Klaus Salchert

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, α4/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 α‐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 α‐subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and α4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis.

A Conserved Domain of the Arabidopsis GNOM Protein Mediates Subunit Interaction and Cyclophilin 5 Binding

The Arabidopsis GNOM protein, a guanine nucleotide exchange factor (GEF) that acts on ADP ribosylation factor (ARF)–type G proteins, is required for coordination of cell polarity along the apical–basal embryo axis. Interallelic complementation of gnom mutants suggested that dimerization is involved in GNOM function. Here, direct interaction between GNOM molecules is demonstrated in vitro and by using a yeast two-hybrid system. Interaction was confined to an N-terminal domain conserved within a subgroup of large ARF GEFs. The same domain mediated in vitro binding to cyclophilin 5 (Cyp5), which was identified as a GNOM interactor in two-hybrid screening. Cyp5 displayed peptidylprolyl cis/trans–isomerase and protein refolding activities that were sensitive to cyclosporin A. Cyp5 protein accumulated in several plant organs and, like GNOM, was partitioned between cytosolic and membrane fractions. Cyp5 protein was also expressed in the developing embryo. Our results suggest that Cyp5 may regulate the ARF GEF function of the GNOM protein during embryogenesis.

Floral inhibition in red fescue (Festuca rubra L.) through expression of a heterologous flowering repressor from Lolium

Extension of the vegetative growth phase through delay of flowering is an important goal in todays breeding programs of both forage and turf grasses. In forage grasses, the stem and inflorescence production comprise a significant reduction in the digestibility, nutritional value and productivity of the crop, and in turf grasses the stems that start to emerge during the growth season suppress the formation of new shoots and affect the quality, density and persistence of the sward. We have tested the potential of the strong floral repressor LpTFL1 from perennial ryegrass (Lolium perenne L.) to manipulate the transition to flowering in red fescue (Festuca rubra L.), a cool-season turf grass. Expression of LpTFL1 from the constitutive maize ubiquitin promoter represses flowering in red fescue, and the flowering repression phenotype correlates well with the level of LpTFL expression. Transgenic lines showing low to intermediate expression of LpTFL1 flowered approximately two weeks later than the controls, and transgenic lines showing very high LpTFL1 expression levels still remained non-flowering after exposure to natural vernalization conditions (Danish winter) in two successive years. There were no other phenotypic effects associated with the LpTFL transgene expression during vegetative growth. However, there was a tendency towards an LpTFL1-mediated reduction in stem length among the flowering lines. Expression of a truncated LpTFL, caused by transgene rearrangements during the transformation, lead to increased flowering and stem production and a decrease in panicle size. This is to our knowledge the first report on full inhibition of floral development in a commercially important grass species.

Inactivation of Plasma Membrane–Localized CDPK-RELATED KINASE5 Decelerates PIN2 Exocytosis and Root Gravitropic Response in Arabidopsis

This work shows that CRK5, a plasma membrane–associated member of the Arabidopsis Ca2+/calmodulin-dependent kinase-related protein family, phosphorylates the hydrophilic loop of PIN2 and is required for proper polar localization of PIN2 in the transition zones of roots. Inactivation of CRK5 inhibits primary root elongation and delays gravitropic bending of roots and shoots. CRK5 is a member of the Arabidopsis thaliana Ca2+/calmodulin-dependent kinase-related kinase family. Here, we show that inactivation of CRK5 inhibits primary root elongation and delays gravitropic bending of shoots and roots. Reduced activity of the auxin-induced DR5–green fluorescent protein reporter suggests that auxin is depleted from crk5 root tips. However, no tip collapse is observed and the transcription of genes for auxin biosynthesis, AUXIN TRANSPORTER/AUXIN TRANSPORTER-LIKE PROTEIN (AUX/LAX) auxin influx, and PIN-FORMED (PIN) efflux carriers is unaffected by the crk5 mutation. Whereas AUX1, PIN1, PIN3, PIN4, and PIN7 display normal localization, PIN2 is depleted from apical membranes of epidermal cells and shows basal to apical relocalization in the cortex of the crk5 root transition zone. This, together with an increase in the number of crk5 lateral root primordia, suggests facilitated auxin efflux through the cortex toward the elongation zone. CRK5 is a plasma membrane–associated kinase that forms U-shaped patterns facing outer lateral walls of epidermis and cortex cells. Brefeldin inhibition of exocytosis stimulates CRK5 internalization into brefeldin bodies. CRK5 phosphorylates the hydrophilic loop of PIN2 in vitro, and PIN2 shows accelerated accumulation in brefeldin bodies in the crk5 mutant. Delayed gravitropic response of the crk5 mutant thus likely reflects defective phosphorylation of PIN2 and deceleration of its brefeldin-sensitive membrane recycling.

Expression of intron modified NPT II genes in monocotyledonous and dicotyledonous plant cells

Intron sequences from monocotyledonous and dicotyledonous origin were used to abolish marker gene expression in prokaryotes (Escherichia coli and Agrobacterium tumefaciens) but permit expression in selected eukaryotic systems using the eukaryotic specific splicing mechanism. A 1014 bp maize Shrunken-1 (Sh 1) intron 1 flanked by exon1 and exon2 sequences was cloned into the N-terminal of the NPT II-coding region. Transient gene expression analysis revealed that the modified neomycin phosphotransferase II (NPT II) gene, driven by the cauliflower mosaic virus (CaMV) 35S promoter, is expressed in barley protoplasts, but poorly expressed in tobacco protoplasts. In dicotyledonous cells AU-rich sequences are known to be important for efficient splicing and therefore an attempt was made to improve expression of the NPT II gene, containing the Sh 1 intron 1, in tobacco by increasing the AU content from 57% to 69%. Reverse transcriptase PCR analysis of RNA from transiently expressed NPT II transcripts from tobacco protoplasts revealed that despite the increase in AU-content, NPT II was still poorly expressed. Cryptic splice sites were identified as one possible cause for missplicing of the Sh1 intron 1 in dicots and poor levels of expression. Alternatively, cloning of the 198 bp intron 2 of the potato STLS 1 gene (81% AU) into the N-terminal part of the NPT II-coding region resulted in proper expression of NPT II in tobacco as well as in barley protoplasts and abolished marker gene expression in prokaryotes. The successful insertion of an intron into a selectable marker gene which completely abolishes gene expression in prokaryotes, without affecting expression of chimeric genes in monocotyledonous and dicotyledonous plant cells provides a suitable system to reduce the number of false-positives in transgenic plant production.