The Ca2+-dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation in Arabidopsis

Plants use different signalling pathways to respond to external stimuli. Intracellular signalling via calcium-dependent protein kinases (CDPKs) or mitogen-activated protein kinases (MAPKs) present two major pathways that are widely used to react to a changing environment. Both CDPK and MAPK pathways are known to be involved in the signalling of abiotic and biotic stresses in animal, yeast and plant cells. Here, we show the essential function of the CDPK CPK3 (At4g23650) for salt stress acclimation in Arabidopsis thaliana, and test crosstalk between CPK3 and the major salt-stress activated MAPKs MPK4 and MPK6 in the salt stress response. CPK3 kinase activity was induced by salt and other stresses after transient overexpression in Arabidopsis protoplasts, but endogenous CPK3 appeared to be constitutively active in roots and leaves in a strictly Ca2+-dependent manner. cpk3 mutants show a salt-sensitive phenotype comparable with mutants in MAPK pathways. In contrast to animal cells, where crosstalk between Ca2+ and MAPK signalling is well established, CPK3 seems to act independently of those pathways. Salt-induced transcriptional induction of known salt stress-regulated and MAPK-dependent marker genes was not altered, whereas post-translational protein phosphorylation patterns from roots of wild type and cpk3 plants revealed clear differences. A significant portion of CPK3 was found to be associated with the plasma membrane and the vacuole, both depending on its N-terminal myristoylation. An initial proteomic study led to the identification of 28 potential CPK3 targets, predominantly membrane-associated proteins

Development of hypertensive complications in oocyte donation pregnancy: protocol for a systematic review and individual participant data meta-analysis (DONOR IPD)

Introduction The assisted reproductive technique of oocyte donation (OD) is comparable to in vitro fertilisation (IVF), with the distinction of using a donated oocyte and thus involving two women. Compared with IVF and naturally conceived (NC) pregnancies, OD pregnancies have a higher risk for pregnancy complications as pregnancy-induced hypertension (PIH) and pre-eclampsia (PE). Various covariates among women pregnant by OD, however, also contribute to an increased risk for developing hypertensive complications. Therefore, we will conduct the DONation of Oocytes in Reproduction individual participant data (DONOR IPD) meta-analysis to determine the risk for the development of hypertensive complications in OD pregnancy, in comparison to autologous oocyte pregnancy (non-donor IVF/intracytoplasmic sperm injection (ICSI) and NC pregnancy). The DONOR IPD meta-analysis will provide an opportunity to adjust for confounders and perform subgroup analyses. Furthermore, IPD will be used to externally validate a prediction model for the development of PE in OD pregnancy.Methods and analysis A systematic literature search will be performed to search for studies that included women pregnant by OD, and documented on hypertensive complications in OD pregnancy. The authors from each study will be asked to collaborate and share IPD. Using the pseudoanonymised combined IPD, we will perform statistical analyses with one-stage and two-stage approaches, subgroup analyses and possibly time-to-event analyses to investigate the risk of developing hypertensive complications in OD pregnancy. Furthermore, we will formally assess a prediction model on its performance in an external validation with the use of IPD.Ethics and dissemination Ethical approval and individual patient consent will not be required in most cases since this IPD meta-analysis will use existing pseudoanonymised data from cohort studies. Results will be disseminated through peer-reviewed journals and international conferences.PROSPERO registration number CRD42021267908

Inhibition of Arabidopsis O-Acetylserine(thiol)lyase A1 by Tyrosine Nitration*

The last step of sulfur assimilation is catalyzed by O-acetylserine(thiol)lyase (OASTL) enzymes. OASTLs are encoded by a multigene family in the model plant Arabidopsis thaliana. Cytosolic OASA1 enzyme is the main source of OASTL activity and thus crucial for cysteine homeostasis. We found that nitrating conditions after exposure to peroxynitrite strongly inhibited OASTL activity. Among OASTLs, OASA1 was markedly sensitive to nitration as demonstrated by the comparative analysis of OASTL activity in nitrated crude protein extracts from wild type and different oastl mutants. Furthermore, nitration assays on purified recombinant OASA1 protein led to 90% reduction of the activity due to inhibition of the enzyme, as no degradation of the protein occurred under these conditions. The reduced activity was due to nitration of the protein because selective scavenging of peroxynitrite with epicatechin impaired OASA1 nitration and the concomitant inhibition of OASTL activity. Inhibition of OASA1 activity upon nitration correlated with the identification of a modified OASA1 protein containing 3-nitroTyr302 residue. The essential role of the Tyr302 residue for the catalytic activity was further demonstrated by the loss of OASTL activity of a Y302A-mutated version of OASA1. Inhibition caused by Tyr302 nitration on OASA1 activity seems to be due to a drastically reduced O-acetylserine substrate binding to the nitrated protein, and also to reduced stabilization of the pyridoxal-5′-phosphate cofactor through hydrogen bonds. This is the first report identifying a Tyr nitration site of a plant protein with functional effect and the first post-translational modification identified in OASA1 enzyme