including Arabidopsis (Arabidopsis thaliana), where soluble sugars accumulate in only a few hours at 2°C. Hence, along with photosynthesis, starch degradation might play a significant role in cold-induced sugar accumulation and enhanced freezing tolerance. Starch-related α-glucan/water dikinase (EC 2.7.9.4), encoded by Arabidopsis STARCH EXCESS 1 (SEX1), is hypothesized to regulate starch degradation in plastids by phosphorylating starch, thereby ensuring better accessibility by starch-degrading enzymes. Here, we show that Arabidopsis sex1 mutants, when incubated at 2°C for 1 d, were unable to accumulate maltooligosaccharides or normal glucose and fructose levels. In addition, they displayed impaired freezing tolerance. After 7 d at 2°C, sex1 mutants did not show any of the above abnormal phenotypes but displayed slightly higher leaf starch contents. The impaired freezing tolerance of sex1 mutants was restored by overexpression of wild-type SEX1 cDNA using the cauliflower mosaic virus 35S promoter. The results demonstrate a genetic link between the SEX1 locus and plant freezing tolerance, and show that starch degradation is important for enhanced freezing tolerance during an early phase of cold acclimation. However, induction of starch degradation was not accompanied by significant changes in α-glucan/water dikinase activity in leaf extracts and preceded cold-induced augmentation of SEX1 transcripts. Therefore, we conclude that augmentation of SEX1 transcripts might be a homeostatic response to low temperature, and that starch degradation during an early phase of cold acclimation could be regulated by a component(s) of a starch degradation pathway(s) downstream of SEX1.

To detect N2-fixing bacteria in a plant without using culture methods, nifH gene segments were amplified with degenerate primers from DNA extracted from stems of sugarcane (Saccharum officinarum L.) and leaves of pineapple (Ananas comosus [L.] Merr.). Sequences of the nifH clones were homologous to those of bacteria in the genera Bradyrhizobium, Serratia, and Klebsiella. The bacteria with nifH sequences homologous to those of the three genera may be promising candidates for predominant endophytic diazotrophs in sugarcane and pineapple, because these sequences were commonly detected in samples from different host plants, which were collected at different locations. On the other hand, no nifH sequence related to Gluconacetobacter diazotrophicus, which is an endophytic N2-fixing bacterium and had been previously isolated from sugarcane, was detected in sugarcane. This indicates the absence or the presence of few G. diazotrophicus in the stems of the sugarcane plants used in the current study.

Non-symbiotic hemoglobins (ns-Hbs) are found in all plants, although their physiological function remains to be determined. The present study was undertaken to explore the mode of induction of ns-Hb genes by metabolites of nitrate assimilation using cultured rice (Oryza sativa L.) cells. Two class-1 ns-Hb genes, ORYsa GLB1a and ORYsa GLB1b, were strongly induced by nitrate, nitrite and nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside. The rapid and transient accumulation of ORYsa GLB1a and ORYsa GLB1b transcripts in response to nitrate, nitrite and NO donors was similar to that of nia1, which encodes NADH-nitrate reductase (NR), although repression by glutamine and asparagines was significant only for nia1. In the mutants defective in NR mRNA expression, nitrate, nitrite and NO donors failed to induce not only nia1 but also ORYsa GLB1a and ORYsa GLB1b transcripts, indicating that the induction of ns-Hb genes is closely associated with that of the NR gene. Although the kinetics of induction by nitrate, nitrite and NO donors are similar for the two ns-Hb genes, an inhibitor study demonstrated that de novo synthesis of the protein in cytoplasm is essential for inducing ORYsa GLB1b. In contrast, ORYsa GLB1a, like nia1, can be induced in the primary response to these signals without de novo protein synthesis. The role of nitrate, nitrite and NO in the induction of ns-Hb gene expression in rice cells and the possible cellar function of ns-Hbs were discussed in relation to nitrate reduction pathways.

