Carroll P. Vance

Legumes: importance and constraints to greater use.

Legumes, broadly defined by their unusual flower structure, podded fruit, and the ability of 88% of the species examined to date to form nodules with rhizobia ([de Faria et al., 1989][1]), are second only to the Graminiae in their importance to humans. The 670 to 750 genera and 18,000 to 19,000

RNA-Seq Atlas of Glycine max : A guide to the soybean transcriptome

BackgroundNext generation sequencing is transforming our understanding of transcriptomes. It can determine the expression level of transcripts with a dynamic range of over six orders of magnitude from multiple tissues, developmental stages or conditions. Patterns of gene expression provide insight into functions of genes with unknown annotation.ResultsThe RNA Seq-Atlas presented here provides a record of high-resolution gene expression in a set of fourteen diverse tissues. Hierarchical clustering of transcriptional profiles for these tissues suggests three clades with similar profiles: aerial, underground and seed tissues. We also investigate the relationship between gene structure and gene expression and find a correlation between gene length and expression. Additionally, we find dramatic tissue-specific gene expression of both the most highly-expressed genes and the genes specific to legumes in seed development and nodule tissues. Analysis of the gene expression profiles of over 2,000 genes with preferential gene expression in seed suggests there are more than 177 genes with functional roles that are involved in the economically important seed filling process. Finally, the Seq-atlas also provides a means of evaluating existing gene model annotations for the Glycine max genome.ConclusionsThis RNA-Seq atlas extends the analyses of previous gene expression atlases performed using Affymetrix GeneChip technology and provides an example of new methods to accommodate the increase in transcriptome data obtained from next generation sequencing. Data contained within this RNA-Seq atlas of Glycine max can be explored at http://www.soybase.org/soyseq.

Phosphorus Deficiency in Lupinus albus (Altered Lateral Root Development and Enhanced Expression of Phosphoenolpyruvate Carboxylase)

The development of clustered tertiary lateral roots (proteoid roots) and the expression of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in roots were studied in white lupin (Lupinus albus L.) grown with either 1 mM P (+P-treated) or without P (-P-treated). The +P-treated plants initiated fewer clustered tertiary meristems and the emergence of these meristems was delayed compared with -P-treated plants. Proteoid root zones could be identified 9 d after emergence in both P treatments. Amounts of PEPC mRNA, PEPC specific activity, and enzyme protein were greater in proteoid roots than in normal roots beginning at 10, 12, and 14 d after emergence, respectively. The increases in PEPC mRNA, PEPC enzyme, and PEPC specific activity suggest that this enzyme is in part under transcriptional regulation. Recovery of organic acids from root exudates coincided with the increases in PEPC specific activity. The -P-treated plants exuded 40-, 20-, and 5-fold more citrate, malate, and succinate, respectively, than did +P-treated plants. Data presented support the hypothesis that white lupin has concerted regulation of proteoid root development, transcriptional regulation of PEPC, and biosynthesis of organic acids for exudation in response to P deficiency.

Overexpression of Malate Dehydrogenase in Transgenic Alfalfa Enhances Organic Acid Synthesis and Confers Tolerance to Aluminum

Al toxicity is a severe impediment to production of many crops in acid soil. Toxicity can be reduced through lime application to raise soil pH, however this amendment does not remedy subsoil acidity, and liming may not always be practical or cost-effective. Addition of organic acids to plant nutrient solutions alleviates phytotoxic Al effects, presumably by chelating Al and rendering it less toxic. In an effort to increase organic acid secretion and thereby enhance Al tolerance in alfalfa (Medicago sativa), we produced transgenic plants using nodule-enhanced forms of malate dehydrogenase and phosphoenolpyruvate carboxylase cDNAs under the control of the constitutive cauliflower mosaic virus 35S promoter. We report that a 1.6-fold increase in malate dehydrogenase enzyme specific activity in root tips of selected transgenic alfalfa led to a 4.2-fold increase in root concentration as well as a 7.1-fold increase in root exudation of citrate, oxalate, malate, succinate, and acetate compared with untransformed control alfalfa plants. Overexpression of phosphoenolpyruvate carboxylase enzyme specific activity in transgenic alfalfa did not result in increased root exudation of organic acids. The degree of Al tolerance by transformed plants in hydroponic solutions and in naturally acid soil corresponded with their patterns of organic acid exudation and supports the concept that enhancing organic acid synthesis in plants may be an effective strategy to cope with soil acidity and Al toxicity.

