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Author contributions KDE and AJM designed the study. FG characterised the gn7 deletion in the Scott lab (Figure 5a). PFD analysed PHY protein content in the gn7 line (Figure 5b). KDE performed all other experiments and data analysis. AJM assembled the data, removed them from Wenden et al. Plant Journal 2011 after peer review to comply with an editorial request for greater focus, and prepared this paper. Acknowledgements We are grateful to Rod Scott and the late Garry Whitelam for supporting early work on gn7, and to Dr. James Lynne (Horticulture Research International, Wellesbourne) for REML analysis. The circadian clock is a fundamental feature of gene regulation and cell physiology in eukaryotes and some prokaryotes, and an exemplar gene regulatory network in Systems Biology. The circadian system in Arabidopsis thaliana is complex in part due to its photo-transduction pathways. Analysis of natural genetic variation between Arabidopsis accessions Cape Verde Islands (Cvi-0) and Landsberg erecta (Ler) identified a major, temperature-specific Quantitative Trait Locus (QTL) on chromosome V that altered the circadian period of leaf movement (Edwards et al., Genetics, 2005). We tested Near-Isogenic Lines (NILs) to confirm that Ler alleles at this PerCv5c QTL lengthened the circadian period at 12°C, with little effect at higher temperatures. The PHYTOCHROME C gene lies within the QTL interval, and contains multiple sequence variants. Plants carrying either a T-DNA-insertion into PHYC or a deletion of PHYC also lengthened circadian period under white light, except at 27°C. phyB and phyABE mutants lengthened period only at 12°C. These results extend recent data showing PhyC effects in red light, confirming the number of photoreceptor proteins implicated in the plant circadian system at eleven. The connection between light input mechanisms and temperature effects on the clock is reinforced. Natural genetic variation within PHYC is likely to underlie the PerCv5c QTL. Our results suggest that functional variation within the PHYC-Ler haplotype group might contribute to the evolution of the circadian system and possibly to clock-related phenotypes such as flowering time. These results have previously passed peer-review, so we provide them in this citable preprint.

The circadian clock is a fundamental feature of gene regulation and cell physiology in eukaryotes and some prokaryotes, and an exemplar gene regulatory network in Systems Biology. The circadian system in Arabidopsis thaliana is complex in part due to its photo-transduction pathways. Analysis of natural genetic variation between Arabidopsis accessions Cape Verde Islands (Cvi-0) and Landsberg erecta (Ler) identified a major, temperature-specific Quantitative Trait Locus (QTL) on chromosome V that altered the circadian period of leaf movement (Edwards et al., Genetics, 2005). We tested Near-Isogenic Lines (NILs) to confirm that Ler alleles at this PerCv5c QTL lengthened the circadian period at 12°C, with little effect at higher temperatures. The PHYTOCHROME C gene lies within the QTL interval, and contains multiple sequence variants. Plants carrying either a T-DNA-insertion into PHYC or a deletion of PHYC also lengthened circadian period under white light, except at 27°C. phyB and phyABE mutants lengthened period only at 12°C. These results extend recent data showing PhyC effects in red light, confirming the number of photoreceptor proteins implicated in the plant circadian system at eleven. The connection between light input mechanisms and temperature effects on the clock is reinforced. Natural genetic variation within PHYC is likely to underlie the PerCv5c QTL. Our results suggest that functional variation within the PHYC-Ler haplotype group might contribute to the evolution of the circadian system and possibly to clock-related phenotypes such as flowering time. These results have previously passed peer-review, so we provide them in this citable preprint.

International audience; Background: PME and PMEI isoforms are co-expressed in Arabidopsis. Their biochemical interaction is yet to be characterized. Results: The processive activity of AtPME3 is regulated by AtPMEI7 in a pH-dependent manner in vitro. Conclusion: AtPMEI7 is a key component of the regulation of AtPME3 activity in planta. Significance: The tuning of AtPME3 activity by AtPMEI7 brings insights into the control of homogalacturonan methylesteri-fication in plant cell walls.

International audience; Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter:: GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3.AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with aPMEligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development