SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase.

Arabidopsis Snf1-related protein kinases (SnRKs) are implicated in pleiotropic regulation of metabolic, hormonal and stress responses through their interaction with the kinase inhibitor PRL1 WD-protein. Here we show that SKP1/ASK1, a conserved SCF (Skp1-cullin-F-box) ubiquitin ligase subunit, which suppresses the skp1-4 mitotic defect in yeast, interacts with the PRL1-binding C-terminal domains of SnRKs. The same SnRK domains recruit an SKP1/ASK1-binding proteasomal protein, alpha4/PAD1, which enhances the formation of a trimeric SnRK complex with SKP1/ASK1 in vitro. By contrast, PRL1 reduces the interaction of SKP1/ASK1 with SnRKs. SKP1/ASK1 is co-immunoprecipitated with a cullin SCF subunit (AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts. SKP1/ASK1, cullin and proteasomal alpha-subunits show nuclear co-localization in differentiated Arabidopsis cells, and are observed in association with mitotic spindles and phragmoplasts during cell division. Detection of SnRK in purified 26S proteasomes and co-purification of epitope- tagged SKP1/ASK1 with SnRK, cullin and proteasomal alpha-subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and alpha4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis.

Regulatory interaction of PRL1 WD protein with Arabidopsis SNF1-like protein kinases.

Mutation of the PRL1 gene, encoding a regulatory WD protein, results in glucose hypersensitivity and derepression of glucose-regulated genes in Arabidopsis. The yeast SNF1 protein kinase, a key regulator of glucose signaling, and Arabidopsis SNF1 homologs AKIN10 and AKIN11, which can complement the Deltasnf1 mutation, were found to interact with an N-terminal domain of the PRL1 protein in the two-hybrid system and in vitro. AKIN10 and AKIN11 suppress the yeast Deltasnf4 mutation and interact with the SNF4p-activating subunit of SNF1. PRL1 and SNF4 bind independently to adjacent C-terminal domains of AKIN10 and AKIN11, and these protein interactions are negatively regulated by glucose in yeast. AKIN10 and AKIN11, purified in fusion with glutathione S-transferase, undergo autophosphorylation and phosphorylate a peptide of sucrose phosphate synthase in vitro. The sucrose phosphate synthase-peptide kinase activity of AKIN complexes detected by immunoprecipitation is stimulated by sucrose in light-grown Arabidopsis plants. In comparison with wild type, the activation level of AKIN immunocomplexes is higher in the prl1 mutant, suggesting that PRL1 is a negative regulator of Arabidopsis SNF1 homologs. This conclusion is supported by the observation that PRL1 is an inhibitor of AKIN10 and AKIN11 in vitro.

Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD protein, in Arabidopsis.

The prl1 mutation localized by T-DNA tagging on Arabidopsis chromosome 4-44 confers hypersensitivity to glucose and sucrose. The prl1 mutation results in transcriptional derepression of glucose responsive genes defining a novel suppressor function in glucose signaling. The prl1 mutation also augments the sensitivity of plants to growth hormones including cytokinin, ethylene, abscisic acid, and auxin; stimulates the accumulation of sugars and starch in leaves; and inhibits root elongation. PRL1 encodes a regulatory WD protein that interacts with ATHKAP2, an alpha-importin nuclear import receptor, and is imported into the nucleus in Arabidopsis. Potential functional conservation of PRL1 homologs found in other eukaryotes is indicated by nuclear localization of PRL1 in monkey COS-1 cells and selective interaction of PRL1 with a nuclear protein kinase C-betaII isoenzyme involved in human insulin signaling.

Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis.

Proline is a common compatible osmolyte in higher plants. Proline accumulation in response to water stress and salinity is preceded by a rapid increase of the mRNA level of delta 1-pyrroline-5-carboxylate synthase (P5CS) controlling the rate-limiting step of glutamate-derived proline biosynthesis. P5CS is encoded by two differentially regulated genes in Arabidopsis. Gene AtP5CS1 mapped to chromosome 2-78.5 is expressed in most plant organs, but silent in dividing cells. Gene AtP5CS2 located close to marker m457 on chromosome 3-101.3 contributes 20-40% of total P5CS mRNA in plant tissues, but is solely responsible for the synthesis of abundant P5CS mRNA in rapidly dividing cell cultures. Accumulation of AtP5CS transcripts is regulated in a tissue specific manner and inducible by drought, salinity, ABA, and to a lesser extent by auxin. Induction of AtP5CS1 mRNA accumulation in salt-treated seedlings involves an immediate early transcriptional response regulated by ABA signalling that is not inhibited by cycloheximide, but abolished by the deficiency of ABA biosynthesis in the aba1 Arabidopsis mutant. However, inhibition of protein synthesis by cycloheximide prevents the induction of AtP5CS2 mRNA accumulation, and blocks further increase of AtP5CS1 mRNA levels during the second, slow phase of salt-induction. Mutations abi1 and axr2, affecting ABA-perception in Arabidopsis, reduce the accumulation of both AtP5CS mRNAs during salt-stress, whereas ABA-signalling functions defined by the abi2 and abi3 mutations have no effect on salt-induction of the AtP5CS genes.

Construction of a basic genetic map for alfalfa using RFLP, RAPD, isozyme and morphological markers.

The genetic map for alfalfa presented here has eight linkage groups representing the haploid chromosome set of the Medicago species. The genetic map was constructed by ordering the linkage values of 89 RFLP, RAPD, isozyme and morphological markers collected from a segregating population of 138 individuals. The segregating population is self-mated progeny of an F1 hybrid plant deriving from a cross between the diploid (2n = 2x = 16) yellow-flowered. Medicago sativa ssp. quasifalcata and the diploid (2n = 2x = 16) blue-flowered M. sativa ssp. coerulea. The inheritance of many traits displayed distorted segregation, indicating the presence of lethal loci in the heterozygotic parent plants. In spite of the lack of uniform segregation, linkage groups could be assigned and the order of the markers spanning > 659 centimorgans could be unambiguously determined. This value and the calculated haploid genome size for Medicago (1n = 1x = 1.0 x 10(9) bp) gives a ratio of < 1500 kb per centimorgan.