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During meiosis, the stepwise release of sister chromatid cohesion is crucial for the equal distribution of genetic material to daughter cells, enabling generation of fertile gametophytes. However, the molecular mechanism that protects centromeric cohesion from release at meiosis I is unclear in Arabidopsis (Arabidopsis thaliana). Here, we report that the protein phosphatase 2A regulatory subunits B'α and B'β participate in the control of sister chromatid separation. The double mutant b'αβ exhibited severe male and female sterility, caused by the lack of a nucleus or presence of an abnormal nucleus in mature microspores and embryo sacs. 4′,6-Diamidino-2-phenylindole staining revealed unequal amounts of DNA in the mononuclear microspores. Transverse sections of the anthers revealed unevenly sized tetrads with or without a nucleus, suggesting a defect in meiocyte meiosis. An analysis of chromosome spreads showed that the sister chromatids separated prematurely at anaphase I in b'αβ. Immunoblotting showed that AtRECOMBINATION DEFECTIVE8 (AtREC8), a key member of the cohesin complex, was hyperphosphorylated in b'αβ anthers and pistils during meiosis but hypophosphorylated in the wild type. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation assays showed that B'α and B'β interact specifically with AtREC8, AtSHUGOSHIN1 (AtSGO1), AtSGO2, and PATRONUS1. Given that B'α was reported to localize to the centromere in meiotic cells, we propose that protein phosphatase 2A B'α and B'β are recruited by AtSGO1/2 and PATRONUS1 to dephosphorylate AtREC8 at the site of centromere cohesion to shield it from cleavage until anaphase II, contributing to the balanced separation of sister chromatids at meiosis.

Heat shock (HS) is a common form of stress suffered by plants. It has been proposed that calmodulin (CaM) is involved in HS signal transduction, but direct evidence has been lacking. To investigate the potential regulatory function of CaM in the HS signal transduction pathway, T-DNA knockout mutants for AtCaM2, AtCaM3, and AtCaM4 were obtained and their thermotolerance tested. Of the three knockout mutant plants, there were no differences compared with wild-type plants under normal conditions. However, the AtCaM3 knockout mutant showed a clear reduction in thermotolerance after heat treatment at 45°C for 50 min. Overexpression of AtCaM3 in either the AtCaM3 knockout or wild-type background significantly rescued or increased the thermotolerance, respectively. Results from electrophoretic mobility-shift assays, real-time quantitative reverse transcription-polymerase chain reaction, and western-blot analyses revealed that, after HS, the DNA-binding activity of HS transcription factors, mRNA transcription of HS protein genes, and accumulation of HS protein were down-regulated in the AtCaM3 knockout mutant and up-regulated in the AtCaM3-overexpressing transgenic lines. Taken together, these results suggest that endogenous AtCaM3 is a key component in the Ca2+-CaM HS signal transduction pathway.