Reversible phosphorylation of thylakoid proteins contributes to photoacclimation responses in photosynthetic organisms, enabling the fine-tuning of light harvesting under changing light conditions and promoting the onset of photoprotective processes. However, the precise functional role of many of the described phosphorylation events on thylakoid proteins remains elusive. The calcium sensor receptor protein (CAS) has previously been indicated as one of the targets of the state transition kinase 8 (STN8). Here we show that in Arabidopsis thaliana , CAS is also phosphorylated by the state transition kinase 7 (STN7), as well as by another, so-far unknown, Ca 2+ -dependent kinase. Phosphoproteomics analysis and in vitro phosphorylation assays on CAS variants identified the phylogenetically conserved residues Thr-376, Ser-378, and Thr-380 as the major phosphorylation sites of the STN kinases. Spectroscopic analyses of chlorophyll fluorescence emission at 77K further showed that, while the cas mutant is not affected in state transition, it displays a persistent strong excitation of PSI under high light exposure, similar to the phenotype previously observed in other mutants defective in photoacclimation mechanisms. Together with the observation of a strong concomitant phosphorylation of light harvesting complex II (LHCII) and photosynthetic core proteins under high irradiance in the cas mutant this suggests a role for CAS in the STN7/STN8/TAP38 network of phosphorylation-mediated photoacclimation processes in Arabidopsis.

HighlightAtCML4 and AtCML5 are single-pass membrane proteins localized to the interphase between Golgi and the endosomal system. They might provide a basis for calcium regulation of endosomal vesicle transport.

Background: T-cell acute lymphoblastic leukemia (T-ALL) is a rare aggressive neoplasm accounting for ∼20% of all ALL cases. It is more common in adults than in children, although the incidence decreases with older age. Subclassification of T-ALL cases according to WHO is so far solely based on the immunophenotype. Although a number of common molecular aberrations have been described in T-ALL, a molecular classification of T-ALLs so far is missing. Aim: (1) Molecular subclassification of T-ALL cases based on whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) data. (2) Analysis of correlations between aberrations, subgroups and other parameters. Methods: WGS and WTS were performed in 114 patients. For WGS, 151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). For WTS, 101 bp paired-end reads were produced on a NovaSeq 6000 system with a yield between 35 and 125 million paired reads per sample. SPSS (version 19.0.0, IBM Corporation, Armonk, NY) was used for statistical analysis. Results: The cohort comprised 114 T-ALL cases (29% female, 71% male) with a median age of 37 years (range 1 - 91 years). Based on mutations (mut), translocations and gene expression, the cases were subdivided into six different groups: group 1 (n=20) was defined by overexpression (oex) of TLX1 (by t(10;14)(q24;q11)/TRAD-TLX1, n=17; t(7;10)(q34;q24)/TRB-TLX1, n=2; inv(10)(q23q24), n=1) and was correlated to a high frequency of mut in NOTCH1 (19/20, 95%, p=0.011 compared to the other T-ALL cases), PHF6 (11/20, 55%, p=0.04) and BCL11B (5/20, 25%, p=0.004). Group 2 (n=9) showed TLX3 oex by t(5;14)(q35;q32)/BCL11B-TLX3 (n=8) or t(5;8)(q35;q24) (n=1). Mut in WT1 (5/9 = 56%, p<0.001) and PHF6 (7/9 = 78%, p=0.005) were frequently detected. Groups 1/2 were further characterized by male predominance (18/20 = 90%, p=0.04 and 8/9 = 89%) and a cortical immunophenotype (20/20 = 100% and 7/9 = 78%). Group 3 (n=23) was defined by oex of HOXA genes, caused by inv(7)(p15q34)/TRB-HOXA (n=6), other HOXA rearrangements (n=2), SET-NUP214 (n=6), NUP98-RAP1GDS1 (n=3) and fusions involving MLLT10 (n=6). Group 4 (n=7) was defined by oex of TAL1 due to the fusion STIL-TAL1 (n=3), t(1;14)(p33;q11)/TRA-TAL1 (n=1), mut in MYB enhancers (n=2) and a MYB-translocation (n=1), and was correlated to the absence of PHF6 mut (p=0.045). The remaining 55 cases did not show oex of TLX1, TLX3, HOXA or TAL1 and were subdivided based on the presence of NOTCH1 aberrations (group 5, n=29, NOTCH1+; group 6, n=26, NOTCH1-). 25/29 NOTCH1+ cases showed at least 1 mut in NOTCH1 (range: 1-3), 2/29 had a t(7;9)(q34;q34)/TRB-NOTCH1, 2/29 showed NOTCH1 deletions. Group 5 correlated to mut in DNMT3A, NRAS and ASXL1 (p=0.001; p=0.006; p<0.001) and comprised a high number of female patients (15/29 = 52%, p=0.004). NOTCH1- cases lacked mut in FBXW7 and DNM2 (p=0.02) and comprised a high number of cases with Pre-T-ALL and mature T-ALL immunophenotype. Other findings include: (1) detection of two cases with a novel type of NOTCH1 mut, termed NOTCH1-ITD, characterized by a duplication event in the NOTCH domain. (2) Mut in DNMT3A, TET2 and ASXL1 were exclusively detected in groups 5 and 6, causing correlation to older age (p=0.011 and p=0.001), whereas the HOXA group was correlated to younger age (p=0.001) (3) NRAS mut were associated with females (6/10 = 60% vs. 27/104 = 26%, p=0.023). (4) Oex of BCL11B and RAG1 was predominantly detected in groups 1 and 4. (5) Mut in DNM2 occurred concomitantly with NOTCH1 mut (p<0.001); NOTCH1 and PHF6 mut were correlated with each other (p=0.008). (6) Female patients were significantly older than male patients (55 vs. 36 years, p=0.005). (7) Finally, analysis of OS (total cohort median: 36 months (m)) in the six groups revealed a significantly more favorable outcome of groups 1 (TLX1; n.r.) and 3 (HOXA; n.r.) compared to the other groups (see table), which might be linked to the high number of cases with cortical immunophenotype. Conclusions: (1) The combination of WGS and WTS successfully subclassified T-ALL cases on a molecular level into six different groups, by oex of TLX1, TLX3, HOXA or TAL1 and presence/absence of NOTCH1 mut. (2) We identified a novel so far unknown type of NOTCH1 aberrations, termed NOTCH1-ITD. (3) Molecular sub-classification of T-ALL cases also impacts on prognosis (TLX1 and HOXA group, longer OS; NOTCH1+ and TAL1 group, shorter OS). Figure Disclosures No relevant conflicts of interest to declare.

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