Developing for nutrient uptake: Induced organogenesis in parasitic plants and root nodule symbiosis.

Plants have evolved diverse strategies to meet their nutritional needs. Parasitic plants employ haustoria, specialized structures that facilitate invasion of host plants and nutrient acquisition. Legumes have adapted to nitrogen-limited conditions by developing nodules that accommodate nitrogen-fixing rhizobia. The formation of both haustoria and nodules is induced by signals originating from the interacting organisms, namely host plants and rhizobial bacteria, respectively. Emerging studies showed that both organogenesis crucially involves plant hormones such as auxin, cytokinins, and ethylene and also integrate nutrient availability, particularly nitrogen. In this review, we discuss recent advances on hormonal and environmental control of haustoria and nodules development with side-by-side comparison. These underscore the remarkable plasticity of plant organogenesis.

The Crotalaria juncea metal transporter CjNRAMP1 has a high Fe uptake activity, even in an environment with high Cd contamination.

Large quantities of Fe and Cd accumulate in the leaves of the metal-accumulating leguminous plant, Crotalaria juncea. A member of the metal transporter NRAMP family was cloned from C. juncea. The amino acid sequence of this clone, designated CjNRAMP1, was similar to the sequence of Arabidopsis AtNRAMP1, which is involved in Fe and Cd transport. Organ-specific analysis showed that CjNRAMP1 mRNA was expressed mainly in the leaves of C. juncea plants, as well as in stems and roots. Use of green fluorescent protein fused to CjNRAMP1 suggested its localization to the plasma membranes of plant cells. Complementation experiments using yeast strains with impaired metal transport systems showed that CjNRAMP1 transported both Fe and Cd in an inward direction within the cells. Transgenic Arabidopsis plants overexpressing CjNRAMP1 showed high tolerance to Cd, with Cd translocation from roots to leaves being substantially greater in transgenic than in wild-type plants. Overexpression of CjNRAMP1 resulted in a greater accumulation of Fe in shoots and roots, suggesting that CjNRAMP1 recognizes Fe and Cd as substrates and that the high Cd tolerance of CjNRAMP1 is due to its strong Fe uptake activity, even in the presence of high Cd concentrations in the rhizosphere.

Identification of putative target genes of bZIP19, a transcription factor essential for Arabidopsis adaptation to Zn deficiency in roots.

Zinc (Zn) depletion adversely affects plant growth. To avoid lethal depletion of cellular Zn, plants have evolved mechanisms to adjust the expression of genes associated with Zn homeostasis, the details of which are poorly understood. In the present study, we isolated an Arabidopsis thaliana T-DNA insertion mutant that exhibited hypersensitivity to Zn depletion. By monitoring root development under Zn-deficient conditions, we isolated a single mutant lacking the basic-region leucine-zipper transcription factor gene bZIP19. To identify proteins whose expression is affected by bZIP19, an iTRAQ-based quantitative proteomics analysis was performed using microsomal proteins from wild-type and the bzip19 mutant A. thaliana roots grown on Basal and Zn-deficient media. Of the 797 proteins identified, expression of two members of the Zrt- and Irt-related protein family, ZIP3 and ZIP9, and three defensin-like family proteins was markedly induced in wild-type but not in the bzip19 mutant under Zn-deficient conditions. Furthermore, selected reaction monitoring and quantitative real-time PCR revealed that ZIP9 expression is mediated by bZIP19 and may be partly supported by bZIP23, a homolog of bZIP19. Mutant analysis revealed that ZIP9 is involved in uptake of Zn by the roots, and the mutant lacking ZIP9 was significantly more sensitive to Zn depletion than the wild-type. These results demonstrate that bZIP19 mainly contributes to expression of genes, such as ZIP9, under Zn-deficient conditions.

Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross-talk between excess zinc and iron deficiency.

