TFIIS is required for reproductive development and thermal adaptation in barley

Abstract

Barley reproductive fitness and efficient heat stress adaptation requires the activity of TFIIS, the elongation cofactor of RNAPII. Regulation of transcriptional machinery and its adaptive role under different stress conditions are studied extensively in the dicot model plant Arabidopsis, but our knowledge on monocot species remains elusive. TFIIS is an RNA polymerase II-associated transcription elongation cofactor. Previously, it was shown that TFIIS ensures efficient transcription elongation that is necessary for heat stress survival in A. thaliana. However, the function of TFIIS has not been analysed in monocots. In the present work, we have generated and studied independent tfIIs-crispr-mutant barley lines. We show that TFIIS is needed for reproductive development and heat stress survival in barley. The molecular basis of HS-sensitivity of tfIIs mutants is the retarded expression of heat stress protein transcripts, which leads to late accumulation of HSP chaperones, enhanced proteotoxicity and ultimately to lethality. We also show that TFIIS is transcriptionally regulated in response to heat, supporting a conserved adaptive function of these control elements for plant thermal adaptation. In sum, our results are a step forward for the better understanding of transcriptional machinery regulation in monocot crops.

Keywords: Barley (Hordeum vulgare L.); Heat stress; Reproductive development; TFIIS; Transcriptional elongation.

Fig. 1

TFIIS is needed for reproductive development in barley. A Schematic representation of HvTFIIS gene locus; protein domain I, II and III location is shown above; exons as black boxes, UTRs as grey boxes, Zn finger domain cysteine residues and DE acidic dipeptide motif as yellow and red line, respectively; HSE cis elements and location of sgRNA guide sites are shown below; B TFIIS mutants of barley were created by CRISPR mutagenesis; insertion or deletion mutation within the TFIIS locus in the selected transgenic lines are shown on chromatograms; C tfIIs-cr1 mutant plants have slightly retarded vegetative growth compared to wild-type plants; D germination rate quantification of wild-type and tfIIs-cr-mutant plants during a timeseries; E pictures of germinating seeds on 2nd day; F the calculated thousand grain weight (TGW) of wild-type and tfIIs-cr-mutant plants; bars represent standard errors based on at least three bio reps; P values based on two-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001, ns; non-significant)

Fig. 2

TFIIS is indispensable for efficient heat stress response. A Schematic representation of non-treated (NT) and thermotolerance to moderately high-temperature regime (TMHT); arrowheads show the time of sampling; B wild-type (wt) and tfIIs CRISPR mutants (tfIIs-cr1, -cr2) were exposed to 1-day (1 d), 2-d or 3-d heat stress TMHT regime or non-treated control (NT); C–F Accumulation of HSR-transcripts is retarded in the absence of TFIIS: relative expressions of C HSP70-4, D HSP90, E HSP101 and F TFIIS mRNA during a timeseries (NT; 1 h, 1 h; 2 h; 1 day, 1 d); bars represent standard errors based on three bio reps; P values based on two-tailed Student’s t test *P < 0.05, **P < 0.01, ***P < 0.001 show differences between wt and mutants; P < 0.05, P < 0.01, P < 0.001 values show differences between NT and heat stress condition within genotypes

Fig. 3

Heat stress causes enhance proteotoxic stress in absence of TFIIS. A Western blot images HSP70, HSP90 and HSP101 proteins; wild-type (wt) and tfIIs CRISPR mutants (tfIIs-cr1, -cr2 and -cr3, respectively) were exposed to NT or TMHT for 1 h (1 h), 2 h or 1 day (1d), as shown; stain-free picture is shown as a loading control; B, C quantification of protein accumulation of HSP90 (B), and HSP101 (C) during the same timeseries; D silver staining gel images of insoluble protein fractions from wt and tfIIs-cr2 plants exposed to non-treated or 1d TMHT heat treatment; Rubisco large subunit (RbcL) is shown as loading control within soluble fraction; treatment conditions are shown; sHSP-CI accumulation is shown at the bottom. E Ratio quantifications of insoluble/soluble protein fractions in wild-type and tfIIs-cr2-mutant plants at non-treated (NT) or TMHT/1 d (1 d) stress conditions; P values based on two-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001) show differences between wild-type and mutant plants. F Working model for TFIIS action during heat stress response: elevated temperature induces expression of HSPs and TFIIS; TFIIS elongation factor positively feedback regulates itself and further enhances expression of HSP transcription; the timely accumulation of HS-transcripts and proteins ensures efficient thermotolerance in barley plants