Additional file 13: of A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16)

A comparison of HS-dependent changes in pollen to 67 multi-stress response genes in vegetative tissues. From a list of 67 multi-stress genes curated by Swindell 2006 (PMCID: PMC1698639 [47]; highlighted in purple), 19 genes showed detectable expression in pollen. Among those, only three genes showed significant changes in pollen HS (red font). (XLSX 596 kb

Additional file 2: of A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16)

Raw expression counts for WT and cngc16 pollen with and without HS. Read counts generated via FeatureCounts, see Methods) for all 12 replicates are shown before normalization and exclusions. (XLSX 4358 kb

Additional file 1: of A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16)

Hot/Cold stress cycle. Diagram showing the Hot/Cold stress-cycle used here for growing plants from which pollen samples for RNA-Seq experiment were harvested at the end of HS-peak at 40 °C. See Methods and [20] for more details. (PPTX 50 kb

Additional file 7: of A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16)

RNA-Seq validation using real-time Q-PCR. a. Comparison of expression values obtained from Q-PCR and RNA-Seq normalized to WT control (normal). The analysis was performed on two different reference genes separately (CYCP2 (AT3G21870) and UBQ7 (AT2G35635)). b. Primer sequences used for real-time Q-PCR. (XLSX 16 kb

Additional file 4: of A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16)

Library size and principal component analysis. a. Table showing library sizes of each sample. b. A principal component analysis (PCA) of the filtered data showing that 87% of the variance of the samples can be explained by differences in the stress states. See methods for more details. Control and heat correspond to normal and HS conditions, respectively. (PPTX 43 kb

Additional file 15: of A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16)

GO analysis on the 192 largest differences between WT and cngc16 under HS. A GO analysis pie chart is shown for Molecular Function (a), Cellular Component (b), and Biological Process (c) generated using an upload of Additional file 3 or Additional file 9e column R listing the differences (≥ 2-fold and adjusted p-value ≤0.01) between WT and cngc16 HS-transcriptomes. Categories were defined using PANTHER Overrepresentation Test (release 2017–04-13 [61]) using a GO Ontology database (released 2017–08-14) with 27,060 reference genes for Arabidopsis thaliana. Gene categories shown displayed enrichments with a p-value of ≤0.05. (PPTX 993 kb

Additional file 5: of A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16)

A transcript profile comparison to evaluate purity of pollen samples used for RNA-Seq. A subset of 12 genes was used to compare relative purities of pollen samples in the current pollen transcriptome study to those from a RNA-Seq study from Loraine et al. [22] (yellow highlights) or a microarray experiment from Qin et al. 2009 [23] (purple highlights). Four references genes were chosen to generate normalization factors that could be used to adjust expression values in Loraine et al. [22] and Qin et al. 2009 [23] to allow a relative comparison of the three data sets for WT pollen under control (normal) conditions. For a control group, three CNGC genes were chosen that displayed low to moderate levels of expression (Tunc-Ozdemir et al. 2013 [24] and Frietsch et al. 2007 [25]). As markers for potential contamination from photosynthetic tissues, five different nuclear encoded genes were chosen that are associated with either photosystems I/II, or chlorophyll A-B binding proteins (Umate 2010 [26]). Average relative ratios are shown for each of the four different pollen samples in comparison to both Loraine et al. [22] and Qin et al. [23]. (XLSX 19 kb

Additional file 6: of A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16)

Integrated Genome Browser (IGB) screenshot showing cngc16 RNA-Seq reads primarily upstream of T-DNA insertion site. The green arrow identifies the position of T-DNA insertion in cngc16–2 (SAIL_726_B04). The observed reads aligning to cngc16 are primarily on the 5′ side of the T-DNA disruption site, with only a few reads observed at two disconnected downstream positions. This suggests that there were no detectable full-length transcripts. (PPTX 66 kb

Additional file 3: of A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16)

Normalized transcript expression counts for WT and cngc16 pollen with and without HS. Expression counts from Additional file 2 were subjected to exclusions/filters and normalized as described in methods. Expression data from other transcriptome studies (microarray and RNA-Seq) were added to the table for comparisons. These included an RNA-Seq data set for WT pollen and seedlings from Loraine et al. 2013 (PMCID: PMC3668042 [22]), two pollen microarray experiments from Qin et al. 2009 (PMCID: PMC2726614 [23]) and Borges et al. 2008 (PMCID: PMC2556834 [72]), and finally HS seedlings from Schmid et al. 2005 (PMID:15806101 [33]), respectively. Ratios of expression between pollen and seedling are based on Loraine et al. 2013 (PMCID: PMC3668042 [22]). In cases where the seedling value was below the limit of detection, a minimal value of 0.0019 was substituted in its place as a denominator (0.0019 was the RPKM for ATCG00860 and was the lowest value reported in Loraine et al. 2013 (PMCID: PMC3668042 [22]). Ratio of expression between semi-in vivo pollen tube over dry pollen is based on Qin et al. 2009 (PMCID: PMC2726614 [23]). HS dependent changes in transcript abundance in shoots were based on publicly available data using the AtGenExpress Visualization Tool (AVT) ( http://jsp.weigelworld.org/expviz/expviz.jsp , Schmid et al. 2005 (PMID:15806101 [33] for seedlings exposed to one hour HS at 38 °C). The log2-fold change was calculated based on a comparison of means of normalized values for two heat-stressed and two non-stressed seedling samples. NA stands for not available. Not Calculated, refers to a value not being calculated because one of the input sample read counts was considered to have an extreme outlier (see AT2G42540 and ATMG01360). (XLSX 8952 kb

Additional file 11: of A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16)

HS-dependent transcript abundance changes corresponding to transcription factors in WT and cngc16 pollen. Differentially expressed transcription factors (TFs) are organized to show HS-dependent differences that are WT-specific (23 changes, yellow highlight), cngc16-specific (98 changes, blue highlight), or common to both mutant and WT (66 changes, white highlight). a A comparison is provided to HS-dependent changes observed for aerial parts of seedlings exposed to a one hour 38 °C HS based on publicly available microarray data in AtGenExpress, ( http://jsp.weigelworld.org/expviz/expviz.jsp ; Schmid et al. 2005 (PMID: 15806101 [34]). The log2-fold change was calculated based on means of normalized values for two heat-stressed seedlings compared to two non-stressed seedlings. NA stands for not available because of absence of probeset on microarray experiment. (XLSX 63 kb