Effect of HsfA7 and HsfA2 knockout on HS-gene expression

To dissect the effect of HsfA7 and HsfA2 loss of function on the overall HSR in vegetative (leaf) tissues, we performed an expression analysis with focus on specific stress induced Hsfs and Hsps.

Four-week-old WT, CR-a7-1 and CR-a2-1 plants were exposed to 35°C or 40°C for 1 hour. The protein levels of HsfA1a, HsfA2, HsfA7 and HsfB1 as well as Hsp101, Hsp70, Hsp90 and Hsp17-CI were analyzed by immunoblotting (Fig. 19). HsfA1a showed higher levels in CR-a7-1 plants at 35 and 40°C compared to the WT and CR-a2-1 plants (Fig. 19). Similar to HsfA1a, HsfA2 is also increased in treated CR-a7-1 plants when compared to WT (Fig. 19). In contrast, HsfA2 KO did not affect the levels of HsfA7. Interestingly, HsfB1 levels were significantly reduced in both HsfA2 and HsfA7 mutants at 40°C compared to WT (Fig. 19).

In terms of Hsp abundance, Hsp101 and Hsp17-CI were differentially expressed (Fig. 19). At 35°C, the levels of both proteins were enhanced in the HsfA7 KO background, while Hsp101 levels were slightly lower in the HsfA2 KO at 40°C. Hsp17-CI protein was very abundant at 40°C where the protein levels were similar in all genotypes (Fig. 19). At 35°C a faint Hsp17-CI signal was detected which was more prominent in the CR-a7-1 sample. Hsp90 was moderately induced at 35 and strongly at 40°C, while Hsp70 accumulated at higher levels only at 40°C. However, neither Hsp90 nor Hsp70 were affected by knockout of HsfA7 or HsfA2.

All in all, the expression analysis confirmed that HsfA7 negatively affects the protein abundance of the master regulator HsfA1a. Moreover, the regulation of HsfA2 as well as HsfB1 protein abundance seems to be affected by knockout of HsfA7. Hsp70 and Hsp90 did not show a significant change in abundance while HS inducible Hsp101 and Hsp17-CI seem to be differentially regulated by HsfA7 or HsfA2 in response to different temperatures.

73

Figure 19. Expression analysis of Hsfs and Hsps in leaves of WT, HsfA7 KO and HsfA2 KO plants.

Temperature dependent induction of HsfA1a, HsfA2, HsfA7, HsfB1, Hsp101, Hsp90, Hsp70 and Hsp17-CI. Four-week old wild-type, CR-a7-1 and CR-a2-1 tomato plants were exposed to a single HS of 35 or 40°C for 1 hour or kept under control conditions (25°C) for the same period of time. Leaf samples were harvested immediately following the treatment. Total protein extract in equal amounts (40 µg) was used for SDS-PAGE and immunoblot detection of HsfA1a, HsfA2, HsfA7, HsfB1, Hsp101, Hsp90, Hsp70, Hsp17-CI and actin (loading control) using specific antibodies. Rbc, Ponceau staining of Rubisco large subunit.

The transcript levels of four HS-induced genes in WT, two HsfA7 and one HsfA2 KO were examined by qPCR. The samples were treated in the same way as for the protein expression analysis (Fig. 19). All genes were expressed at similar levels to WT in the mutants under non-stress conditions. APX3 and Hsa32 showed a stronger induction in both HsfA7 KO lines at 35°C and APX3 a weaker induction in CR-a2-1 at 35°C compared to WT (Fig. 20). Similarly, Hsp101 and Hsp17.7A-CI were expressed at higher levels in CR-a7 lines at 35°C, in agreement with immunoblot findings for the two proteins (Fig. 19), and lower levels in the CR-a2 line at 35°C compared to WT (Fig. 20).

74

Figure 20. Expression analysis of putative HS-inducible target genes in leaves of WT, HsfA7 KO and HsfA2 KO plants.

Temperature dependent induction of APX3, Hsa32, Hsp101 and Hsp17.7A-CI. Four-week old wild-type, CR-a7-1, CR-a7-2 and CR-a2-1 tomato plants were exposed to a single HS of 35 or 40°C for 1 hour or kept under control conditions (25°C) for the same period of time. Leaf samples were harvested immediately following the treatment. Following RNA extraction and cDNA synthesis, relative transcript abundance (2^-ΔΔCt) of APX3, Hsa32, Hsp101 and Hsp17.7A-CI was determined using gene specific primers. The Ct value of each gene was normalized to the Ct value of EF1α housekeeping gene and to the control sample. Vertical bars represent the average ± SD of three replicates. Asterisks depict statistical significance (p<0.05) based on one–way ANOVA followed by Duncan's Multiple Range Test.

To confirm that the KO of HsfA7 is the cause for the increased accumulation of HS-inducible transcripts in the CR-a7 background, we performed a transient complementation of HsfA7 KO using mesophyll protoplasts. In brief, protoplasts from CR-a7-2 plants were transformed with either the pRT-Neo mock plasmid or a construct containing the coding sequence for HsfA7-I or HsfA7-II under the control of the endogenous SlHsfA7 promoter (1.2 kb promoter fragment). For comparison, WT protoplasts were transformed only with the mock plasmid. Following an incubation of 6 hours the cells were exposed to 35 or 40°C for 1 hour. qPCR analysis confirmed the higher accumulation of APX3 and Hsa32 in CR-a7 protoplasts compared to WT at 35°C and for APX3 at 40°C as well (Fig 21), thereby by large matching the expression data obtained for leaves described above (Fig. 20). APX3 and Hsa32 transcripts accumulated at lower levels in CR-a7 protoplasts expressing HsfA7 isoforms compared to the mock control, and similar to WT at

75

35°C (Fig. 21). The weaker gene induction in complemented CR-a7 protoplasts was confirmed for APX3 at 40°C as well (Fig. 21). These results clearly demonstrate that the accumulation of HsfA7 results in weaker induction of HS-induced genes.

Figure 21. Expression analysis of APX3 and Hsa32 in WT and HsfA7 KO protoplasts.

Temperature-dependent induction of APX3 and Hsa32. Tomato mesophyll protoplasts having a WT (cv.

Moneymaker) background were transformed with the mock plasmid pRT-Neo while HsfA7 KO (CR-a7) protoplasts were transformed either with pRT-Neo or with plasmid constructs harbouring HsfA7-I or HsfA7-II under the control of the endogenous HsfA7 promoter (1.2 kb fragment). Protoplasts were incubated at 25°C for 6 hours to allow expression, exposed to HS at 35 or 40°C for 1 hour and harvested immediately following the stress treatment. After RNA extraction and cDNA synthesis, relative transcript abundance (2^-ΔΔCt) of APX3 and Hsa32 was determined using gene specific primers. Vertical bars represent the average ± SD of three replicates.