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.

Sequencing of Medicago truncatula, a model organism of legume biology, shows that genome duplications had a role in the evolution of endosymbiotic nitrogen fixation. Legumes are unusual among plants in that they can carry out endosymbiotic nitrogen fixation with rhizobial bacteria. The genome of Medicago truncatula (also known as barrel medic or barrel clover), a well-established model for the study of legume biology, has now been sequenced. Genome analysis shows that M. truncatula has undergone several rounds of whole-genome duplication, and that the duplication that took place approximately 58 million years ago played an important part in the evolution of endosymbiotic nitrogen fixation. Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation1. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species2. Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing ∼94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa’s genomic toolbox.

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.

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.

Since 1999, various forward- and reverse-genetic approaches have uncovered nearly 200 genes required for symbiotic nitrogen fixation (SNF) in legumes. These discoveries advanced our understanding of the evolution of SNF in plants and its relationship to other beneficial endosymbioses, signaling between plants and microbes, the control of microbial infection of plant cells, the control of plant cell division leading to nodule development, autoregulation of nodulation, intracellular accommodation of bacteria, nodule oxygen homeostasis, the control of bacteroid differentiation, metabolism and transport supporting symbiosis, and the control of nodule senescence. This review catalogs and contextualizes all of the plant genes currently known to be required for SNF in two model legume species, Medicago truncatula and Lotus japonicus, and two crop species, Glycine max (soybean) and Phaseolus vulgaris (common bean). We also briefly consider the future of SNF genetics in the era of pan-genomics and genome editing.

Summary: Legumes played central roles in the development of agriculture and civilization, and today account for approximately one‐third of the world’s 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.