We investigated the effects of a four-day treatment with 0.3 mM glutathione (GSH) and 10 mM O-acetyl-L- serine (OAS), negative and positive regulators of sulfur-responsive gene expression, respectively, alone or in combination, on the sulfur-responsive gene expression in ten-day-old Arabidopsis thaliana plants. We determined the relationship between the concentrations of GSH, OAS and sulfate, and expression of sulfur-responsive genes in rosette leaves. The concentrations of GSH and OAS correlated negatively and positively with expression of genes, respectively, suggesting that GSH and OAS independently affect expression of sulfur-responsive genes.

A field study was conducted to investigate the long-term effect of surface application of sewage sludge composts vs chemical N fertilizer on total N, total C, soluble organic C, pH, EC, microbial biomass C and N, protease activity, deaminase activity, urease activity, gross and net rates of N mineralization and nitrification, CO2 evolution, and N2O production. Soil samples were taken from five depths (0–15, 15–20, 20–30, 30–40, and 40–50 cm) of a long-term experiment at the University of Tokyo, Japan. Three fields have been receiving sewage sludge composted with rice husk (RH), sawdust (SD), or mixed chemical fertilizer NPK (CF), applied at the rate of 240 kg N ha−1 each in split applications in summer and autumn since 1978. Significantly higher amounts of total N and C and soluble organic C were found in the compost than in the CF treatments up to the 40-cm soil depth, indicating improved soil quality in the former. In the CF treatment, soil pH values were significantly lower and electrical conductivity values were significantly higher than those of compost-treated soils of up to 50 cm depth. Soil microbial biomass C and N, CO2 evolution, protease, deaminase, and urease activities were significantly higher in the compost than in the CF treatments due to greater availability of organic substrates that stimulated microbial activity. Gross N mineralization rates determined by 15N dilution technique were eight and five times higher in the SD and RH treatments than in the CF treatment, respectively, probably due to high levels of microbial and enzyme activities. Net N mineralization rates were also significantly higher in the compost treatments and were negative in the CF treatment indicating immobilization. Net nitrification rates were higher in compost treatments and negative in the CF treatment. Nitrous oxide productions from compost treatments were higher than the CF treatment due to the greater availability of mineral N as a result of higher mineralization and nitrification rates and soluble organic C in the former. Most of the measured parameters were highest in the surface soil (0–15 cm) and were significantly higher in the SD treatment than in the RH treatment.

We present a method for instant DNA extraction fromArabidopsis thaliana based on a simple DNA extraction method (Edwards et al., 1991). A piece of rosette leaf (typically 3–5 mg) was ground in a centrifuge tube in extraction solution. Extracted DNA was suitable for PCR analysis, without centrifugation. The feasibility of this method was confirmed by testing 24 primer sets. This method requires less than 1 mg of plant tissue and is useful for genetic mapping, transgene detection, and other experiments.

Volcanic ash soils (including Andisols) have been known to accumulate extremely large amounts of humic acids in their thick surface horizons, and their accumulation mechanism is of interest. Two mechanisms have been proposed: (1) active metals like Al and Fe, supplied from weathered volcanic materials, stabilize humic acids through complexation reactions; (2) cultivated Japanese pampas grasses ( Miscanthus sinensis A.) and its charred materials are the major carbon source of humic acids. In the present study, contribution ratio of the pampas grass (C4-plant) on the carbons of the humic acids was determined by measuring their stable isotopic ratio of carbon (δ 13 C). In Japanese volcanic ash soils, humic acids were originated from both C3- and C4-plants, and the contribution ratio of C4-plants (mostly pampas grass) ranged from 18% to 52%. Highly humified (dark-colored) humic acids tended to show higher contribution ratio of C4-plants among volcanic ash soils, although the major part of the carbon had originated from C3-plants. It was also clarified that the δ 13 C values of crude soil samples correlated well with those of humic and fulvic acids. Therefore, reported δ 13 C values of crude soil samples in the literature would be useful for estimating the carbon source of soil humic substances. Literature survey of the δ 13 C values of crude soil samples also indicated that a large part of the carbon in humic substances has originated from C3-plants rather than C4-plants, implying the importance of the active metals (such as Al and Fe) on the formation and accumulation of the humic acids in volcanic ash soils.

