N-myristoylation and S-acylation promote protein membrane association, allowing regulation of membrane proteins. However, how widespread this targeting mechanism is in plant signaling processes remains unknown. Through bioinformatics analyses, we determined that among plant protein kinase families, the occurrence of motifs indicative for dual lipidation by N-myristoylation and S-acylation is restricted to only five kinase families, including the Ca -regulated CDPK-SnRK and CBL protein families. We demonstrated N-myristoylation of CDPK-SnRKs and CBLs by incorporation of radiolabeled myristic acid. We focused on CPK6 and CBL5 as model cases and examined the impact of dual lipidation on their function by fluorescence microscopy, electrophysiology and functional complementation of Arabidopsis mutants. We found that both lipid modifications were required for proper targeting of CBL5 and CPK6 to the plasma membrane. Moreover, we identified CBL5-CIPK11 complexes as phosphorylating and activating the guard cell anion channel SLAC1. SLAC1 activation by CPK6 or CBL5-CIPK11 was strictly dependent on dual lipid modification, and loss of CPK6 lipid modification prevented functional complementation of cpk3 cpk6 guard cell mutant phenotypes. Our findings establish the general importance of dual lipid modification for Ca signaling processes, and demonstrate their requirement for guard cell anion channel regulation.

HighlightsThe bacterial metabolic pathway of trans-3-hydroxy-l-proline (T3LHyp) has been identified.Azospirillum brasilense can grow on T3LHyp as a sole carbon source.The T3LHyp pathway comprises T3LHyp dehydratase and Δ1-pyrroline-2-carboxylate reductase.This pathway is similar to one found in mammals.

SummaryThe rgp7 gene, originally isolated from rice seedlings, encodes a small GTP-binding protein which is related to the product of the human proto-oncogene, ras-p21. To determine the physiological role of the rgpl protein, rgpl-p25, the coding region of rgpl was introduced into tobacco plants in both sense and antisense orientations. Transformants, which were found to contain the rgpi gene at up to three loci, showed distinct phenotypic changes. The most notable was a reduction in apical dominance with increased tillering, together with dwarfism or abnormal flower development or both. These effects were similarly observed in both sense and antisense transformants. Northern hybridization analysis showed that rgp7 was expressed only in phenotypically abnormal transformants and not in the apparently normal phenotypes. Furthermore, the R1 progenies from most transformants co-segregated into a 31 ratio for both kanamycin resistance and tillering. The expression of tgp7, a presumed tobacco homologue of rgpl, was markedly reduced in transformants expressing the antisense rgpi, whereas it was apparently unaffected in transformants with sense rgpl. These observations suggest that the phenotypic changes in antisense transformants may be mediated by an effect on native tgp7 mRNA, whereas in sense transformants the changes may be induced by over-production of rgplp25. The possibility that the increased tillering may be related to abnormal phytohormone metabolism or response pathways, and that rgpl -p25 may mediate the transmission of signals in these pathways is discussed.

Protein N‐myristoylation plays key roles in various cellular functions in eukaryotic organisms. To clarify the relationship between the efficiency of protein N‐myristoylation and the amino acid sequence of the substrate in plants, we have applied a wheat germ cell‐free translation system with high protein productivity to examine the N‐myristoylation of various wild‐type and mutant forms of Arabidopsis thaliana proteins. Evaluation of the relationship between removal of the initiating Met and subsequent N‐myristoylation revealed that constructs containing Pro at position 3 do not undergo N‐myristoylation, primarily because of an inhibitory effect of this amino acid on elimination of the initiating Met by methionyl aminopeptidase. Our analysis of the consensus sequence for N‐myristoylation in plants focused on the variability of amino acids at positions 3, 6 and 7 of the motif. We found that not only Ser at position 6 but also Lys at position 7 affects the selectivity for the amino acid at position 3. The results of our analyses allowed us to identify several A. thaliana proteins as substrates for N‐myristoylation that had previously been predicted not to be candidates for such modification with a prediction program. We have thus shown that a wheat germ cell‐free system is a useful tool for plant N‐myristoylome analysis. This in vitro approach will facilitate comprehensive determination of N‐myristoylated proteins in plants.

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