The Compact Root Architecture 2 systemic pathway is required for the induction of cytokinins and of the miR399 in Medicago truncatula N-satisfied plants.

Plants use a combination of sophisticated local and systemic pathways to optimize growth depending on heterogeneous nutrient availability in the soil. Legume plants can acquire mineral nitrogen (N) either through their roots or via a symbiotic interaction with N-fixing rhizobia bacteria housed in so-called root nodules. To identify shoot-to-root systemic signals acting in Medicago truncatula plants at N-deficit or N-satiety, plants were grown in a split-root experimental design, in which either high or low N was provided to a half of the root system, allowing the analysis of systemic pathways independently of any local N response. Among the plant hormone families analyzed, the cytokinin trans-Zeatin accumulated in plants at N-satiety. Cytokinin application by petiole feeding led to an inhibition of both root growth and nodulation. In addition, an exhaustive analysis of miRNAs revealed that miR2111 accumulates systemically under N-deficit in both shoots and non-treated distant roots, whereas a miRNA related to inorganic Phosphate (Pi)-acquisition, the miR399, does so in plants grown at N-satiety. These two accumulation patterns are dependent on CRA2 (Compact Root Architecture 2), a receptor required for CEP (C-terminally Encoded Peptide) signaling. Constitutive ectopic expression of the miR399 reduced nodule numbers and root biomass depending on Pi availability, suggesting that the miR399-dependent Pi-acquisition regulatory module controlled by N-availability affects the development of the whole legume plant root system.

An allelic series of starch-branching enzyme mutants in pea (Pisum sativum L.) reveals complex relationships with seed starch phenotypes.

A set of mutant pea lines carrying induced mutations within the major seed-expressed starch-branching enzyme gene has been characterised at the molecular, chemical and agronomic levels. Eight of the induced mutations, three of which predicted a premature stop codon, were compared with the naturally occurring starch-branching enzyme mutation within the same genetic background. Starch, amylose and sugar measurements, coupled with analysis by ultra-high performance liquid chromatography-size exclusion chromatography of starches, identified a range of phenotypes which were grouped according to the nature of the mutation. Homology modelling of proteins supported the differences in phenotypes observed. Differences in field performance were evident for selected mutants, particularly in seed yield and mean seed weight traits for early compared with late spring sowings. The data show the potential of an allelic series of mutants at this locus for nutritional studies. CHEMICAL COMPOUNDS: starch, amylose, amylopectin, raffinose, stachyose, verbascose.

An Integrated Linkage Map of Three Recombinant Inbred Populations of Pea (Pisum sativum L.).

Biparental recombinant inbred line (RIL) populations are sets of genetically stable lines and have a simple population structure that facilitates the dissection of the genetics of interesting traits. On the other hand, populations derived from multiparent intercrosses combine both greater diversity and higher numbers of recombination events than RILs. Here, we describe a simple population structure: a three-way recombinant inbred population combination. This structure was easy to produce and was a compromise between biparental and multiparent populations. We show that this structure had advantages when analyzing cultivar crosses, and could achieve a mapping resolution of a few genes.

Identification of Stipules reduced, a leaf morphology gene in pea (Pisum sativum).

Pea (Pisum sativum) is one of relatively few genetically amenable plant species with compound leaves. Pea leaves have a variety of specialized organs: leaflets, tendrils, pulvini and stipules, which enable the identification of mutations that transform or affect distinct parts of the leaf. Characterization of these mutations offers insights into the development and evolution of novel leaf traits. The previously characterized morphological gene Cochleata, conferring stipule identity, was known to interact with Stipules reduced (St), which conditions stipule size in pea, but the St gene remained unknown. Here we analysed Fast Neutron irradiated pea mutants by restriction site associated DNA sequencing. We identified St as a gene encoding a C2H2 zinc finger transcription factor that is regulated by Cochleata. St regulates both cell division and cell expansion in the stipule. Our approach shows how systematic genome-wide screens can be used successfully for the analysis of traits in species for which whole genome sequences are not available.

Genetic Variation Controlling Wrinkled Seed Phenotypes in Pisum: How Lucky Was Mendel?

One of the traits studied by Mendel in pea (Pisum sativum L.) was the wrinkled-seeded phenotype, and the molecular basis for a mutation underlying this phenotype was discovered in the 1990s. Although the starch-branching enzyme gene mutation identified at the genetic locus r is most likely to be that in seeds available to Mendel in the mid-1800s, it has remained an open question as to whether or not additional natural mutations in this gene exist within Pisum germplasm collections. Here, we explore this question and show that all but two wrinkled-seeded variants in one such collection correspond to either the mutant allele described previously for the r locus or a mutation at a second genetic locus, rb, affecting the gene encoding the large subunit of Adenosine diphosphoglucose (ADP-glucose) pyrophosphorylase; the molecular basis for the rb mutation is described here. The genetic basis for the phenotype of one (JI 2110) of the two lines which are neither r nor rb has been studied in crosses with a round-seeded variant (JI 281); for which extensive genetic marker data were expected. In marked contrast to the trait studied by Mendel and the rb phenotype; the data suggest that the wrinkled-seeded phenotype in JI 2110 is maternally determined, controlled by two genetic loci, and the extent to which it is manifested is very sensitive to the environment. Metabolite analysis of the cotyledons of JI 2110 revealed a profile for sucrose and sucrose-derived compounds that was more similar to that of wild-type round-seeded, than that of wrinkled-seeded r, pea lines. However, the metabolite profile of the seed coat (testa) of JI 2110 was distinct from that of other round-seeded genotypes tested which, together with analysis of recombinant inbred progeny lines, suggests an explanation for the seed phenotype.

