One Heat Shock Transcription Factor Confers High Thermal Tolerance in Clematis Plants

Abstract

Clematis plants play an important role in botanical gardens. Heat stress can destroy the activity, state and conformation of plant proteins, and its regulatory pathway has been well characterized in Arabidopsis and some crop plants. However, the heat resistance response mechanism in horticultural plants including Clematis has rarely been reported. Here, we identified a heat-tolerant clematis species, Clematis vitalba. The relative water loss and electrolytic leakage were significantly lower under heat treatment in Clematis vitalba compared to Stolwijk Gold. Differential expression heat-tolerant genes (HTGs) were identified based on nonparametric transcriptome analysis. For validation, one heat shock transcription factor, CvHSF30-2, extremely induced by heat stimuli in Clematis vitalba, was identified to confer tolerance to heat stress in Escherichia coli and Saccharomyces cerevisiae. Furthermore, silencing of HSF30-2 by virus-induced gene silencing (VIGS) led to heat sensitivity in tobacco and Clematis, suggesting that the candidate heat-resistant genes identified in this RNA-seq analysis are credible and offer significant utility. We also found that CvHSF30-2 improved heat tolerance of Clematis vitalba by elevating heat shock protein (HSP) expression, which was negatively regulated by CvHSFB2a. Taken together, this study provides insights into the mechanism of Clematis heat tolerance and the findings can be potentially applied in horticultural plants to improve economic efficiency through genetic approaches.

Keywords: Clematis; CvHSF30-2; CvHSFB2a; VIGS; heat stress; transcriptome analysis.

Figure 1

Two Clematis varieties and physiological changes after high-temperature treatment. (A) Two Clematis plants: Clematis vitalba (Cv, left) and Clematis “Stolwijk Gold” (SG, right). (B) Phenotype of the two Clematis plants after heat treatment. (C) Change of relative water loss and relative electrolytic leakage after high-temperature treatment (42 °C for 2 h) in Clematis varieties. (D) Diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining of the two Clematis varieties’ leaves after high-temperature treatment (42 °C for 2 h).

Figure 2

RNA-seq analysis of Clematis vitalba after high-temperature treatment. (A) Heat map of differentially expressing heat-related genes (HRGs). The genes chosen for the qPCR assay are marked by arrows. The descriptions of the names of HRGs are shown in Supplementary Table S2. (B) qPCR analysis of some HRGs. * p < 0.05; ** p < 0.01. *** p < 0.001.

Figure 3

Phylogenetic and motifs analysis of heat shock transcription factors (HSFs). (A) Phylogenetic analysis of HSF30-2 in different species. The accession numbers of HSFs are shown top to bottom as follows: XP_008220492.1, Prupe.1G410400.7, PQM38437.1, NP_001290015.1, Gorai.013G220400.5, Ciclev10008618m, Glyma.14G096800.3, Solyc08g062960.2.1, AT2G26150.1, Cagra.7662s0001.1, XP_010262066.1, KAF9613066.1, Cv, Aco005862.1, Zm00008a004344_T01, evm.model.supercontig_44.122, LOC_Os07g08140.1, Traes_2DS_B6872CB84.1, PRW33971.1 and GBF94047.1. (B) Motifs of CvHSF30-2 and CvHSFB2a. Motif prediction and visualization were performed in HEATSTER (https://applbio.biologie.uni-frankfurt.de/hsf/heatster/home.php (accessed on 25 July 2020)).

Figure 4

Phenotype of HSF30-2 silencing in tobacco under heat stress. (A) Phenotype of HSF30-2-silencing in tobacco by virus-induced gene silencing (VIGS) before and after heat shock. TRV-HSF30-2 means the silencing of the CvHSF30-2 homologous gene in tobacco. TRV-HSF30-2① and TRV-HSF30-2② represent two different HSF30-2-silencing lines. (B) Silencing effect of TRV-HSF30-2 plants based on qPCR. (C,D) DAB and NBT staining of HSF30-2-silenced tobacco after heat treatment. (E,F) DAB and NBT staining quantified using the ImageJ2x program. The unit on the x-axis is the average grey value. The staining intensity was quantified using the ImageJ2x program. The image (inverse phase) was changed into an 8-bit grey image, and the average grey values from the upper, middle and lower areas of root tips calculated by ImageJ2x reflect the staining intensity. Error bars represent the standard error of three biological replicates. * p < 0.05; ** p < 0.01. TRV: tobacco rattle virus; CK: control check (normal temperature).

Figure 5

Silencing of CvHSF30-2 on Clematis. (A) Operation flow chart of VIGS in Cv. (a–c) Four-week-old tobacco, N. benthamiana, was injected with GV3101 harboring TRV2-CvHSF30-2, and the leaves were taken three days later. (d–f) The leaves injected with TRV2-NBHSF30-2 virus were thoroughly ground. Then, juice was taken and placed on the clematis leaves with quartz sand added. The clematis leaves were inoculated by rubbing by hand. (g,h) The clematis inoculated by friction was marked and placed in a constant-temperature incubator for cultivation until new branches and leaves had grown. (B) Silencing effect of CvHSF30-2 in control (GFP) and virus-silenced (VIGS: HSF30-2) clematis leaves based on qPCR. (C) Heat-shock phenotype of CvHSF30-2-silenced Cv by VIGS. (D) Staining of Cv leaves after heat shock by DAB and NBT. DAB- and NBT-stained pictures of each group of clematis leaves; the parts of each picture marked with red squares and labelled 1, 2, 3 and 4 correspond to the enlarged pictures below. (E) DAB and NBT staining quantified using the ImageJ2x program. Error bars represent the standard error of three biological replicates. *** p < 0.001.

Figure 6

Expressions of CvHSP17.3, CvHSP17.6, CvHSP17.8, CvHSP20, CvHSP26.5 and CvHSP30.1 in CvHSF30-2-silenced plants by VIGS. Error bars represent the standard error of three biological replicates. Asterisks indicate a significant difference between TRV-GFP and TRV-CvHSF30-2 by t-test. * p < 0.05; ** p < 0.01.

Figure 7

Silencing of CvHSFB2a in Clematis. (A) Phenotype of CvHSFB2a-silenced Clematis under heat treatment. (B) DAB and NBT staining of TRV-GFP and TRV-CvHSFB2a Clematis before and after heat treatment. (C) Silencing effect of CvHSFB2a. (D) The expression of CvHSF30-2 in TRV-CvHSFB2a plants. (E,F) DAB and NBT staining as quantified using the ImageJ2x program. Error bars represent the standard error of three biological replicates. * p < 0.05; ** p < 0.01.

Figure 8

Working model for Clematis vitalba thermotolerance.