Metagenomic Anlaysis of microsymbiont selection by the legume plant host

Rhizobium leguminosarum bv.viciae is able to establish nitrogen-fixing symbioses with legumes of the genera Pisum, Lens, Lathyrus and Vicia. Classic studies using trap plants (Laguerre et al., Young et al.) provided evidence that different plant hosts are able to select different rhizobial genotypes among those available in a given soil. However, these studies were necessarily limited by the paucity of relevant biodiversity markers. We have now reappraised this problem with the help of genomic tools. A well-characterized agricultural soil (INRA Bretennieres) was used as source of rhizobia. Plants of Pisum sativum, Lens culinaris, Vicia sativa and V. faba were used as traps. Isolates from 100 nodules were pooled, and DNA from each pool was sequenced (BGI-Hong Kong; Illumina Hiseq 2000, 500 bp PE libraries, 100 bp reads, 12 Mreads). Reads were quality filtered (FastQC, Trimmomatic), mapped against reference R. leguminosarum genomes (Bowtie2, Samtools), and visualized (IGV). An important fraction of the filtered reads were not recruited by reference genomes, suggesting that plant isolates contain genes that are not present in the reference genomes. For this study, we focused on three conserved genomic regions: 16S-23S rDNA, atpD and nodDABC, and a Single Nucleotide Polymorphism (SNP) analysis was carried out with meta / multigenomes from each plant. Although the level of polymorphism varied (lowest in the rRNA region), polymorphic sites could be identified that define the specific soil population vs. reference genomes. More importantly, a plant-specific SNP distribution was observed. This could be confirmed with many other regions extracted from the reference genomes (data not shown). Our results confirm at the genomic level previous observations regarding plant selection of specific genotypes. We expect that further, ongoing comparative studies on differential meta / multigenomic sequences will identify specific gene components of the plant-selected genotypes

Population genomics of host specificity in Rhizobium leguminosarum bv. viciae

Legumes establish a root-nodule symbiosis with soil bacteria collectively known as rhizobia. This symbiosis allows legumes to benefit from the nitrogen fixation capabilities of rhizobia and thus to grow in the absence of any fixed nitrogen source. This is especially relevant for Agriculture, where intensive plant growth depletes soils of useable, fixed nitrogen sources. One of the main features of the root nodule symbiosis is its specificity. Different rhizobia are able to nodulate different legumes. Rhizobium leguminosarum bv. viciae is able to establish an effective symbiosis with four different plant genera (Pisum, Lens, Vicia, Lathyrus), and any given isolate will nodulate any of the four plant genera. A population genomics study with rhizobia isolated from P. sativum, L. culinaris, V. sativa or V. faba, all originating in the same soil, showed that plants select specific genotypes from those available in that soil. This was demonstrated at the genome-wide level, but also for specific genes. Accelerated mesocosm studies with successive plant cultures provided additional evidence on this plant selection and on the nature of the genotypes selected. Finally, representatives from the major rhizobial genotypes isolated from these plants allowed characterization of the size and nature of the respective pangenome and specific genome compartments. These were compared to the different genotypes ?symbiotic and non-symbiotic?present in rhizobial populations isolated directly from the soil without plant intervention.

Effect of the Legume Plant Host on Rhizobial Soil Population Dynamics

Rhizobium leguminosarum bv viciae (Rlv) is a soil bacterium able to establish specific root-nodule symbioses with legumes of four different genera: Pisum, Vicia, Lens and Lathyrus. Rlv isolates from nodules of any of these legumes can nodulate any of them; however, it has been shown that plants select specific rhizobial genotypes from those present in the soil (1,2). We have previously shown this at the genomic level by following a population genomics approach. Pool genomic sequences from 100 isolates from each of four plant species: P. sativum, L. culinaris, V. faba and V. sativa, show different, specific profiles at the single nucleotide polymorphism (SNP) level for relevant genes. In this work, the extent of Rlv selection from a well-characterized soil population by different legume plant hosts: P. sativum, L. culinaris, V. faba and V. sativa, after a medium-term mesocosm study is described. Direct soil isolates from each of these mesocosm studies have been tested for specific rhizobial genes (glnII and fnrN) and symbiotic genes (nodC and nifH). Different populations were characterized further by Sanger sequencing of both the rpoB phylogenetic marker gene and the symbiotic genes nodC and nifH. The distribution and size of the rhizobial population for each legume host showed changes during the medium-term mesocosm study. Particularly, a non-symbiotic group of rhizobia was enriched by all four hosts, in contrast to the symbiotic rhizobia profile, which was specific for each legume plant host.

