Spectinomycin resistance mutations in the rrn16 gene are new plastid markers in Medicago sativa.

We report here the isolation of spectinomycin-resistant mutants in cultured cells of Medicago sativa line RegenSY-T2. Spectinomycin induces bleaching of cultured alfalfa cells due to inhibition of protein synthesis on the prokaryotic type 70S plastid ribosomes. Spontaneous mutants resistant to spectinomycin bleaching were identified by their ability to form green shoots on plant regeneration medium containing selective spectinomycin concentrations in the range of 25-50 mg/l. Sequencing of the plastid rrn16 gene revealed that spectinomycin resistance is due to mutations in a conserved stem structure of the 16S rRNA. Resistant plants transferred to the greenhouse developed normally and produced spectinomycin-resistant seed progeny. In light of their absence in soybean, a related leguminous plant, the isolation of spectinomycin-resistant mutants in M. sativa was unexpected. The new mutations are useful for the study of plastid inheritance, as demonstrated by detection of predominantly paternal plastid inheritance in the RegenSY-T2 × Szapko57 cross, and can be used as selective markers in plastid transformation vectors to obtain cisgenic plants.

Hose in Hose, an S locus-linked mutant of Primula vulgaris, is caused by an unstable mutation at the Globosa locus.

Hose in Hose mutants of primrose and cowslip have been cultivated since the early 17th century and show dominant homeotic conversion of sepals to petals. The phenotype shows variable penetrance and expressivity and is linked to the S locus, which controls floral heteromorphy in Primula species. Here we demonstrate that the homeotic conversion of sepals to petals in Hose in Hose is associated with up-regulation of both Primula B-function MADS box genes PvDef and PvGlo in the first floral whorl. We have defined a restriction fragment length polymorphism associated with PvGlo that cosegregates with the Hose in Hose phenotype and have also identified and characterized a retrotransposon insertion in the PvGlo promoter which is associated with the up-regulated expression of PvGlo. Excision of this retrotransposon, associated with epigenetic changes at the locus, causes reversion toward normal calyces and restores wild-type flower development. These data define the molecular basis of the Hose in Hose mutation and provide an explanation for its long-documented phenotypic instability.

The S locus-linked Primula homeotic mutant sepaloid shows characteristics of a B-function mutant but does not result from mutation in a B-function gene.

Floral homeotic and flower development mutants of Primula, including double, Hose in Hose, Jack in the Green and Split Perianth, have been cultivated since the late 1500s as ornamental plants but until recently have attracted limited scientific attention. Here we describe the characterization of a new mutant phenotype, sepaloid, that produces flowers comprising only sepals and carpels. The sepaloid mutation is recessive, and is linked to the S locus that controls floral heteromorphy. The phenotype shows developmental variability, with flowers containing three whorls of sepals surrounding fertile carpels, two whorls of sepals with a diminished third whorl of sepals surrounding a fourth whorl of carpels, or three whorls of sepals surrounding abnormal carpels. In some respects, these phenotypes resemble the Arabidopsis and Antirrhinum homeotic B-function mutants apetala3/deficiens (ap3/def) and pistillata/globosa (pi/glo). We have isolated the Primula vulgaris B-function genes PvDEFICIENS (PvDEF) and PvGLOBOSA (PvGLO), expression of both of which is affected in the sepaloid mutant. PvGLO, like sepaloid, is linked to the S locus, whereas PvDEF is not. However, our analyses reveal that sepaloid and PvGLO represent different genes. We conclude that SEPALOID is an S-linked independent regulator of floral organ identity genes including PvDEF and PvGLO.

An ERF transcription factor in Medicago truncatula that is essential for Nod factor signal transduction.

Rhizobial bacteria activate the formation of nodules on the appropriate host legume plant, and this requires the bacterial signaling molecule Nod factor. Perception of Nod factor in the plant leads to the activation of a number of rhizobial-induced genes. Putative transcriptional regulators in the GRAS family are known to function in Nod factor signaling, but these proteins have not been shown to be capable of direct DNA binding. Here, we identify an ERF transcription factor, ERF Required for Nodulation (ERN), which contains a highly conserved AP2 DNA binding domain, that is necessary for nodulation. Mutations in this gene block the initiation and development of rhizobial invasion structures, termed infection threads, and thus block nodule invasion by the bacteria. We show that ERN is necessary for Nod factor-induced gene expression and for spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase, DMI3, which is a component of the Nod factor signaling pathway. We propose that ERN is a component of the Nod factor signal transduction pathway and functions downstream of DMI3 to activate nodulation gene expression.