Analysis of transcriptome in moss Physcomitrella patens CNGCb null mutant at 25 and 34 degrees C for 30 minutes [Unpublished data]. Physcomitrium patens

Analysis of transcriptome in moss Physcomitrella patens CNGCb null mutant at 25 and 34 degrees C for 30 minutes. Results provide insight into role of CNGCb in acquired thermotolerance induced by non-lethal heat treatment. Typically at dawn of a hot summer day, land plants need precise molecular thermometers to sense harmless increments in the ambient temperature to timely develop a heat-shock response (HSR) and accumulate protective heat shock proteins (Hsps), in anticipation of upcoming harmful temperatures at mid-day. Here, we found that the CNGCb gene from Physcomitrella patens and its Arabidopsis ortholog CNGC2, encode for a component of cyclic nucleotide gated Ca2+ channels acting as the primary thermosensors of land plant cells. Disruption of CNGCb or CNGC2 produced a hyper-thermosensitive phenotype, giving rise to a HSR and acquired thermotolerance at significantly milder heat-priming treatments than in wild type plants. In an aequorin-expressing moss, CNGCb loss-of-function caused altered Ca2+ signaling and a sustained Ca2+ influx. Patch clamp recordings on moss protoplasts showed the presence of three distinct thermo-responsive Ca2+-channels in wild type cells. Deletion of CNGCb led to a total absence of one, and it increased the open probability of the remaining two thermo-responsive Ca2+ channels. Thus, both in Arabidopsis and moss, CNGC2 and CNGCb are expected to form with other related CNGCs, heteromeric Ca2+ channels in the plasma membrane that respond to mild increments in the ambient temperature by triggering an optimal HSR, leading to the onset of plant acquired thermotolerance. Overall design: The WT moss tissues were heat-shocked for a half an hour at 34°C and 38°C and CNGCb at 25and 34°C followed by liquid nitrogen freezing. Total RNA was isolated using RNeasy Mini Kit (QIAGEN, Hilden, Germany) and two biological replicate samples for each treatment, were extracted. An Agilent-certified microarray service lab (MOgene, LC, St. Louis, MO, USA) was used to verify the integrity of the RNA and perform the microarray experiments. Two biological replicates were performed.