John W. Gronwald

Inhibition of plant acetyl-coenzyme a carboxylase by the herbicides sethoxydim and haloxyfop

Incorporation of [14C]acetate or [14C]pyruvate into fatty acids in isolated corn seedling chloroplasts was inhibited 90% or greater by 10 microM sethoxydim or 1 microM haloxyfop. At these concentrations, neither sethoxydim nor haloxyfop inhibited [14C]acetate incorporation into fatty acids in isolated pea chloroplasts. Sethoxydim (10 microM) and haloxyfop (1 microM) did not inhibit incorporation of [14C]malonyl-CoA into fatty acids in cell free extracts from corn tissue cultures. Acetyl coenzyme A carboxylase (EC 6.4.1.2) from corn seedling chloroplasts was inhibited by both sethoxydim and haloxyfop, with I50 values of 2.9 and 0.5 microM, respectively. This enzyme in pea was not inhibited by 10 microM sethoxydim or 1 microM haloxyfop.

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.

Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems

BackgroundAlfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling.ResultsCell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. In addition, 341,984 ESTs were generated from ES and PES internodes of genotype 773 using the GS FLX Titanium platform. The first alfalfa (Medicago sativa) gene index (MSGI 1.0) was assembled using the Sanger ESTs available from GenBank, the GS FLX Titanium EST sequences, and the de novo assembled Illumina sequences. MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1,294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Out of 55 SNPs randomly selected for experimental validation, 47 (85%) were polymorphic between the two genotypes. We also identified numerous allelic variations within each genotype. Digital gene expression analysis identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes.ConclusionsOur results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a forage crop and cellulosic feedstock.

Reduction in plasma membrane ATPase activity of tomato roots by salt stress

Abstract ATPase activity was characterized in a plasma membrane-enriched fraction isolated from tomato ( Lycopersicon exculentum Mill., cv. Heinz 1350) roots grown in the absence or presence of salinity stress (−4 bars, 60 mM NaCl plus 12 mM CaCl 2 ). The enzyme exhibited the following properties in both non-stressed and salt-stressed roots: (1) activated by divalent cations (Mg 2+ ≻ Mn 2+ ≻ Co 2+ ≻ Ni 2+ ≻ Ca 2+ ≻ Zn 2+ ) and further stimulated by monovalent cations (Na + = K + ≻ Rb + ≻ Li + ); (2) pH optima for Mg 2+ activation and KCl-stimulation of 7.0 and 6.5, respectively; (3) selective for Mg 2+ -ATP as substrate; (4) sensitive to N , N ′-dicyclohexylcarbodiimide and vanadate but insensitive to azide and oligomycin; (5) not stimulated synergistically by Na + plus K + . Exposing roots to salt-stress altered the kinetics of Mg 2+ -ATPase activity. Simple Michaelis-Menten kinetics were observed when Mg 2+ -ATP was used as substrate for both control and salt-treated roots. Salt-stress had little effect on the apparent K m for Mg 2+ -ATP. The predominant effect of salt-stress was reduce V max of Mg 2+ -ATPase activity from 69 μmol P i (mg protein) −1 h −1 in control roots to 39 μmol P i (mg protein) −1 h −1 in salt-treated roots.

Transcript profiling of common bean (Phaseolus vulgaris L.) using the GeneChip®Soybean Genome Array: optimizing analysis by masking biased probes

BackgroundCommon bean (Phaseolus vulgaris L.) and soybean (Glycine max) both belong to the Phaseoleae tribe and share significant coding sequence homology. This suggests that the GeneChip® Soybean Genome Array (soybean GeneChip) may be used for gene expression studies using common bean.ResultsTo evaluate the utility of the soybean GeneChip for transcript profiling of common bean, we hybridized cRNAs purified from nodule, leaf, and root of common bean and soybean in triplicate to the soybean GeneChip. Initial data analysis showed a decreased sensitivity and accuracy of measuring differential gene expression in common bean cross-species hybridization (CSH) GeneChip data compared to that of soybean. We employed a method that masked putative probes targeting inter-species variable (ISV) regions between common bean and soybean. A masking signal intensity threshold was selected that optimized both sensitivity and accuracy of measuring differential gene expression. After masking for ISV regions, the number of differentially-expressed genes identified in common bean was increased by 2.8-fold reflecting increased sensitivity. Quantitative RT-PCR (qRT-PCR) analysis of 20 randomly selected genes and purine-ureide pathway genes demonstrated an increased accuracy of measuring differential gene expression after masking for ISV regions. We also evaluated masked probe frequency per probe set to gain insight into the sequence divergence pattern between common bean and soybean. The sequence divergence pattern analysis suggested that the genes for basic cellular functions and metabolism were highly conserved between soybean and common bean. Additionally, our results show that some classes of genes, particularly those associated with environmental adaptation, are highly divergent.ConclusionsThe soybean GeneChip is a suitable cross-species platform for transcript profiling in common bean when used in combination with the masking protocol described. In addition to transcript profiling, CSH of the GeneChip in combination with masking probes in the ISV regions can be used for comparative ecological and/or evolutionary genomics studies.