Iron (Fe) deficiency significantly effects plant growth and development. Plant symptoms under excess zinc (Zn) resemble symptoms of Fe-deficient plants. To understand cross-talk between excess Zn and Fe deficiency, we investigated physiological parameters of Arabidopsis plants and applied iTRAQ-OFFGEL quantitative proteomic approach to examine protein expression changes in microsomal fraction from Arabidopsis shoots under those physiological conditions. Arabidopsis plants manifested shoot inhibition and chlorosis symptoms when grown on Fe-deficient media compared to basal MGRL solid medium. iTRAQ-OFFGEL approach identified 909 differentially expressed proteins common to all three biological replicates; the majority were transporters or proteins involved in photosynthesis, and ribosomal proteins. Interestingly, protein expression changes between excess Zn and Fe deficiency showed similar pattern. Further, the changes due to excess Zn were dramatically restored by the addition of Fe. To obtain biological insight into Zn and Fe cross-talk, we focused on transporters, where STP4 and STP13 sugar transporters were predominantly expressed and responsive to Fe-deficient conditions. Plants grown on Fe-deficient conditions showed significantly increased level of sugars. These results suggest that Fe deficiency might lead to the disruption of sugar synthesis and utilization.

Correlation analysis of proteins responsive to Zn, Mn, or Fe deficiency in Arabidopsis roots based on iTRAQ analysis.

KEY MESSAGE: For discovering the functional correlation between the identified and quantified proteins by iTRAQ analysis, here we propose a correlation analysis method with cosine correlation coefficients as a powerful tool. iTRAQ analysis is a quantitative proteomics approach that enables identification and quantification of a large number of proteins. In order to obtain proteins responsive to Zn, Mn, or Fe mineral deficiency, we conducted iTRAQ analysis using a microsomal fraction of protein extractions from Arabidopsis root tissues. We identified and quantified 730 common proteins in three biological replicates with less than 1 % false discovery rate. To determine the role of these proteins in tolerating mineral deficiencies and their relation to each other, we calculated cosine correlation coefficients and represented the outcomes on a correlation map for visual understanding of functional relations among the identified proteins. Functionally similar proteins were gathered into the same clusters. Interestingly, a cluster of proteins (FRO2, IRT1, AHA2, PDR9/ABCG37, and GLP5) highly responsive to Fe deficiency was identified, which included both known and unknown novel proteins involved in tolerating Fe deficiency. We propose that the correlation analysis with the cosine correlation coefficients is a powerful method for finding important proteins of interest to several biological processes through comprehensive data sets.

Unraveling the iron deficiency responsive proteome in Arabidopsis shoot by iTRAQ-OFFGEL approach.

Iron (Fe) is required by plants for basic redox reactions in photosynthesis and respiration, and for many other key enzymatic reactions in biological processes. Fe homeostatic mechanisms have evolved in plants to enable the uptake and sequestration of Fe in cells. To elucidate the network of proteins that regulate Fe homeostasis and transport, we optimized the iTRAQ-OFFGEL method to identify and quantify the number of proteins that respond to Fe deficiency in the model plant Arabidopsis. In this study, Fe deficiency was created using Fe-deficient growth conditions, excess zinc (Zn), and use of the irt1-1 mutant in which the IRT1 Fe transporter is disrupted. Using the iTRAQ-OFFGEL approach, we identified 1139 proteins, including novel Fe deficiency-responsive proteins, in microsomal fractions isolated from 3 different types of Fe-deficient shoots compared with just 233 proteins identified using conventional iTRAQ-CEX. Further analysis showed that greater numbers of low-abundance proteins could be identified using the iTRAQ-OFFGEL method and that proteins could be identified from numerous cellular compartments. The improved iTRAQ-OFFGEL method used in this study provided an efficient means for identifying greater numbers of proteins from microsomal fractions of Arabidopsis shoots. The proteome identified in this study provides new insight into the regulatory cross talk between Fe-deficient and excess Zn conditions.

N(2)O emission from degraded soybean nodules depends on denitrification by Bradyrhizobium japonicum and other microbes in the rhizosphere.