Fungal Development and Induction of Defense Response Genes During Early Infection of Wheat Spikes by Fusarium graminearum

Fusarium head blight (FHB) of wheat is a crippling disease that causes severe economic losses in many of the wheat-growing regions of the world. Temporal patterns of fungus development and transcript accumulation of defense response genes were studied in Fusarium graminearum-inoculated wheat spikes within the first 48 to 76 h after inoculation (hai). Microscopy of inoculated glumes revealed that the fungus appeared to penetrate through stomata, exhibited subcuticular growth along stomatal rows, colonized glume parenchyma cells, and sporulated within 48 to 76 hai. No major differences in the timing of these events were found between Sumai 3 (resistant) and Wheaton (susceptible) genotypes. In complementary experiments, RNA was extracted from spikes at several time intervals up to 48 hai and temporal expression patterns were determined for defense response genes encoding peroxidase, PR-1, PR-2 (beta-1,3-glucanase), PR-3 (chitinase), PR-4, and PR-5 (thaumatin-like protein). In both genotypes, transcripts for the six defense response genes accumulated as early as 6 to 12 hai during F. graminearum infection and peaked at 36 to 48 hai. Greater and earlier PR-4 and PR-5 transcript accumulation was observed in Sumai 3, compared with Wheaton. Our results show that the timing of defense response gene induction is correlated with F. graminearum infection.

Phosphorus Stress in Common Bean: Root Transcript and Metabolic Responses

Phosphorus (P) is an essential element for plant growth. Crop production of common bean (Phaseolus vulgaris), the most important legume for human consumption, is often limited by low P in the soil. Functional genomics were used to investigate global gene expression and metabolic responses of bean plants grown under P-deficient and P-sufficient conditions. P-deficient plants showed enhanced root to shoot ratio accompanied by reduced leaf area and net photosynthesis rates. Transcript profiling was performed through hybridization of nylon filter arrays spotted with cDNAs of 2,212 unigenes from a P deficiency root cDNA library. A total of 126 genes, representing different functional categories, showed significant differential expression in response to P: 62% of these were induced in P-deficient roots. A set of 372 bean transcription factor (TF) genes, coding for proteins with Inter-Pro domains characteristic or diagnostic for TF, were identified from The Institute of Genomic Research/Dana Farber Cancer Institute Common Bean Gene Index. Using real-time reverse transcription-polymerase chain reaction analysis, 17 TF genes were differentially expressed in P-deficient roots; four TF genes, including MYB TFs, were induced. Nonbiased metabolite profiling was used to assess the degree to which changes in gene expression in P-deficient roots affect overall metabolism. Stress-related metabolites such as polyols accumulated in P-deficient roots as well as sugars, which are known to be essential for P stress gene induction. Candidate genes have been identified that may contribute to root adaptation to P deficiency and be useful for improvement of common bean.

Genome-Wide Identification of Nodule-Specific Transcripts in the Model Legume Medicago truncatula