The Pangenome of the Plant-Selected Rhizobium leguminosarum bv viciae Sub-popuations

Rhizobium leguminosarum bv viciae (Rlv) is a bacterium able to establish effective symbioses with four different legume genera: Pisum, Lens, Lathyrus and Vicia. Classic studies using trap plants have previously shown that, given a choice, different plants prefer specific genotypes of rhizobia, which are adapted to the host (1, 2). In previous work we have performed a Pool-Seq analysis bases on pooled DNA samples from Rlv nodule isolates obtained from Pisum sativum, Lens culinaris, Vicia fava and V. sativa plants, used as rhizobial traps. This experiment allowed us to test the host preference hypothesis: different plant hosts select specific sub-populations of rhizobia from the available population present in a given soil. We have observed that plant-selected sub-populations are different at the single nucleotide polymorphism (SNP) level. We have selected individual isolates from each sub-population (9 fava-bean isolates, 14 pea isolates 9 vetch isolates and 9 lentil isolates) and sequenced their genomes at draft level (ca. 30x, 90 contigs). Genomic analyses have been carried out using J-species and CMG-Biotools. All the isolates had similar genome size (7.5 Mb) and number of genes (7,300). The resulting Average Nucleotide Identity (ANIm) tree showed that Rhizobium leguminosarum bv viciae is a highly diverse group. Each plant-selected subpopulation showed a closed pangenome and core genomes of similar size (11,500 and 4,800 genes, respectively). The addition of all four sub-population results in a larger, closed pangenome of 19,040 genes and a core genome of similar size (4,392 genes). Each sub-population contains a characteristic set of genes but no universal, plant-specific genes were found. The core genome obtained from all four sub-populations is probably a representative core genome for Rhizobium leguminosarum, given that the reference genome (Rhizobium leguminosarum bv. viciae strain 3841) contains most of the core genome. We have also analyzed the symbiotic cluster (nod), and different nod cluster genotypes were found in each sub-population. Supported by MINECO (Consolider-Ingenio 2010, MICROGEN Project, CSD2009-00006).

Mendel’s dwarfing gene: cDNAs from the Le alleles and function of the expressed proteins

The major gibberellin (GA) controlling stem elongation in pea (Pisum sativum L.) is GA1, which is formed from GA20 by 3β-hydroxylation. This step, which limits GA1 biosynthesis in pea, is controlled by the Le locus, one of the original Mendelian loci. Mutations in this locus result in dwarfism. We have isolated cDNAs encoding a GA 3β-hydroxylase from lines of pea carrying the Le, le, le-3, and led alleles. The cDNA sequences from le and le-3 each contain a base substitution resulting in single amino acid changes relative to the sequence from Le. The cDNA sequence from led, a mutant derived from an le line, contains both the le “mutation” and a single-base deletion, which causes a shift in reading frame and presumably a null mutation. cDNAs from each line were expressed in Escherichia coli. The expression product for the clone from Le converted GA9 to GA4, and GA20 to GA1, with Km values of 1.5 μM and 13 μM, respectively. The amino acid substitution in the clone from le increased Km for GA9 100-fold and reduced conversion of GA20 to almost nil. Expression products from le and le-3 possessed similar levels of 3β-hydroxylase activity, and the expression product from led was inactive. Our results suggest that the 3β-hydroxylase cDNA is encoded by Le. Le transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in internodes and leaves of established seedlings, and in developing seeds.

