Susan S. Miller

An RNA-Seq Transcriptome Analysis of Orthophosphate-Deficient White Lupin Reveals Novel Insights into Phosphorus Acclimation in Plants

Summary: Analysis of all the expressed genes in white lupin roots and leaves shows that acclimation to phosphorous deficiency involves changes in root development and modifications in metabolism. Phosphorus, in its orthophosphate form (Pi), is one of the most limiting macronutrients in soils for plant growth and development. However, the whole-genome molecular mechanisms contributing to plant acclimation to Pi deficiency remain largely unknown. White lupin (Lupinus albus) has evolved unique adaptations for growth in Pi-deficient soils, including the development of cluster roots to increase root surface area. In this study, we utilized RNA-Seq technology to assess global gene expression in white lupin cluster roots, normal roots, and leaves in response to Pi supply. We de novo assembled 277,224,180 Illumina reads from 12 complementary DNA libraries to build what is to our knowledge the first white lupin gene index (LAGI 1.0). This index contains 125,821 unique sequences with an average length of 1,155 bp. Of these sequences, 50,734 were transcriptionally active (reads per kilobase per million reads ≥ 3), representing approximately 7.8% of the white lupin genome, using the predicted genome size of Lupinus angustifolius as a reference. We identified a total of 2,128 sequences differentially expressed in response to Pi deficiency with a 2-fold or greater change and P ≤ 0.05. Twelve sequences were consistently differentially expressed due to Pi deficiency stress in three species, Arabidopsis (Arabidopsis thaliana), potato (Solanum tuberosum), and white lupin, making them ideal candidates to monitor the Pi status of plants. Additionally, classic physiological experiments were coupled with RNA-Seq data to examine the role of cytokinin and gibberellic acid in Pi deficiency-induced cluster root development. This global gene expression analysis provides new insights into the biochemical and molecular mechanisms involved in the acclimation to Pi deficiency.

Molecular characterization of NADH-dependent glutamate synthase from alfalfa nodules.

Alfalfa NADH-dependent glutamate synthase (NADH-GOGAT), together with glutamine synthetase, plays a central role in the assimilation of symbiotically fixed nitrogen into amino acids in root nodules. Antibodies previously raised against purified NADH-GOGAT were employed to screen a cDNA library prepared using RNA isolated from nodules of 20-day-old alfalfa plants. A 7.2-kb cDNA clone was obtained that contained the entire protein coding region of NADH-GOGAT. Analysis of this cDNA and determination of the amino-terminal amino acids of the purified protein revealed that NADH-GOGAT is synthesized as a 2194-amino acid protein that includes a 101-amino acid presequence. The deduced amino acid sequence shares significant identity with maize ferredoxin-dependent GOGAT, and with both large and small subunits of Escherichia coli NADPH-GOGAT. DNA gel blot analysis of alfalfa genomic DNA suggests the presence of a single NADH-GOGAT gene or a small gene family. The expression of NADH-GOGAT mRNA, enzyme protein, and enzyme activity was developmentally regulated in root nodules. A dramatic increase in gene expression occurred coincidentally with the onset of nitrogen fixation in the bacteroid, and was absent in both ineffective plants that were nodulated with effective Rhizobium meliloti and effective plants that had been nodulated with ineffective R. meliloti strains. Maximum NADH-GOGAT expression, therefore, appears to require an effective, nitrogen-fixing symbiosis.

Primary assimilation of nitrogen in alfalfa nodules: molecular features of the enzymes involved

Abstract The primary assimilation of symbiotically fixed nitrogen (N) in alfalfa root nodules involves complex intermingling with carbon (C) metabolism. Integrated functioning of both cytosolic and organelle-associated enzymes is required to link N assimilation with C metabolism. Understanding how N and C metabolism are controlled in root nodules requires fundamental knowledge of how the plant genes involved are regulated. While significant progress has been made in understanding the regulation of glutamine synthetase (GS), much less is known about the genes controlling other enzymatic steps in this process. To that end we have isolated, purified and characterized the root nodules enzymes aspartate aminotransferase (AAT), phosphoenolpyruvate carboxylase (PEPC) and glutamate synthase (NADH-GOGAT). Moreover the cDNAs encoding these crucial enzymes were isolated and characterized. While the most prominent forms of GS associated with N assimilation in nodules are located in the cytosol, AAT and NADH-GOGAT appears to be organelle-associated. The deduced amino acid sequence suggested and immunogold labeling showed that nodule-enhanced AAT-2 is located in amyloplasts. Comparison of the deduced amino acid sequence of nodule-enhanced NADH-GOGAT to the N-terminal sequence of the processed protein indicated that NADH-GOGAT has a 101 amino acid presequence. However, it is unclear as to which organelle ADH-GOGAT is targeted. Cytosolic phosphoenolpyruvate carboxylase (PEPC), which can be expressed in legume root nodules at levels comparable to those detected in leaves of C4 plants, provides a substantial amount of carbon for malate, aspartate and asparagine biosyntheses. RNA blots showed that GS, AAT, PEPC, and NADH-GOGAT mRNAs were enhanced about 15-fold during the development of effective alfalfa nodules. By comparison, the expression of GS, AAT and PEPC mRNAs was reduced by 65% in ineffective nodules. NADH-GOGAT was different from GS, AAT, and PEPC in that expression had an absolute requirement for a factor(s) related to effective nodules. The data suggest that NADH-GOGAT plays a key role in regulating N assimilation. Moreover, plastids in nodules play a major role not only in C metabolism but also in N metabolism.

