Michael K. Udvardi

Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis

Gene transcripts with invariant abundance during development and in the face of environmental stimuli are essential reference points for accurate gene expression analyses, such as RNA gel-blot analysis or quantitative reverse transcription-polymerase chain reaction (PCR). An exceptionally large set of data from Affymetrix ATH1 whole-genome GeneChip studies provided the means to identify a new generation of reference genes with very stable expression levels in the model plant species Arabidopsis (Arabidopsis thaliana). Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions. Most of these were expressed at much lower levels than traditional reference genes, making them very suitable for normalization of gene expression over a wide range of transcript levels. Specific and efficient primers were developed for 22 genes and tested on a diverse set of 20 cDNA samples. Quantitative reverse transcription-PCR confirmed superior expression stability and lower absolute expression levels for many of these genes, including genes encoding a protein phosphatase 2A subunit, a coatomer subunit, and an ubiquitin-conjugating enzyme. The developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels in Arabidopsis in the future.

Genome-Wide Reprogramming of Primary and Secondary Metabolism, Protein Synthesis, Cellular Growth Processes, and the Regulatory Infrastructure of Arabidopsis in Response to Nitrogen

Transcriptome analysis, using Affymetrix ATH1 arrays and a real-time reverse transcription-PCR platform for >1,400 transcription factors, was performed to identify processes affected by long-term nitrogen-deprivation or short-term nitrate nutrition in Arabidopsis. Two days of nitrogen deprivation led to coordinate repression of the majority of the genes assigned to photosynthesis, chlorophyll synthesis, plastid protein synthesis, induction of many genes for secondary metabolism, and reprogramming of mitochondrial electron transport. Nitrate readdition led to rapid, widespread, and coordinated changes. Multiple genes for the uptake and reduction of nitrate, the generation of reducing equivalents, and organic acid skeletons were induced within 30 min, before primary metabolites changed significantly. By 3 h, most genes assigned to amino acid and nucleotide biosynthesis and scavenging were induced, while most genes assigned to amino acid and nucleotide breakdown were repressed. There was coordinate induction of many genes assigned to RNA synthesis and processing and most of the genes assigned to amino acid activation and protein synthesis. Although amino acids involved in central metabolism increased, minor amino acids decreased, providing independent evidence for the activation of protein synthesis. Specific genes encoding expansin and tonoplast intrinsic proteins were induced, indicating activation of cell expansion and growth in response to nitrate nutrition. There were rapid responses in the expression of many genes potentially involved in regulation, including genes for trehalose metabolism and hormone metabolism, protein kinases and phosphatases, receptor kinases, and transcription factors.

A gene expression atlas of the model legume Medicago truncatula

Legumes played central roles in the development of agriculture and civilization, and today account for approximately one-third of the worlds primary crop production. Unfortunately, most cultivated legumes are poor model systems for genomic research. Therefore, Medicago truncatula, which has a relatively small diploid genome, has been adopted as a model species for legume genomics. To enhance its value as a model, we have generated a gene expression atlas that provides a global view of gene expression in all major organ systems of this species, with special emphasis on nodule and seed development. The atlas reveals massive differences in gene expression between organs that are accompanied by changes in the expression of key regulatory genes, such as transcription factor genes, which presumably orchestrate genetic reprogramming during development and differentiation. Interestingly, many legume-specific genes are preferentially expressed in nitrogen-fixing nodules, indicating that evolution endowed them with special roles in this unique and important organ. Comparative transcriptome analysis of Medicago versus Arabidopsis revealed significant divergence in developmental expression profiles of orthologous genes, which indicates that phylogenetic analysis alone is insufficient to predict the function of orthologs in different species. The data presented here represent an unparalleled resource for legume functional genomics, which will accelerate discoveries in legume biology.

Repressor- and Activator-Type Ethylene Response Factors Functioning in Jasmonate Signaling and Disease Resistance Identified via a Genome-Wide Screen of Arabidopsis Transcription Factor Gene Expression

To identify transcription factors (TFs) involved in jasmonate (JA) signaling and plant defense, we screened 1,534 Arabidopsis (Arabidopsis thaliana) TFs by real-time quantitative reverse transcription-PCR for their altered transcript at 6 h following either methyl JA treatment or inoculation with the incompatible pathogen Alternaria brassicicola. We identified 134 TFs that showed a significant change in expression, including many APETALA2/ethylene response factor (AP2/ERF), MYB, WRKY, and NAC TF genes with unknown functions. Twenty TF genes were induced by both the pathogen and methyl JA and these included 10 members of the AP2/ERF TF family, primarily from the B1a and B3 subclusters. Functional analysis of the B1a TF AtERF4 revealed that AtERF4 acts as a novel negative regulator of JA-responsive defense gene expression and resistance to the necrotrophic fungal pathogen Fusarium oxysporum and antagonizes JA inhibition of root elongation. In contrast, functional analysis of the B3 TF AtERF2 showed that AtERF2 is a positive regulator of JA-responsive defense genes and resistance to F. oxysporum and enhances JA inhibition of root elongation. Our results suggest that plants coordinately express multiple repressor- and activator-type AP2/ERFs during pathogen challenge to modulate defense gene expression and disease resistance.