A model system developed to produce N(2)O emissions from degrading soybean nodules in the laboratory was used to clarify the mechanism of N(2)O emission from soybean fields. Soybean plants inoculated with nosZ-defective strains of Bradyrhizobium japonicum USDA110 (ΔnosZ, lacking N(2)O reductase) were grown in aseptic jars. After 30 days, shoot decapitation (D, to promote nodule degradation), soil addition (S, to supply soil microbes), or both (DS) were applied. N(2)O was emitted only with DS treatment. Thus, both soil microbes and nodule degradation are required for the emission of N(2)O from the soybean rhizosphere. The N(2)O flux peaked 15 days after DS treatment. Nitrate addition markedly enhanced N(2)O emission. A (15)N tracer experiment indicated that N(2)O was derived from N fixed in the nodules. To evaluate the contribution of bradyrhizobia, N(2)O emission was compared between a nirK mutant (ΔnirKΔnosZ, lacking nitrite reductase) and ΔnosZ. The N(2)O flux from the ΔnirKΔnosZ rhizosphere was significantly lower than that from ΔnosZ, but was still 40% to 60% of that of ΔnosZ, suggesting that N(2)O emission is due to both B. japonicum and other soil microorganisms. Only nosZ-competent B. japonicum (nosZ+ strain) could take up N(2)O. Therefore, during nodule degradation, both B. japonicum and other soil microorganisms release N(2)O from nodule N via their denitrification processes (N(2)O source), whereas nosZ-competent B. japonicum exclusively takes up N(2)O (N(2)O sink). Net N(2)O flux from soybean rhizosphere is likely determined by the balance of N(2)O source and sink.

Autoregulation of nodulation interferes with impacts of nitrogen fertilization levels on the leaf-associated bacterial community in soybeans.

The diversities leaf-associated bacteria on nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans were evaluated by clone library analyses of the 16S rRNA gene. To analyze the impact of nitrogen fertilization on the bacterial leaf community, soybeans were treated with standard nitrogen (SN) (15 kg N ha(-1)) or heavy nitrogen (HN) (615 kg N ha(-1)) fertilization. Under SN fertilization, the relative abundance of Alphaproteobacteria was significantly higher in Nod(-) and Nod(++) soybeans (82% to 96%) than in Nod(+) soybeans (54%). The community structure of leaf-associated bacteria in Nod(+) soybeans was almost unaffected by the levels of nitrogen fertilization. However, differences were visible in Nod(-) and Nod(++) soybeans. HN fertilization drastically decreased the relative abundance of Alphaproteobacteria in Nod(-) and Nod(++) soybeans (46% to 76%) and, conversely, increased those of Gammaproteobacteria and Firmicutes in these mutant soybeans. In the Alphaproteobacteria, cluster analyses identified two operational taxonomic units (OTUs) (Aurantimonas sp. and Methylobacterium sp.) that were especially sensitive to nodulation phenotypes under SN fertilization and to nitrogen fertilization levels. Arbuscular mycorrhizal infection was not observed on the root tissues examined, presumably due to the rotation of paddy and upland fields. These results suggest that a subpopulation of leaf-associated bacteria in wild-type Nod(+) soybeans is controlled in similar ways through the systemic regulation of autoregulation of nodulation, which interferes with the impacts of N levels on the bacterial community of soybean leaves.

Community shifts of soybean stem-associated bacteria responding to different nodulation phenotypes and N levels.