Malate plays a central role in plant nutrition

Malate occupies a central role in plant metabolism. Its importance in plant mineral nutrition is reflected by the role it plays in symbiotic nitrogen fixation, phosphorus acquisition, and aluminum tolerance. In nitrogen-fixing root nodules, malate is the primary substrate for bacteroid respiration, thus fueling nitrogenase. Malate also provides the carbon skeletons for assimilation of fixed nitrogen into amino acids. During phosphorus deficiency, malate is frequently secreted from roots to release unavailable forms of phosphorus. Malate is also involved with plant adaptation to aluminum toxicity. To define the genetic and biochemical regulation of malate formation in plant nutrition we have isolated and characterized genes involved in malate metabolism from nitrogen-fixing root nodules of alfalfa and those involved in organic acid excretion from phosphorus-deficient proteoid roots of white lupin. Moreover, we have overexpressed malate dehydrogenase in alfalfa in attempts to improve nutrient acquisition. This report is an overview of our efforts to understand and modify malate metabolism, particularly in the legumes alfalfa and white lupin.

Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules.

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N2 fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.

An RNA-Seq based gene expression atlas of the common bean

BackgroundCommon bean (Phaseolus vulgaris) is grown throughout the world and comprises roughly 50% of the grain legumes consumed worldwide. Despite this, genetic resources for common beans have been lacking. Next generation sequencing, has facilitated our investigation of the gene expression profiles associated with biologically important traits in common bean. An increased understanding of gene expression in common bean will improve our understanding of gene expression patterns in other legume species.ResultsCombining recently developed genomic resources for Phaseolus vulgaris, including predicted gene calls, with RNA-Seq technology, we measured the gene expression patterns from 24 samples collected from seven tissues at developmentally important stages and from three nitrogen treatments. Gene expression patterns throughout the plant were analyzed to better understand changes due to nodulation, seed development, and nitrogen utilization. We have identified 11,010 genes differentially expressed with a fold change ≥ 2 and a P-value < 0.05 between different tissues at the same time point, 15,752 genes differentially expressed within a tissue due to changes in development, and 2,315 genes expressed only in a single tissue. These analyses identified 2,970 genes with expression patterns that appear to be directly dependent on the source of available nitrogen. Finally, we have assembled this data in a publicly available database, The Phaseolus vulgaris Gene Expression Atlas (Pv GEA), http://plantgrn.noble.org/PvGEA/ . Using the website, researchers can query gene expression profiles of their gene of interest, search for genes expressed in different tissues, or download the dataset in a tabular form.ConclusionsThese data provide the basis for a gene expression atlas, which will facilitate functional genomic studies in common bean. Analysis of this dataset has identified genes important in regulating seed composition and has increased our understanding of nodulation and impact of the nitrogen source on assimilation and distribution throughout the plant.

White Lupin Cluster Root Acclimation to Phosphorus Deficiency and Root Hair Development Involve Unique Glycerophosphodiester Phosphodiesterases

White lupin (Lupinus albus) is a legume that is very efficient in accessing unavailable phosphorus (Pi). It develops short, densely clustered tertiary lateral roots (cluster/proteoid roots) in response to Pi limitation. In this report, we characterize two glycerophosphodiester phosphodiesterase (GPX-PDE) genes (GPX-PDE1 and GPX-PDE2) from white lupin and propose a role for these two GPX-PDEs in root hair growth and development and in a Pi stress-induced phospholipid degradation pathway in cluster roots. Both GPX-PDE1 and GPX-PDE2 are highly expressed in Pi-deficient cluster roots, particularly in root hairs, epidermal cells, and vascular bundles. Expression of both genes is a function of both Pi availability and photosynthate. GPX-PDE1 Pi deficiency-induced expression is attenuated as photosynthate is deprived, while that of GPX-PDE2 is strikingly enhanced. Yeast complementation assays and in vitro enzyme assays revealed that GPX-PDE1 shows catalytic activity with glycerophosphocholine while GPX-PDE2 shows highest activity with glycerophosphoinositol. Cell-free protein extracts from Pi-deficient cluster roots display GPX-PDE enzyme activity for both glycerophosphocholine and glycerophosphoinositol. Knockdown of expression of GPX-PDE through RNA interference resulted in impaired root hair development and density. We propose that white lupin GPX-PDE1 and GPX-PDE2 are involved in the acclimation to Pi limitation by enhancing glycerophosphodiester degradation and mediating root hair development.