The Medicago sativa gene index 1.2: a web-accessible gene expression atlas for investigating expression differences between Medicago sativa subspecies

BackgroundAlfalfa (Medicago sativa L.) is the primary forage legume crop species in the United States and plays essential economic and ecological roles in agricultural systems across the country. Modern alfalfa is the result of hybridization between tetraploid M. sativa ssp. sativa and M. sativa ssp. falcata. Due to its large and complex genome, there are few genomic resources available for alfalfa improvement.ResultsA de novo transcriptome assembly from two alfalfa subspecies, M. sativa ssp. sativa (B47) and M. sativa ssp. falcata (F56) was developed using Illumina RNA-seq technology. Transcripts from roots, nitrogen-fixing root nodules, leaves, flowers, elongating stem internodes, and post-elongation stem internodes were assembled into the Medicago sativa Gene Index 1.2 (MSGI 1.2) representing 112,626 unique transcript sequences. Nodule-specific and transcripts involved in cell wall biosynthesis were identified. Statistical analyses identified 20,447 transcripts differentially expressed between the two subspecies. Pair-wise comparisons of each tissue combination identified 58,932 sequences differentially expressed in B47 and 69,143 sequences differentially expressed in F56. Comparing transcript abundance in floral tissues of B47 and F56 identified expression differences in sequences involved in anthocyanin and carotenoid synthesis, which determine flower pigmentation. Single nucleotide polymorphisms (SNPs) unique to each M. sativa subspecies (110,241) were identified.ConclusionsThe Medicago sativa Gene Index 1.2 increases the expressed sequence data available for alfalfa by ninefold and can be expanded as additional experiments are performed. The MSGI 1.2 transcriptome sequences, annotations, expression profiles, and SNPs were assembled into the Alfalfa Gene Index and Expression Database (AGED) at http://plantgrn.noble.org/AGED/, a publicly available genomic resource for alfalfa improvement and legume research.

Medicago truncatula as a Model for Dicot Cell Wall Development

We have initiated a genome-wide transcript profiling study using the model legume Medicago truncatula to identify putative genes related to cell wall biosynthesis and regulatory function in legumes. We used the GeneChip® Medicago Genome Array to compare transcript abundance in elongating versus postelongation stem internode segments of two M. truncatula accessions and two Medicago sativa (alfalfa) clones with contrasting stem cell wall concentration and composition. Hundreds of differentially expressed probe sets between elongating and postelongation stem segments showed similar patterns of gene expression in the model legume and cultivated alfalfa. Differentially expressed genes included genes with putative functions associated with primary and secondary cell wall biosynthesis and growth. Mining of public microarray data for coexpressed genes with two marker genes for secondary cell wall synthesis identified additional candidate secondary cell wall-related genes. Coexpressed genes included protein kinases, transcription factors, and unclassified groups that were not previously reported with secondary cell wall-associated genes. M. truncatula has been recognized as an excellent model plant for legume genomics. The stem tissue transcriptome analysis, described here, indicates that M. truncatula has utility as a model plant for cell wall genomics in legumes in general and shows excellent potential for translating gene discoveries to its close relative, cultivated alfalfa, in particular. The natural variation for stem cell wall traits in Medicago may offer a new tool to study an expanded repertoire of valuable agronomic traits in related species, including woody dicots in the eurosid I clade.

Transcript profiling of two alfalfa genotypes with contrasting cell wall composition in stems using a cross-species platform: optimizing analysis by masking biased probes