SGRL can regulate chlorophyll metabolism and contributes to normal plant growth and development in Pisum sativum L.

Among a set of genes in pea (Pisum sativum L.) that were induced under drought-stress growth conditions, one encoded a protein with significant similarity to a regulator of chlorophyll catabolism, SGR. This gene, SGRL, is distinct from SGR in genomic location, encoded carboxy-terminal motif, and expression through plant and seed development. Divergence of the two encoded proteins is associated with a loss of similarity in intron/exon gene structure. Transient expression of SGRL in leaves of Nicotiana benthamiana promoted the degradation of chlorophyll, in a manner that was distinct from that shown by SGR. Removal of a predicted transmembrane domain from SGRL reduced its activity in transient expression assays, although variants with and without this domain reduced SGR-induced chlorophyll degradation, indicating that the effects of the two proteins are not additive. The combined data suggest that the function of SGRL during growth and development is in chlorophyll re-cycling, and its mode of action is distinct from that of SGR. Studies of pea sgrL mutants revealed that plants had significantly lower stature and yield, a likely consequence of reduced photosynthetic efficiencies in mutant compared with control plants under conditions of high light intensity.

Exploiting a fast neutron mutant genetic resource in Pisum sativum (pea) for functional genomics.

A fast neutron (FN)-mutagenised population was generated in Pisum sativum L. (pea) to enable the identification and isolation of genes underlying traits and processes. Studies of several phenotypic traits have clearly demonstrated the utility of the resource by associating gene deletions with phenotype followed by functional tests exploiting additional mutant sources, from both induced and natural variant germplasm. For forward genetic screens, next generation sequencing methodologies provide an opportunity for identifying genes associated with deletions rapidly and systematically. The application of rapid reverse genetic screens of the fast neutron mutant pea population supports conclusions on the frequency of deletions based on phenotype alone. These studies also suggest that large deletions affecting one or more loci can be non-deleterious to the pea genome, yielding mutants that could not be obtained by other means. Deletion mutants affecting genes associated with seed metabolism and storage are providing unique opportunities to identify the products of complex and related gene families, and to study the downstream consequences of such deletions.

Conserved genetic determinant of motor organ identity in Medicago truncatula and related legumes.

Plants exhibit various kinds of movements that have fascinated scientists and the public for centuries. Physiological studies in plants with the so-called motor organ or pulvinus suggest that cells at opposite sides of the pulvinus mediate leaf or leaflet movements by swelling and shrinking. How motor organ identity is determined is unknown. Using a genetic approach, we isolated a mutant designated elongated petiolule1 (elp1) from Medicago truncatula that fails to fold its leaflets in the dark due to loss of motor organs. Map-based cloning indicated that ELP1 encodes a putative plant-specific LOB domain transcription factor. RNA in situ analysis revealed that ELP1 is expressed in primordial cells that give rise to the motor organ. Ectopic expression of ELP1 resulted in dwarf plants with petioles and rachises reduced in length, and the epidermal cells gained characteristics of motor organ epidermal cells. By identifying ELP1 orthologs from other legume species, namely pea (Pisum sativum) and Lotus japonicus, we show that this motor organ identity is regulated by a conserved molecular mechanism.

The B gene of pea encodes a defective flavonoid 3',5'-hydroxylase, and confers pink flower color.

The inheritance of flower color in pea (Pisum sativum) has been studied for more than a century, but many of the genes corresponding to these classical loci remain unidentified. Anthocyanins are the main flower pigments in pea. These are generated via the flavonoid biosynthetic pathway, which has been studied in detail and is well conserved among higher plants. A previous proposal that the Clariroseus (B) gene of pea controls hydroxylation at the 5' position of the B ring of flavonoid precursors of the anthocyanins suggested to us that the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the enzyme that hydroxylates the 5' position of the B ring, was a good candidate for B. In order to test this hypothesis, we examined mutants generated by fast neutron bombardment. We found allelic pink-flowered b mutant lines that carried a variety of lesions in an F3'5'H gene, including complete gene deletions. The b mutants lacked glycosylated delphinidin and petunidin, the major pigments present in the progenitor purple-flowered wild-type pea. These results, combined with the finding that the F3'5'H gene cosegregates with b in a genetic mapping population, strongly support our hypothesis that the B gene of pea corresponds to a F3'5'H gene. The molecular characterization of genes involved in pigmentation in pea provides valuable anchor markers for comparative legume genomics and will help to identify differences in anthocyanin biosynthesis that lead to variation in pigmentation among legume species.