Effect of the difference in nitrogen media on the salt stress response and contents of nitrogen compounds (amino acids, polyamines, and protein) was investigated in rice seedlings (Oryza sativa L. cv. Nipponbare) at the 2nd leaf stage. Plant growth and chlorophyll fluorescence (ΦII), reflecting the efficiency of photosynthetic energy conversion, markedly decreased during the 3-d period of the 100 mM NaCl treatment. Response to salt stress, however, varied with the difference in the nitrogen media. Rice seedlings grown in a low ammonium medium, containing a sub-optimal concentration of ammonium for seedling growth, showed the lowest sensitivity to the salt stress treatment. In these seedlings, free amino acids, especially amide glutamine and arginine, strikingly accumulated in the 2nd leaf blades with the NaCl treatment. Additionally, the leaf polyamine contents in the seedlings grown in a low ammonium medium increased under salt stress, while the leaf polyamine contents in the seedlings grown in ...

The N-terminal amino-acid sequence of a major rice phloem-sap protein, designated as RPP23, was determined. The complete amino-acid sequence of RPP23 was deduced from the corresponding rice EST- clone and contained an extra 46 amino acids at the N-terminus, that was apparently cleaved off to form mature RPP23 in sieve tubes. RPP23 shared a similarity to plant small heat-shock proteins (smHSPs), though the N-terminal region of RPP23 was distinct from that of known smHSPs. Immunocytological analyses using leaf sections showed that RPP23 was located only in the phloem regions of leaves, and was present in non-stressed plants. In mature leaves, stronger immunocytological signals were detected in sieve elements than in companion cells.

Rice plants were grown for 4 weeks in nutrient solutions containing 0, 0.015, 0.15, and 1.5 mm silicate. Si content in leaves was lower in the plants grown at low silicate concentrations. RNA was extracted from leaves and subjected to microarray analysis. Statistical analysis of the data from two independent hybridizations enabled to identify a number of candidate clones regulated by Si nutritional conditions. Quantitative reverse transcriptionme diated-PCR enabled to identify-five genes whose transcript accumulation was affected by Si nutrition.

Microarray analysis was carried out with mRNAs isolated from aerial portions of rice plants subjected to sulfur deficiency. Among the candidate clones regulated by sulfur deficiency, three genes were identified by northern analysis that showed altered levels of mRNA accumulation. Genes similar to those for ferritin, S-like ribonuclease, and DnaJ-like protein were down-, down-, and upregulated by sulfate deficiency, respectively. These genes were found for the first time to be responsive to sulfur nutrition in the present study.

The influence of P deficiency on the utilization of two sources of N, mineral N (exogenous N) and reserved N (endogenous N), for regrowth of Italian ryegrass ( Lolium multiflorum ) was studied. P-sufficient (+P) or P-free (−P) nutrition solution was applied from 7 days before defoliation to 24 days of regrowth and the N flows derived from two different N sources within the plant were quantified by 15 N pulse-chase labeling. Shoot regrowth significantly reduced by 12 days of regrowth, while root growth was more in −P plants. Inorganic P (P i ) concentration was highly reduced by P deprivation more in the stubble and regrowing shoots and less in the roots. At defoliation, P deprivation had induced a higher accumulation for all N compounds in the stubble and for amino acids in the roots. The previously incorporated 15 N in stubble and roots as nitrate and amino acids was much decreased in −P plants especially for the first 6 days of regrowth. Total N content in the regrowing leaves was not significantly different between +P and −P plants, but percentage contribution of remobilized N for total leaf N formation was significantly higher in −P plants (78%) than in +P plants (69%) at 6 days of regrowth. From day 12, the utilization of both endogenous and exogenous N was apparently inhibited in −P plants.