BackgroundThe GeneChip®Medicago Genome Array, developed for Medicago truncatula, is a suitable platform for transcript profiling in tetraploid alfalfa [Medicago sativa (L.) subsp. sativa]. However, previous research involving cross-species hybridization (CSH) has shown that sequence variation between two species can bias transcript profiling by decreasing sensitivity (number of expressed genes detected) and the accuracy of measuring fold-differences in gene expression.ResultsTranscript profiling using the Medicago GeneChip® was conducted with elongating stem (ES) and post-elongation stem (PES) internodes from alfalfa genotypes 252 and 1283 that differ in stem cell wall concentrations of cellulose and lignin. A protocol was developed that masked probes targeting inter-species variable (ISV) regions of alfalfa transcripts. A probe signal intensity threshold was selected that optimized both sensitivity and accuracy. After masking for both ISV regions and previously identified single-feature polymorphisms (SFPs), the number of differentially expressed genes between the two genotypes in both ES and PES internodes was approximately 2-fold greater than the number detected prior to masking. Regulatory genes, including transcription factor and receptor kinase genes that may play a role in development of secondary xylem, were significantly over-represented among genes up-regulated in 252 PES internodes compared to 1283 PES internodes. Several cell wall-related genes were also up-regulated in genotype 252 PES internodes. Real-time quantitative RT-PCR of differentially expressed regulatory and cell wall-related genes demonstrated increased sensitivity and accuracy after masking for both ISV regions and SFPs. Over 1,000 genes that were differentially expressed in ES and PES internodes of genotypes 252 and 1283 were mapped onto putative orthologous loci on M. truncatula chromosomes. Clustering simulation analysis of the differentially expressed genes suggested co-expression of some neighbouring genes on Medicago chromosomes.ConclusionsThe problems associated with transcript profiling in alfalfa stems using the Medicago GeneChip as a CSH platform were mitigated by masking probes targeting ISV regions and SFPs. Using this masking protocol resulted in the identification of numerous candidate genes that may contribute to differences in cell wall concentration and composition of stems of two alfalfa genotypes.

Effects of the fungal protein Nep1 and Pseudomonas syringae on growth of Canada thistle (Cirsium arvense), common ragweed (Ambrosia artemisiifolia), and common dandelion (Taraxacum officinale)

Abstract The effects of the fungal protein Nep1 and Pseudomonas syringae pv. tagetis (Pst) applied separately or in combination on Canada thistle, common ragweed, and common dandelion were examined in growth chamber experiments. Experiments examined five treatments: (1) untreated control, (2) Silwet L-77 (0.3%, v/v) control, (3) Nep1 (5 μg ml−1) plus Silwet L-77 (0.3%, v/v), (4) Pst (109 colony-forming units [cfu] ml−1) plus Silwet L-77 (0.3%, v/v), and (5) Pst (109 cfu ml−1) and Nep1 (5 μg ml−1) plus Silwet L-77 (0.3%, v/v). Foliar treatments were applied at 28, 26, and 21 d after planting for Canada thistle, common dandelion, and common ragweed, respectively. For all three species, foliar application of Nep1 alone or in combination with Pst caused rapid desiccation and necrosis of leaves, with the greatest effect on recent, fully expanded (RFE) leaves. Within 4 to 8 h after treatment (HAT), 60 to 80% of RFE leaves of all three species were necrotic. Measured 72 HAT, Pst populations in Canada thistle leaves treated with Nep1 plus Pst were approximately 105 cfu cm−2 compared with 107 cfu cm−2 for leaves treated with Pst alone. Measured 2 wk after treatment, foliar application of Nep1 reduced shoot dry weight of the three weeds by 30 to 41%. Treatment with Pst reduced shoot growth of common ragweed, Canada thistle, and common dandelion by 82, 31, and 41%, respectively. The large suppression of common ragweed shoot growth caused by Pst treatment was associated with a high percentage (60%) of leaf area exhibiting chlorosis. Treatment with Pst plus Nep1 did not result in significant decreases in shoot dry weight for Canada thistle and common dandelion compared with either treatment alone. For common ragweed, shoot growth reduction caused by applying Pst and Nep1 together was not greater than that caused by Pst alone. Nomenclature: Canada thistle, Cirsium arvense L. (Scop.) CIRAR; common ragweed, Ambrosia artemisiifolia L. AMBEL; common dandelion, Taraxacum officinale Weber in Wiggers TAROF.

Arabidopsis UDP-sugar pyrophosphorylase: Evidence for two isoforms

Arabidopsis UDP-sugar pyrophosphorylase (AtUSP, EC 2.7.7.64) is a broad substrate pyrophosphorylase that exhibits activity with GlcA-1-P, Gal-1-P and Glc-1-P. Immunoblots using polyclonal antibodies raised to recombinant AtUSP demonstrated the presence of two USP isoforms of approximately 70 kDa (USP1) and 66 kDa (USP2) in crude extracts of Arabidopsis tissues. The 66 kDa isoform was not the result of proteolytic cleavage of USP1 during extraction. Trypsin digestion of bands on SDS gels corresponding to the location of the two isoforms followed by tandem mass spectrometry confirmed that USP peptides were present in both bands. Both USP isoforms were detected in the cytosol as determined by immunoblots of cellular fractions obtained by differential centrifugation. However, some USP1 was also detected in the microsomal fraction. Immunoprecipitation assays demonstrated that AtUSP antibodies removed USP activity (UDP-GlcA-->GlcA-1-P) measured in floret extracts. These results indicate that USP is the only pyrophosphorylase that utilizes UDP-GlcA as a substrate and suggest that it serves as the terminal enzyme of the myo-inositol oxidation pathway.