A reversibly glycosylated polypeptide (RGP1) possibly involved in plant cell wall synthesis: Purification, gene cloning, and trans-Golgi localization

We purified from pea (Pisum sativum) tissue an ≈40 kDa reversibly glycosylated polypeptide (RGP1) that can be glycosylated by UDP-Glc, UDP-Xyl, or UDP-Gal, and isolated a cDNA encoding it, apparently derived from a single-copy gene (Rgp1). Its predicted translation product has 364 aminoacyl residues and molecular mass of 41.5 kDa. RGP1 appears to be a membrane-peripheral protein. Immunogold labeling localizes it specifically to trans-Golgi dictyosomal cisternae. Along with other evidence, this suggests that RGP1 is involved in synthesis of xyloglucan and possibly other hemicelluloses. Corn (Zea mays) contains a biochemically similar and structurally homologous RGP1, which has been thought (it now seems mistakenly) to function in starch synthesis. The expressed sequence database also reveals close homologs of pea Rgp1 in Arabidopsis and rice (Oryza sativa). Rice possesses, in addition, a distinct but homologous sequence (Rgp2). RGP1 provides a polypeptide marker for Golgi membranes that should be useful in plant membrane studies.

Vipp1 deletion mutant of Synechocystis: A connection between bacterial phage shock and thylakoid biogenesis?

Plant chloroplasts originated from an endosymbiotic event by which an ancestor of contemporary cyanobacteria was engulfed by an early eukaryotic cell and then transformed into an organelle. Oxygenic photosynthesis is the specific feature of cyanobacteria and chloroplasts, and the photosynthetic machinery resides in an internal membrane system, the thylakoids. The origin and genesis of thylakoid membranes, which are essential for oxygenic photosynthesis, are still an enigma. Vipp1 (vesicle-inducing protein in plastids 1) is a protein located in both the inner envelope and the thylakoids of Pisum sativum and Arabidopsis thaliana. In Arabidopsis disruption of the VIPP1 gene severely affects the plant's ability to form properly structured thylakoids and as a consequence to carry out photosynthesis. In contrast, Vipp1 in Synechocystis appears to be located exclusively in the plasma membrane. Yet, as in higher plants, disruption of the VIPP1 gene locus leads to the complete loss of thylakoid formation. So far VIPP1 genes are found only in organisms carrying out oxygenic photosynthesis. They share sequence homology with a subunit encoded by the bacterial phage shock operon (PspA) but differ from PspA by a C-terminal extension of about 30 amino acids. In two cyanobacteria, Synechocystis and Anabaena, both a VIPP1 and a pspA gene are present, and phylogenetic analysis indicates that VIPP1 originated from a gene duplication of the latter and thereafter acquired its new function. It also appears that the C-terminal extension that discriminates VIPP1 proteins from PspA is important for its function in thylakoid formation.

Cloning and Characterization of TPE4A, a Thiol-Protease Gene Induced during Ovary Senescence and Seed Germination in Pea1

A cDNA clone encoding a thiol-protease (TPE4A) was isolated from senescent ovaries of pea (Pisum sativum) by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of TPE4A has the conserved catalytic amino acids of papain. It is very similar to VSCYSPROA, a thiol-protease induced during seed germination in common vetch. TPE4A mRNA levels increase during the senescence of unpollinated pea ovaries and are totally suppressed by treatment with gibberellic acid. In situ hybridization indicated that TPE4A mRNA distribution in senescent pea ovaries is different from that of previously reported thiol-proteases induced during senescence, suggesting the involvement of different proteases in the mobilization of proteins from senescent pea ovaries. TPE4A is also induced during the germination of pea seeds, indicating that a single protease gene can be induced during two different physiological processes, senescence and germination, both of which require protein mobilization.

