PHOSPHO-REGULATION OF ACA8, A PLASMA MEMBRANE CA2+-ATPASE OF ARABIDOPSIS THALIANA

ACA8 is a plasma membrane-localized isoform of calmodulin (CaM)-regulated Ca2+-ATPase of Arabidopsis thaliana. Phospho-proteomic studies identified several phosphopeptides corresponding to portions of its regulatory N-terminus. Each of the Ser found to be phosphorylated in those studies (S19, S22, S27, S29, S57, and S99) has been mutated to Asp, to mimic phosphorylation of the ACA8 N-terminus, and to Ala to prevent phosphorylation. Mutants have been expressed in Saccharomyces cerevisiae and characterized: as shown by the low activation by CaM, mutants S19D, S57D, S22D and S27D are deregulated. Moreover, the low response to CaM of ACA8 mutants S22A, S27A, and S29A points the relevance of these serine residues per se in determining the amplitude of the response of ACA8 to CaM. To analyse the effect of S to D mutation on the kinetic of CaM binding, His-tagged N-termini of wild-type and mutant ACA8 (6His-1M-I116) were expressed in Escherichia coli, affinity-purified and used in surface plasmon resonance experiments. All the analysed mutations affect the kinetics of interaction with CaM to some extent: in most cases, the altered kinetics result in marginal changes in affinity, with the exception of mutants S57D (KD 10-fold higher than wild-type ACA8) and S99D (KD about half that of wild-type ACA8). Since S19 is in a consensus motive for calcium-dependent protein kinases (CDPKs) the ACA8 N-terminus has been subjected to in vitro phosphorylation assays with two isoforms of A. thaliana CDPKs: CDPK1, that phosphorylates ACA2 (an endoplasmic reticulum localised isoform of A. thaliana ACA) and CDPK16, a plasma membrane localised isoform of CDPK. Results show that both kinases are able to phosphorylate ACA8 N-terminus, but CDPK16 with higher extent. Phosphorylation of mutant 6His-1M-I116 peptides mapped CDPK16 phosphorylation site at S19 and at S22. Furthermore, we identified by two-hybrid screening two isoforms of CBL-interacting protein kinases (CIPKs) as putative interactors of ACA8 N-terminus region: CIPK9 and CIPK14. BiFC analysis in Nicotiana benthamiana confirmed the two-hybrid results, showing that interaction between ACA8 full length and CIPK9 or CIPK14 occurs in planta at the plasma membrane. Moreover, phosphorylation assay demonstrate that both kinases phosphorylate ACA8 N-terminus in vitro. Implications of these results are discussed

Maize 16-kD γ-zein forms very unusual disulfide-bonded polymers in the endoplasmic reticulum : implications for prolamin evolution

In the lumen of the endoplasmic reticulum (ER), prolamin storage proteins of cereal seeds form very large, ordered heteropolymers termed protein bodies (PBs), which are insoluble unless treated with alcohol or reducing agents. In maize PBs, 16-kD \u3b3-zein locates at the interface between a core of alcohol-soluble \u3b1-zeins and the outermost layer mainly composed of the reduced-soluble 27-kD \u3b3-zein. 16-kD \u3b3-zein originates from 27-kD \u3b3-zein upon whole-genome duplication and is mainly characterized by deletions in the N-terminal domain that eliminate most Pro-rich repeats and part of the Cys residues involved in inter-chain bonds. 27-kD \u3b3-zein also forms insoluble PBs when expressed in transgenic vegetative tissues. We show that in Arabidopsis leaves, 16-kD \u3b3-zein assembles into disulfide-linked polymers that fail to efficiently become insoluble. Instead of forming PBs, these polymers accumulate as very unusual threads that markedly enlarge the ER lumen, resembling amyloid-like fibers. Domain-swapping between the two \u3b3-zeins indicates that the N-terminal region of 16-kD \u3b3-zein has a dominant effect in preventing full insolubilization. Therefore, a newly evolved prolamin has lost the ability to form homotypic PBs, and has acquired a new function in the assembly of natural, heteropolymeric PBs

High-quality chromosome-scale assembly of the walnut (Juglans regia L.) reference genome

The release of the first reference genome of walnut (Juglans regia L.) enabled many achievements in the characterization of walnut genetic and functional variation. However, it is highly fragmented, preventing the integration of genetic, transcriptomic, and proteomic information to fully elucidate walnut biological processes. Findings: Here, we report the new chromosome-scale assembly of the walnut reference genome (Chandler v2.0) obtained by combining Oxford Nanopore long-read sequencing with chromosome conformation capture (Hi-C) technology. Relative to the previous reference genome, the new assembly features an 84.4-fold increase in N50 size, with the 16 chromosomal pseudomolecules assembled and representing 95% of its total length. Using full-length transcripts from single-molecule real-time sequencing, we predicted 37,554 gene models, with a mean gene length higher than the previous gene annotations. Most of the new protein-coding genes (90%) present both start and stop codons, which represents a significant improvement compared with Chandler v1.0 (only 48%). We then tested the potential impact of the new chromosome-level genome on different areas of walnut research. By studying the proteome changes occurring during male flower development, we observed that the virtual proteome obtained from Chandler v2.0 presents fewer artifacts than the previous reference genome, enabling the identification of a new potential pollen allergen in walnut. Also, the new chromosome-scale genome facilitates in-depth studies of intraspecies genetic diversity by revealing previously undetected autozygous regions in Chandler, likely resulting from inbreeding, and 195 genomic regions highly differentiated between Western and Eastern walnut cultivars. Conclusion: Overall, Chandler v2.0 will serve as a valuable resource to better understand and explore walnut biology

Cooperative effects on the compaction of DNA fragments by the nucleoid protein H-NS and the crowding agent PEG probed by Magnetic Tweezers

Background: DNA bridging promoted by the H-NS protein, combined with the compaction induced by cellular crowding, plays a major role in the structuring of the E. coli genome. However, only few studies consider the effects of the physical interplay of these two factors in a controlled environment. Methods: We apply a single molecule technique (Magnetic Tweezers) to study the nanomechanics of compaction and folding kinetics of a 6 kb DNA fragment, induced by H-NS bridging and/or PEG crowding. Results: In the presence of H-NS alone, the DNA shows a step-wise collapse driven by the formation of multiple bridges, and little variations in the H-NS concentration-dependent unfolding force. Conversely, the DNA collapse force observed with PEG was highly dependent on the volume fraction of the crowding agent. The two limit cases were interpreted considering the models of loop formation in a pulled chain and pulling of an equilibrium globule respectively. Conclusions: We observed an evident cooperative effect between H-NS activity and the depletion of forces induced by PEG. General Significance: Our data suggest a double role for H-NS in enhancing compaction while forming specific loops, which could be crucial in vivo for defining specific mesoscale domains in chromosomal regions in response to environmental changes