Behavior of Cd and Zn in the rhizosphere soil and their uptake by two species of Brassica plants were investigated by using a rhizobox system filled with an Andosol contaminated with Cd and Zn (4 .24 Cd and 398 Zn mg kg−1 dry soil). The Brassica plants used were hybrid 60 / 31 (BT, Brassica juncea × Thlaspi caerulescens) and komatsuna (Brassica campestris var. rapa). By 34 d of plant cultivation, the soil pH values in the root-growing zone compartment (R.C.) and 0–1 mm zone compartment (0–1 mm Z.C.) decreased from 5.5 to around 4.6 in BT and to around 4.9 in komatsuna. The 1 M NH4Cl-extractable Cd concentrations in the soils of R.C. and 0–1 mm Z.C. of the rhizobox decreased, while the NH4Cl-extractable Zn concentrations in the soils of these compartments increased. Under the present experimental conditions, it is considered that Zn moved from the outer compartments toward R.C. by mass flow. On the other hand, it was considered that Cd in R.C. and 0–1 mm Z.C. moved mainly by diffusion because the ...

The changes in the cAMP and cGMP contents in root nodules were investigated using pea (Pisum sativum L. cv. Izusen) and soybeans (Glycine max L. (Merr.) cv. Enrei and its supernodulating mutant En6500). The concentration of cAMP increased in the nodules from pea and soybean cv. Enrei with nodule development, but not in the nodules from soybean En6500. The concentrations of cGMP remained low in pea and soybean cv. Enrei, while the levels of cGMP in the roots and nodules from soybean En6500 showed a temporal increase after rhizobium infection. The external application of dibutyryl-cAMP (db-cAMP) significantly decreased nodule production in these leguminous plants, indicating the possible involvement of cAMP in the regulation of nodule formation. On the other hand, dibutyryl-cGMP (db-cGMP) did not significantly affect the nodule fresh weight of these legumes. The data further suggest the existence of a difference of the functional role of cGMP from that of cAMP in the regulation of root nodule forma...

The effects of NaCl-induced stress on physiochemical factors such as inorganic cations, proline, malondialdehyde (MDA) and polyamines were investigated in the gramineous weed, Echinochloa crus-galli Beauv. var. formosensis Ohwi (E. crus-galli) and rice (Oryza sativa L. cv. Nipponbare). Growth inhibition at the 2nd leaf stage under salt stress was more severe in rice than in E. crus-galli. Water content in the 2nd leaves was also more severely decreased in rice, indicating that E. crus-galli was more salt-tolerant. After NaCl treatment, Na+ accumulated in the 2nd leaves of both plant species but not in their roots. Proline accumulation in the 2nd leaves was significantly higher in salt stressed E. crus-galli than in rice, suggesting the significance of proline production in the salt tolerance of this weed. Content of MDA of the rice increased more greatly with NaCl treatment than that in E. crus-galli. NaCl treatment affected polyamine metabolism of both plant species, but the response of each plant to salt stress was somewhat different, especially in the leaves. Leaf putrescine and spermidine contents were high in non-stressed plants in salt-sensitive rice, although rather lower in E. crus-galli in response to NaCl concentrations. These results indicate that an increase in proline and changes in polyamines relates to the salt tolerance of E. crus-galli.

Fused potassium silicate (FPS), which contains K2Ca2Si2O7, has been prepared as a slow-releasing potassium fertilizer. Moreover, it is difficult to estimate the proportion of nutrients utilized by plants that come from the soil versus the slow-releasing fertilizer applied. To trace the uptake of potassium (K) by plants from FPS supplied to the soil, the fertilizer K was partially replaced with rubidium (Rb). The growth and K+Rb uptake (moles) of Chinese cabbage (Brassica pekinensis Rupy. cv. Kekkyu) plants in sand culture experiments were not affected by the replacement of K with Rb. In pot experiments using a volcanic ash Ando soil, Chinese cabbage was grown with no application of K, with K and Rb salts, or with Rb-containing FPS for three cycles of 40 days each. The amounts of fertilizer-derived K in the shoots estimated by the Rb-tracer method were smaller than those estimated by the difference of K accumulation between K fertilized and unfertilized plants. Such result suggests the involvement of `K priming', a process by which the addition of K fertilizers enhances plant K uptake from the soil. The amount of K absorbed from FPS, calculated by the Rb-tracer method, indicated that the sparingly soluble K, that is soluble in 0.2 g L−1 citric acid solution but not in water, was absorbed at least partly through direct contact with the roots without prior exchange with soil K. Moreover, the plant absorption of sparingly soluble K from FPS was also confirmed by the difference method in K uptake.