Identification of a Ca2+/H+ Antiport in the Plant Chloroplast Thylakoid Membrane1

To assess the availability of Ca2+ in the lumen of the thylakoid membrane that is required to support the assembly of the oxygen-evolving complex of photosystem II, we have investigated the mechanism of 45Ca2+ transport into the lumen of pea (Pisum sativum) thylakoid membranes using silicone-oil centrifugation. Trans-thylakoid Ca2+ transport is dependent on light or, in the dark, on exogenously added ATP. Both light and ATP hydrolysis are coupled to Ca2+ transport through the formation of a transthylakoid pH gradient. The H+-transporting ionophores nigericin/K+ and carbonyl cyanide 3-chlorophenylhydrazone inhibit the transport of Ca2+. Thylakoid membranes are capable of accumulating up to 30 nmol Ca2+ mg−1 chlorophyll from external concentrations of 15 μm over the course of a 15-min reaction. These results are consistent with the presence of an active Ca2+/H+ antiport in the thylakoid membrane. Ca2+ transport across the thylakoid membrane has significant implications for chloroplast and plant Ca2+ homeostasis. We propose a model of chloroplast Ca2+ regulation whereby the activity of the Ca2+/H+ antiporter facilitates the light-dependent uptake of Ca2+ by chloroplasts and reduces stromal Ca2+ levels.

A Calcium-Selective Channel from Root-Tip Endomembranes of Garden Cress1

A Ca2+ channel from root-tip endomembranes of garden cress (Lepidium sativum L.) (LCC1) was characterized using the planar lipid-bilayer technique. Investigation of single-channel recordings revealed that LCC1 is voltage gated and strongly rectifying. In symmetrical 50 mm CaCl2 solutions, the single-channel conductance was 24 picosiemens. LCC1 showed a moderate selectivity for Ca2+ over K+ (9.4:1) and was permeable for a range of divalent cations (Ca2+, Ba2+, and Sr2+). In contrast to Bryonia dioica Ca2+ channel 1, a Ca2+-selective channel from the endoplasmic reticulum of touch-sensitive tendrils, LCC1 showed no bursting channel activity and had a low open probability and mean open time (2.83 ms at 50 mV). Inhibitor studies demonstrated that LCC1 is blocked by micromolar concentrations of erythrosin B (inhibitor concentration for 50% inhibition [IC50] = 1.8 μm) and the trivalent cations La3+ (IC50 = 5 μm) and Gd3+ (IC50 = 10 μm), whereas verapamil showed no blocking effect. LCC1 may play an important role in the regulation of the cytoplasmic free Ca2+ concentration in root-tip and/or root-cap cells. The question of whether this ion channel is part of the gravitropic signal transduction pathway deserves further investigation.

Brassinosteroid/Sterol Synthesis and Plant Growth as Affected by lka and lkb Mutations of Pea1

The dwarf pea (Pisum sativum) mutants lka and lkb are brassinosteroid (BR) insensitive and deficient, respectively. The dwarf phenotype of the lkb mutant was rescued to wild type by exogenous application of brassinolide and its biosynthetic precursors. Gas chromatography-mass spectrometry analysis of the endogenous sterols in this mutant revealed that it accumulates 24-methylenecholesterol and isofucosterol but is deficient in their hydrogenated products, campesterol and sitosterol. Feeding experiments using 2H-labeled 24-methylenecholesterol indicated that the lkb mutant is unable to isomerize and/or reduce the Δ24(28) double bond. Dwarfism of the lkb mutant is, therefore, due to BR deficiency caused by blocked synthesis of campesterol from 24-methylenecholesterol. The lkb mutation also disrupted sterol composition of the membranes, which, in contrast to those of the wild type, contained isofucosterol as the major sterol and lacked stigmasterol. The lka mutant was not BR deficient, because it accumulated castasterone. Like some gibberellin-insensitive dwarf mutants, overproduction of castasterone in the lka mutant may be ascribed to the lack of a feedback control mechanism due to impaired perception/signal transduction of BRs. The possibility that castasterone is a biologically active BR is discussed.

Study of the 3-Hydroxy Eicosanoyl-Coenzyme A Dehydratase and (E)-2,3 Enoyl-Coenzyme A Reductase Involved in Acyl-Coenzyme A Elongation in Etiolated Leek Seedlings1

(R,S)-[1-14C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-14C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-14C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-14C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-14C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-14C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-14C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg−1 h−1, which is the highest ever reported for a plant system.