Identification of Mendel's white flower character.

BACKGROUND: The genetic regulation of flower color has been widely studied, notably as a character used by Mendel and his predecessors in the study of inheritance in pea. METHODOLOGY/PRINCIPAL FINDINGS: We used the genome sequence of model legumes, together with their known synteny to the pea genome to identify candidate genes for the A and A2 loci in pea. We then used a combination of genetic mapping, fast neutron mutant analysis, allelic diversity, transcript quantification and transient expression complementation studies to confirm the identity of the candidates. CONCLUSIONS/SIGNIFICANCE: We have identified the pea genes A and A2. A is the factor determining anthocyanin pigmentation in pea that was used by Gregor Mendel 150 years ago in his study of inheritance. The A gene encodes a bHLH transcription factor. The white flowered mutant allele most likely used by Mendel is a simple G to A transition in a splice donor site that leads to a mis-spliced mRNA with a premature stop codon, and we have identified a second rare mutant allele. The A2 gene encodes a WD40 protein that is part of an evolutionarily conserved regulatory complex.

High-throughput retrotransposon-based fluorescent markers: improved information content and allele discrimination.

BACKGROUND: Dense genetic maps, together with the efficiency and accuracy of their construction, are integral to genetic studies and marker assisted selection for plant breeding. High-throughput multiplex markers that are robust and reproducible can contribute to both efficiency and accuracy. Multiplex markers are often dominant and so have low information content, this coupled with the pressure to find alternatives to radio-labelling, has led us to adapt the SSAP (sequence specific amplified polymorphism) marker method from a 33P labelling procedure to fluorescently tagged markers analysed from an automated ABI 3730 xl platform. This method is illustrated for multiplexed SSAP markers based on retrotransposon insertions of pea and is applicable for the rapid and efficient generation of markers from genomes where repetitive element sequence information is available for primer design. We cross-reference SSAP markers previously generated using the 33P manual PAGE system to fluorescent peaks, and use these high-throughput fluorescent SSAP markers for further genetic studies in Pisum. RESULTS: The optimal conditions for the fluorescent-labelling method used a triplex set of primers in the PCR. These included a fluorescently labelled specific primer together with its unlabelled counterpart, plus an adapter-based primer with two bases of selection on the 3' end. The introduction of the unlabelled specific primer helped to optimise the fluorescent signal across the range of fragment sizes expected, and eliminated the need for extensive dilutions of PCR amplicons. The software (GeneMarker Version 1.6) used for the high-throughput data analysis provided an assessment of amplicon size in nucleotides, peak areas and fluorescence intensity in a table format, so providing additional information content for each marker. The method has been tested in a small-scale study with 12 pea accessions resulting in 467 polymorphic fluorescent SSAP markers of which 260 were identified as having been mapped previously using the radio-labelling technique. Heterozygous individuals from pea cultivar crosses were identifiable after peak area data analysis using the fluorescent SSAP method. CONCLUSION: As well as developing a rapid, and high-throughput marker method for genetic studies, the fluorescent SSAP system improved the accuracy of amplicon scoring, increased the available marker number, improved allele discrimination, and was sensitive enough to identify heterozygous loci in F1 and F2 progeny, indicating the potential to develop high-throughput codominant SSAPs.

Tendril-less regulates tendril formation in pea leaves.

Tendrils are contact-sensitive, filamentous organs that permit climbing plants to tether to their taller neighbors. Tendrilled legume species are grown as field crops, where the tendrils contribute to the physical support of the crop prior to harvest. The homeotic tendril-less (tl) mutation in garden pea (Pisum sativum), identified almost a century ago, transforms tendrils into leaflets. In this study, we used a systematic marker screen of fast neutron-generated tl deletion mutants to identify Tl as a Class I homeodomain leucine zipper (HDZIP) transcription factor. We confirmed the tendril-less phenotype as loss of function by targeting induced local lesions in genomes (TILLING) in garden pea and by analysis of the tendril-less phenotype of the t mutant in sweet pea (Lathyrus odoratus). The conversion of tendrils into leaflets in both mutants demonstrates that the pea tendril is a modified leaflet, inhibited from completing laminar development by Tl. We provide evidence to show that lamina inhibition requires Unifoliata/LEAFY-mediated Tl expression in organs emerging in the distal region of the leaf primordium. Phylogenetic analyses show that Tl is an unusual Class I HDZIP protein and that tendrils evolved either once or twice in Papilionoid legumes. We suggest that tendrils arose in the Fabeae clade of Papilionoid legumes through acquisition of the Tl gene.