Recruitment of Novel Calcium-Binding Proteins for Root Nodule Symbiosis in Medicago truncatula

Legume rhizobia symbiotic nitrogen (N2) fixation plays a critical role in sustainable nitrogen management in agriculture and in the Earths nitrogen cycle. Signaling between rhizobia and legumes initiates development of a unique plant organ, the root nodule, where bacteria undergo endocytosis and become surrounded by a plant membrane to form a symbiosome. Between this membrane and the encased bacteria exists a matrix-filled space (the symbiosome space) that is thought to contain a mixture of plant- and bacteria-derived proteins. Maintenance of the symbiosis state requires continuous communication between the plant and bacterial partners. Here, we show in the model legume Medicago truncatula that a novel family of six calmodulin-like proteins (CaMLs), expressed specifically in root nodules, are localized within the symbiosome space. All six nodule-specific CaML genes are clustered in the M. truncatula genome, along with two other nodule-specific genes, nodulin-22 and nodulin-25. Sequence comparisons and phylogenetic analysis suggest that an unequal recombination event occurred between nodulin-25 and a nearby calmodulin, which gave rise to the first CaML, and the gene family evolved by tandem duplication and divergence. The data provide striking evidence for the recruitment of a ubiquitous Ca2+-binding gene for symbiotic purposes.

Nodule Carbon Metabolism: Organic Acids for N2 Fixation

The energy burden (carbon (C) cost) imposed on plants for symbiotic nitrogen (N2) fixation is approximately 6 mg C · mg N reduced (Day, Copeland, 1991). Photosynthate, in the form of sucrose, is the ultimate source of carbon required for both N2 fixation and assimilation. Labeling studies show that sucrose derived from the shoot is transported to the nodule within 15 min, reaching steady state concentrations of 3.6 mg · gfw (Streeter, 1991). Although sucrose is the initial nodule product with the greatest amount of label derived from shoot CO2 fixation, it is rapidly metabolized to the organic acids malate and succinate accompanied by subsequent 14CO2 evolution. Several lines of evidence make it apparent, however, that C4-dicarboxylic acids rather than sucrose provide the energy for nitrogenase activity and C skeletons for N assimilation. Rhlzobium/Bradyrhizobium mutants incapable of utilizing glucose and fructose continue to form effective nodules, while mutants unable to take up malate and succinate form ineffective nodules (Ronson et al., 1981; Finan et al., 1983; Vance, Heichel, 1991). Bacteroids have a high affinity uptake system for C4-dicarboxylic acids but not sugars (Udvardi, Day, 1997). Bacterial mutants lacking malic enzyme are Fix (Driscoll, Finan, 1993). Symbiosome membranes have organic acid transporters capable of mediating a high flux of malate and succinate but lack comparable systems for sugars and amino acids (Udvardi, Day, 1997). Furthermore 14C-malate and - succinate synthesized through nodule 14CO2 fixation are directly incorporated into effective bacteroids and respired while concomitant metabolism does not occur with ineffective nodules (Rosendahl et al., 1990). Lastly, nodules incapable of N2 fixation have strikingly reduced concentrations of organic acids but not sucrose or other sugars (Anthon, Emerich 1990; Rosendahl et al., 1990; Romanov et al., 1995). The enhanced metabolism of sucrose to C4-dicarboxylic acids coupled to their uptake and use by bacteroids reflect exquisite symbiotic adaptations in carbon metabolism for energy production in a low O2 environment. These plant adaptations involve coordinated expression and control of three critical enzymes, sucrose synthase (SS; EC 2.4.1.13), phosphoenolpyruvate carboxylase (PEPC; 4.1.1.31), and malate dehydrogenase (MDH; EC 1.1.1.82).

Genomic structure, expression and evolution of the alfalfa aspartate aminotransferase genes.

Genomic clones encoding two isozymes of aspartate aminotransferase (AAT) were isolated from an alfalfa genomic library and their DNA sequences were determined. The AAT1 gene contains 12 exons that encode a cytosolic protein expressed at similar levels in roots, stems and nodules. In nodules, the amount of AAT1 mRNA was similar at all stages of development, and was slightly reduced in nodules incapable of fixing nitrogen. The AAT1 mRNA is polyadenylated at multiple sites differing by more than 250 bp. The AAT2 gene contains 11 exons, with 5 introns located in positions identical to those found in animal AAT genes, and encodes a plastid-localized isozyme. The AAT2 mRNA is polyadenylated at a very limited range of sites. The transit peptide of AAT2 is encoded by the first two and part of the third exon. AAT2 mRNA is much more abundant in nodules than in other organs, and increases dramatically during the course of nodule development. Unlike AAT1, expression of AAT2 is significantly reduced in nodules incapable of fixing nitrogen. Phylogenetic analysis of deduced AAT proteins revealed 4 separate but related groups of AAT proteins; the animal cytosolic AATs, the plant cytosolic AATs, the plant plastid AATs, and the mitochondrial AATs.