Oxaloacetate Transport into Plant Mitochondria1

The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake of 14C-labeled OA into liposomes with incorporated mitochondrial membrane proteins preloaded with various dicarboxylates or citrate. OA was found to be transported in an obligatory counterexchange with malate, 2-oxoglutarate, succinate, citrate, or aspartate. Phtalonate inhibited all of these countertransports. OA-malate countertransport was inhibited by 4,4′-dithiocyanostilbene-2,2′-disulfonate and pyridoxal phosphate, and also by p-chloromercuribenzene sulfonate and mersalyl, indicating that a lysine and a cysteine residue of the translocator protein are involved in the transport. From these and other inhibition studies, we concluded that plant mitochondria contain an OA translocator that differs from all other known mitochondrial translocators. Major functions of this translocator are the export of reducing equivalents from the mitochondria via the malate-OA shuttle and the export of citrate via the citrate-OA shuttle. In the cytosol, citrate can then be converted either into 2-oxoglutarate for use as a carbon skeleton for nitrate assimilation or into acetyl-coenzyme A for use as a precursor for fatty acid elongation or isoprenoid biosynthesis.

A Multisubunit Acetyl Coenzyme A Carboxylase from Soybean1

A multisubunit form of acetyl coenzyme A (CoA) carboxylase (ACCase) from soybean (Glycine max) was characterized. The enzyme catalyzes the formation of malonyl CoA from acetyl CoA, a rate-limiting step in fatty acid biosynthesis. The four known components that constitute plastid ACCase are biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and the α- and β-subunits of carboxyltransferase (α- and β-CT). At least three different cDNAs were isolated from germinating soybean seeds that encode BC, two that encode BCCP, and four that encode α-CT. Whereas BC, BCCP, and α-CT are products of nuclear genes, the DNA that encodes soybean β-CT is located in chloroplasts. Translation products from cDNAs for BC, BCCP, and α-CT were imported into isolated pea (Pisum sativum) chloroplasts and became integrated into ACCase. Edman microsequence analysis of the subunits after import permitted the identification of the amino-terminal sequence of the mature protein after removal of the transit sequences. Antibodies specific for each of the chloroplast ACCase subunits were generated against products from the cDNAs expressed in bacteria. The antibodies permitted components of ACCase to be followed during fractionation of the chloroplast stroma. Even in the presence of 0.5 m KCl, a complex that contained BC plus BCCP emerged from Sephacryl 400 with an apparent molecular mass greater than about 800 kD. A second complex, which contained α- and β-CT, was also recovered from the column, and it had an apparent molecular mass of greater than about 600 kD. By mixing the two complexes together at appropriate ratios, ACCase enzymatic activity was restored. Even higher ACCase activities were recovered by mixing complexes from pea and soybean. The results demonstrate that the active form of ACCase can be reassembled and that it could form a high-molecular-mass complex.

Organellar and Cytosolic Localization of Four Phosphoribosyl Diphosphate Synthase Isozymes in Spinach

Four cDNAs encoding phosphoribosyl diphosphate (PRPP) synthase were isolated from a spinach (Spinacia oleracea) cDNA library by complementation of an Escherichia coli Δprs mutation. The four gene products produced PRPP in vitro from ATP and ribose-5-phosphate. Two of the enzymes (isozymes 1 and 2) required inorganic phosphate for activity, whereas the others were phosphate independent. PRPP synthase isozymes 2 and 3 contained 76 and 87 amino acid extensions, respectively, at their N-terminal ends in comparison with other PRPP synthases. Isozyme 2 was synthesized in vitro and shown to be imported and processed by pea (Pisum sativum) chloroplasts. Amino acid sequence analysis indicated that isozyme 3 may be transported to mitochondria and that isozyme 4 may be located in the cytosol. The deduced amino acid sequences of isozymes 1 and 2 and isozymes 3 and 4 were 88% and 75% identical, respectively. In contrast, the amino acid identities of PRPP synthase isozyme 1 or 2 with 3 or 4 was modest (22%–25%), but the sequence motifs for binding of PRPP and divalent cation-nucleotide were identified in all four sequences. The results indicate that PRPP synthase isozymes 3 and 4 belong to a new class of PRPP synthases that may be specific to plants.