The major findings of many years of research into plant N cycling are summarised in this review, firstly as revealed by 15N-enriched methods and secondly, in relation to natural 15N abundance (δ15N) in plants and their metabolites. This work has mainly been done in an agricultural context. As many groups especially attempt to relate δ15N to N cycling, atmospheric N deposition and the interactions of N with carbon budgets, we deem it useful to synthesize these major findings. Primary assimilation and distribution of N within plants were investigated from the 15N enrichment in individual plant organs and in individual amino acids after feeding them 15N-labelled compounds. In both roots and leaves, NH4 + and NO3 − were assimilated into amino acids, largely by a combination of glutamine synthetase (GS) and glutamate synthase (GOGAT). In the leaves, the transfer of glutamine (amide) N to glutamic acid was accelerated in the light, and amino N in some amino acids was deaminated to ammonia in the dark, followed by its incorporation into glutamine. The N in the growing parts such as growing leaves, filling grains and growing root parts were from two sources: re-allocation (phloem supply) of reserved N (amino acids), and currently-absorbed N. The metabolites from the mature parts may perform the roles of substrates for plant growth and signals for gene expression. δ15N values, measured for plants/soils and plant metabolites (inorganic N, amino acids, polyamines) were related with the acquisition, metabolism and distribution of N in plants. Small 15N/14N fractionation in the acquisition of N2 and NO3 − and large 15N/14N fractionation in NH4 + uptake were found. The δ15N values of whole shoots or grains from field-grown crops were largely reflected major sources of N. In some legumes, 15N was enriched in their nodules and an extremely 15N-enriched compound was homospermidine. Nitrate reduction to ammonia (NR) and ammonia assimilation to glutamine (GS) showed large 15N/14N fractionations. Specific attention was paid to the δ15N values in xylem and phloem exudates compared to those of plant organs.

Boron deficiency hampers the productivity of 132 crops in more than 80 countries1. Boron is essential in higher plants primarily for maintaining the integrity of cell walls2,3,4 and is also beneficial and might be essential in animals5 and in yeast6. Understanding the molecular mechanism(s) of boron transport is crucial for alleviating boron deficiency. Here we describe the molecular identification of boron transporters in biological systems. The Arabidopsis thaliana mutant bor1-1 is sensitive to boron deficiency7,8. Uptake studies indicated that xylem loading is the key step for boron accumulation in shoots with a low external boron supply and that the bor1-1 mutant is defective in this process. Positional cloning identified BOR1 as a membrane protein with homology to bicarbonate transporters in animals. Moreover, a fusion protein of BOR1 and green fluorescent protein (GFP) localized to the plasma membrane in transformed cells. The promoter of BOR1 drove GFP expression in root pericycle cells. When expressed in yeast, BOR1 decreased boron concentrations in cells. We show here that BOR1 is an efflux-type boron transporter for xylem loading and is essential for protecting shoots from boron deficiency.

Nitrification and denitrification processes of inorganic nitrogen (N) from several fertilizers placed in an acid soil (pH 4.1) of tea fields were examined in two experiments based on the NO3-N leaching pattern from soil columns (20 cm depth) and on the δ15N values of leached N. In the first experiment, ammonium nitrate (AN) was placed in the soil columns with and without dicyandiamide (DCD), a nitrification inhibitor. The NO3-N content in the leachate was more than 10 times higher than the NH4-N content and DCD application decreased the NO3-N content in the leachate. The δ15N values of the leached N with and without DCD initially decreased, with minimum values of − 3.8‰and − 1.5\%, respectively, which were lower than that of applied AN (+ 0.7\%). These results showed that 15N depletion occurred by the conversion of NH4-N to NO3-N through nitrification under acidic soil conditions (pH 4.1) and larger isotopic fractionation in the presence of DCD. In the second experiment, four slow-release fertili...