The diversity of stem-associated bacteria of non-nodulated (Nod(-)), wild-type nodulated (Nod(+)) and hypernodulated (Nod(++)) soybeans were evaluated by clone library analyses of the 16S ribosomal RNA gene. Soybeans were dressed with standard nitrogen (SN) fertilization (15 kg N ha(-1)) and heavy nitrogen (HN) fertilization (615 kg N ha(-1)). The relative abundance of Alphaproteobacteria in Nod(+) soybeans (66%) was smaller than that in Nod(-) and Nod(++) soybeans (75-76%) under SN fertilization, whereas that of Gammaproteobacteria showed the opposite pattern (23% in Nod(+) and 12-16% in Nod(-) and Nod(++) soybeans). Principal coordinate analysis showed that the bacterial communities of Nod(-) and Nod(++) soybeans were more similar to each other than to that of Nod(+) soybeans under SN fertilization. HN fertilization increased the relative abundance of Gammaproteobacteria in all nodulation phenotypes (33-57%) and caused drastic shifts of the bacterial community. The clustering analyses identified a subset of operational taxonomic units (OTUs) at the species level in Alpha- and Gammaproteobacteria responding to both the nodulation phenotypes and nitrogen fertilization levels. Meanwhile, the abundance of Betaproteobacteria was relatively constant in all libraries constructed under these environmental conditions. The relative abundances of two OTUs in Alphaproteobacteria (Aurantimonas sp. and Methylobacterium sp.) were especially sensitive to nodulation phenotype and were drastically decreased under HN fertilization. These results suggested that a subpopulation of proteobacteria in soybeans is controlled in a similar manner through both the regulation systems of plant-rhizobia symbiosis and the nitrogen signaling pathway in plants.

Nitrous oxide emission and microbial community in the rhizosphere of nodulated soybeans during the late growth period.

We examined N(2)O emissions from the rhizosphere of field-grown soybeans during the late growth stage (99-117 days after sowing). Marked emissions were detected from the nodulated root systems of field-grown soybeans, whereas a non-nodulating soybean mutant showed no emission. Degraded nodules exclusively generated the N(2)O. A culture-independent analysis of microbial communities showed Bradyrhizobium sp., Acidvorax facilis, Salmonella enterica, Xanthomonas sp., Enterobacter cloacae, Pseudomonas putida, Fusarium sp., nematodes, and other protozoans to be more abundant in the degraded nodules, suggesting that some of these organisms participate in the N(2)O emission process in the soybean rhizosphere.

Microbial community analysis of field-grown soybeans with different nodulation phenotypes.

Microorganisms associated with the stems and roots of nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans [Glycine max (L.) Merril] were analyzed by ribosomal intergenic transcribed spacer analysis (RISA) and automated RISA (ARISA). RISA of stem samples detected no bands specific to the nodulation phenotype, whereas RISA of root samples revealed differential bands for the nodulation phenotypes. Pseudomonas fluorescens was exclusively associated with Nod(+) soybean roots. Fusarium solani was stably associated with nodulated (Nod(+) and Nod(++)) roots and less abundant in Nod(-) soybeans, whereas the abundance of basidiomycetes was just the opposite. The phylogenetic analyses suggested that these basidiomycetous fungi might represent a root-associated group in the Auriculariales. Principal-component analysis of the ARISA results showed that there was no clear relationship between nodulation phenotype and bacterial community structure in the stem. In contrast, both the bacterial and fungal community structures in the roots were related to nodulation phenotype. The principal-component analysis further suggested that bacterial community structure in roots could be classified into three groups according to the nodulation phenotype (Nod(-), Nod(+), or Nod(++)). The analysis of root samples indicated that the microbial community in Nod(-) soybeans was more similar to that in Nod(++) soybeans than to that in Nod(+) soybeans.

Effects of dissolved organic matter on toxicity and bioavailability of copper for lettuce sprouts.

It is well known that dissolved organic matter in soil solution may affect the toxicity or bioavailability of heavy metals to plants, but existing information on various organic substances is insufficient for treating problems with heavy metal-contaminated soils. To clarify how dissolved organic matter alters the toxicity and bioavailability of metals, we germinated lettuce seeds exposed to solutions containing Cu and several kinds of dissolved organic matters. Low molecular weight organic acids (citric, malic, and oxalic acids) increased the toxicity and bioavailability of Cu, but low concentrations of the synthetic chelators ethylenediamine tetra-acetic acid (EDTA) and diethylenetriamine penta-acetic acid (DTPA) decreased the toxicity and bioavailability of Cu. In contrast, humic acid appeared to be the most effective organic substance for detoxifying Cu, even though it did not significantly decrease the bioavailability of Cu. Consequently, the bioavailability and toxic effects of Cu in soil depend on the nature of coexisting organic substances in the soil solution.