Eleven golden rules of quantitative RT-PCR.

Reverse transcription followed by quantitative polymerase chain reaction analysis, or qRT-PCR, is an extremely sensitive, cost-effective method for quantifying gene transcripts from plant cells. The availability of nonspecific double-stranded DNA (dsDNA) binding fluorophors, such as SYBR Green, and

Transport and Metabolism in Legume-Rhizobia Symbioses

Symbiotic nitrogen fixation by rhizobia in legume root nodules injects approximately 40 million tonnes of nitrogen into agricultural systems each year. In exchange for reduced nitrogen from the bacteria, the plant provides rhizobia with reduced carbon and all the essential nutrients required for bacterial metabolism. Symbiotic nitrogen fixation requires exquisite integration of plant and bacterial metabolism. Central to this integration are transporters of both the plant and the rhizobia, which transfer elements and compounds across various plant membranes and the two bacterial membranes. Here we review current knowledge of legume and rhizobial transport and metabolism as they relate to symbiotic nitrogen fixation. Although all legume-rhizobia symbioses have many metabolic features in common, there are also interesting differences between them, which show that evolution has solved metabolic problems in different ways to achieve effective symbiosis in different systems.

Medicago truncatula and Glomus intraradices gene expression in cortical cells harboring arbuscules in the arbuscular mycorrhizal symbiosis

BackgroundMost vascular flowering plants have the capacity to form symbiotic associations with arbuscular mycorrhizal (AM) fungi. The symbiosis develops in the roots where AM fungi colonize the root cortex and form arbuscules within the cortical cells. Arbuscules are enveloped in a novel plant membrane and their establishment requires the coordinated cellular activities of both symbiotic partners. The arbuscule-cortical cell interface is the primary functional interface of the symbiosis and is of central importance in nutrient exchange. To determine the molecular events the underlie arbuscule development and function, it is first necessary to identify genes that may play a role in this process. Toward this goal we used the Affymetrix GeneChip® Medicago Genome Array to document the M. truncatula transcript profiles associated with AM symbiosis, and then developed laser microdissection (LM) of M. truncatula root cortical cells to enable analyses of gene expression in individual cell types by RT-PCR.ResultsThis approach led to the identification of novel M. truncatula and G. intraradices genes expressed in colonized cortical cells and in arbuscules. Within the arbuscule, expression of genes associated with the urea cycle, amino acid biosynthesis and cellular autophagy was detected. Analysis of gene expression in the colonized cortical cell revealed up-regulation of a lysine motif (LysM)-receptor like kinase, members of the GRAS transcription factor family and a symbiosis-specific ammonium transporter that is a likely candidate for mediating ammonium transport in the AM symbiosis.ConclusionTranscript profiling using the Affymetrix GeneChip® Medicago Genome Array provided new insights into gene expression in M. truncatula roots during AM symbiosis and revealed the existence of several G. intraradices genes on the M. truncatula GeneChip®. A laser microdissection protocol that incorporates low-melting temperature Steedmans wax, was developed to enable laser microdissection of M. truncatula root cortical cells. LM coupled with RT-PCR provided spatial gene expression information for both symbionts and expanded current information available for gene expression in cortical cells containing arbuscules.

Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development

Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.

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 reprogramming of regulatory networks, transport, cell wall and membrane biogenesis during arbuscular mycorrhizal symbiosis in Lotus japonicus.

* Arbuscular mycorrhizas (AMs) contribute significantly to soil nutrient uptake in plants. As a consequence of the fungal colonization and of the deep reorganization shown by arbusculated cells, important impacts on root transcriptome are expected. * An Affymetrix GeneChip with 50,000 probe-sets and real-time RT-PCR allowed us to detect transcriptional changes triggered in Lotus japonicus by the AM fungus Gigaspora margarita, when arbuscules are at their maximum (28 d postinoculation (dpi)). An early time (4 dpi) was selected to differentiate genes potentially involved in signaling and/or in colonization of outer tissues. * A large number (75 out of 558) of mycorrhiza-induced genes code for proteins involved in protein turnover, membrane dynamics and cell wall synthesis, while many others are involved in transport (47) or transcription (24). Induction of a subset (24 genes) of these was tested and confirmed by qRT-PCR, and transcript location in arbusculated cells was demonstrated for seven genes using laser-dissected cells. * When compared with previously published papers, the transcript profiles indicate the presence of a core set of responsive genes (25) that seem to be conserved irrespective of the symbiotic partner identity.