The Medicago truncatula expressed sequence tag (EST) database (Gene Index) contains over 140,000 sequences from 30 cDNA libraries. This resource offers the possibility of identifying previously uncharacterized genes and assessing the frequency and tissue specificity of their expression in silico. BecauseM. truncatula forms symbiotic root nodules, unlike Arabidopsis, this is a particularly important approach in investigating genes specific to nodule development and function in legumes. Our analyses have revealed 340 putative gene products, or tentative consensus sequences (TCs), expressed solely in root nodules. These TCs were represented by two to 379 ESTs. Of these TCs, 3% appear to encode novel proteins, 57% encode proteins with a weak similarity to the GenBank accessions, and 40% encode proteins with strong similarity to the known proteins. Nodule-specific TCs were grouped into nine categories based on the predicted function of their protein products. Besides previously characterized nodulins, other examples of highly abundant nodule-specific transcripts include plantacyanin, agglutinin, embryo-specific protein, and purine permease. Six nodule-specific TCs encode calmodulin-like proteins that possess a unique cleavable transit sequence potentially targeting the protein into the peribacteroid space. Surprisingly, 114 nodule-specific TCs encode small Cys cluster proteins with a cleavable transit peptide. To determine the validity of the in silico analysis, expression of 91 putative nodule-specific TCs was analyzed by macroarray and RNA-blot hybridizations. Nodule-enhanced expression was confirmed experimentally for the TCs composed of five or more ESTs, whereas the results for those TCs containing fewer ESTs were variable.

Phosphorus Stress-Induced Proteoid Roots Show Altered Metabolism in Lupinus albus

Proteoid roots develop in Lupinus albus L. in response to nutrient stress, especially P. Proteoid roots excrete citrate and thus increase the availability of P, Fe, and Mn in the rhizosphere. In an effort to understand citrate synthesis and organic acid metabolism in proteoid roots of lupin, we have evaluated in vitro enzyme activities of citrate synthase (CS), malate dehydrogenase (MDH), and phosphoenolpyruvate carboxylase (PEPC) in proteoid and normal roots of plants grown with or without P. Organic acid concentrations, respiration rates, and dark 14CO2-labeling patterns were also determined. The in vitro specific activities of CS, MDH, and PEPC and in vivo dark 14CO2 fixation were higher in proteoid roots compared to normal roots, particularly under P stress. Western blot analysis showed that PEPC enzyme protein was more highly expressed in -P proteoid roots compared to other tissues. The majority of the fixed 14C was found in organic acids, predominantly malate and citrate. A larger fraction of citrate was labeled in P- stressed proteoid roots compared to other root tissue. Respiration rates of proteoid roots were 31% less than those of normal roots. The data provide evidence for increased synthesis of citrate in proteoid roots compared to normal roots, particularly under P stress. A portion of the carbon for citrate synthesis is derived from nonautotrophic CO2 fixation via PEPC in proteoid roots.

Root Carbon Dioxide Fixation by Phosphorus-Deficient Lupinus albus (Contribution to Organic Acid Exudation by Proteoid Roots)

When white lupin (Lupinus albus L.) is subjected to P deficiency lateral root development is altered and densely clustered, tertiary lateral roots (proteoid roots) are initiated. These proteoid roots exude large amounts of citrate, which increases P solubilization. In the current study plants were grown with either 1 mM P (+P-treated) or without P (-P-treated). Shoots or roots of intact plants from both P treatments were labeled independently with 14CO2 to compare the relative contribution of C fixed in each with the C exuded from roots as citrate and other organic acids. About 25-fold more acid-stable 14C, primarily in citrate and malate, was recovered in exudates from the roots of -P-treated plants compared with +P-treated plants. The rate of in vivo C fixation in roots was about 4-fold higher in -P-treated plants than in +P-treated plants. Evidence from labeling intact shoots or roots indicates that synthesis of citrate exuded by -P-treated roots is directly related to nonphotosynthetic C fixation in roots. C fixed in roots of -P-treated plants contributed about 25 and 34% of the C exuded as citrate and malate, respectively. Nonphotosynthetic C fixation in white lupin roots is an integral component in the exudation of large amounts of citrate and malate, thus increasing the P available to the plant.

Glutamate synthase and nitrogen assimilation

The assimilation of ammonia by a wide variety of organisms is the primary route for the introduction of nitrogen into the biosphere. The assimilatory enzymes glutamine synthetase and glutamate synthase catalyze reactions that convert α-ketoglutarate and ammonia to glutamate, which is then used in a wide variety of biosynthetic reactions. These enzymes also play a major role in the reassimilation of ammonia derived from photorespiration in C 3 plants. Recent biochemical, molecular and genetic studies are leading to a better understanding of the factors that determine the activity and function of glutamate synthase.