Stress memory gene FaHSP17.8-CII controls thermotolerance via remodeling PSII and ROS signaling in tall fescue

Abstract

High temperature is the most limiting factor in the growth of cool-season turfgrass. To cope with high-temperature stress, grass often adopt a memory response by remembering one past recurring stress and preparing a quicker and more robust reaction to the next stress exposure. However, little is known about how stress memory genes regulate the thermomemory response in cool-season turfgrass. Here, we characterized a transcriptional memory gene, Fa-heat shock protein 17.8 Class II (FaHSP17.8-CII) in a cool-season turfgrass species, tall fescue (Festuca arundinacea Schreb.). The thermomemory of FaHSP17.8-CII continued for more than 4 d and was associated with a high H3K4me3 level in tall fescue under heat stress (HS). Furthermore, heat acclimation or priming (ACC)-induced reactive oxygen species (ROS) accumulation and photosystem II (PSII) electron transport were memorable, and this memory response was controlled by FaHSP17.8-CII. In the fahsp17.8-CII mutant generated using CRISPR/Cas9, ACC+HS did not substantially block the ROS accumulation, the degeneration of chloroplast ultra-structure, and the inhibition of PSII activity compared with HS alone. However, overexpression of FaHSP17.8-CII in tall fescue reduced ROS accumulation and chloroplast ultra-structure damage, and improved chlorophyll content and PSII activity under ACC+HS compared with that HS alone. These findings unveil a FaHSP17.8-CII-PSII-ROS module regulating transcriptional memory to enhance thermotolerance in cool-season turfgrass.

Figure 1

Memory responses of sHSP transcription under ACC and HS. A, The ACC and HS experimental set-up; arrows indicate the time of sampling. B, Transcript levels of four sHSP genes during ACC phases. Error bars indicate means ± se (n = 4). Asterisks indicate significant differences relative to S1 based on least significant difference (LSD) test (*P < 0.05, **P < 0.01). C, Transcript levels of four sHSP genes for HS 0, 2, 4, 8, 12, 24, and 36 h. YT-521B was used as an internal control. Vertical bars represent means ± se (n = 4). Asterisks indicate significant differences relative to 0 h treatment based on LSD test (*P < 0.05, **P < 0.01).

Figure 2

Isolation, subcellular localization, and homology analysis of HS memory-related genes FaHSP17.8-CII and FaHSP17.9-CI. A, Phylogenetic analysis of FaHSP17.8-CII and FaHSP17.9-CI with its orthologous genes based on their deduced amino acid sequences. B, Protein sequence multiple alignment of FaHSP17.8-CII with its orthologous genes in other plant species. Solid black boxes indicate the conserved residues, such as methionine (M). The number and * indicate the amino-acid positions in its protein sequence. The sequences were listed in Supplemental Table S4. C, Subcellular localization of FaHSP17.8-CII and FaHSP17.9-CI in Nicotiana benthamiana leaf epidermis cells. Scale bars: 25 μm. D and E, Relative quantification of FaHSP17.8-CII and FaHSP17.9-CI expression in different tall fescue tissues. Vertical bars indicate means ± se (n = 4). Means followed by different letters were significantly different based on the LSD test at P < 0.05.

Figure 3

Profile of H3K4me3 at HS memory-related gene FaHSP17.8-CII and its transcriptional level under ACC+HS. A, Schematic diagram of FaHSP17.8-CII indicating the regions analyzed by ChIP-qPCR. Amplicon positions relative to TSS are: 12,356 bp before exons “ATG” sequence in promoter region, 2, at the beginning sequence in exons (dark gray box). B and D, ChIP-qPCR analysis of the amounts of specific modifications present at regions 1 and 2 for FaHSP17.8-CII under ACC or HS. C, Transcript levels of FaHSP17.8-CII under ACC+HS and HS relative to YT-521B. Vertical bars represent means ± se (n = 4) using LSD test (*P < 0.05, **P < 0.01). B, Asterisks indicate significant differences between control (S0) and S1, R3, or S4 treatments. C and D, Asterisks indicate significant differences between ACC and HS when grasses exposed to the same treatment time.

Figure 4

fahsp17.8-CII mutant reduces HS memory and thermotolerance, but overexpressing FaHSP17.8-CII enhances them. A, Sanger sequencing of site-specific mutations in fahsp17.8-CII generated by CRISPR/Cas9. B, The phenotype of fahsp17.8-CII and FaHSP17.8-CII-OE under ACC+HS and HS. C–H, The quantification analysis of physiological trait in fahsp17.8-CII and FaHSP17.8-CII-OE under ACC+HS and HS. Vertical bars indicate means ± se (n = 4). Different letters above the bars indicate significant difference based on the LSD test at P < 0.05. fahsp17.8-CII and FaHSP17.8-CII-OE were simplified as fahsp17.8 and FaHSP17.8-OE and the same below. Scale bars: 7 cm. ACC, heat acclimation or priming; OE, overexpression; PAM, protospacer-adjacent motif.

Figure 5

FaHSP17.8-CII leads to the change of chloroplast structure and PSII function during the thermomemory phase. A, Electron microscopy pictures of chloroplasts in WT, fahsp17.8-CII, and FaHSP17.8-CII-OE under ACC+HS and HS. B and C, Comparison of Chlorophyll a fluorescence (OJIP) transients changed in different tall fescue transgenic plants under ACC+HS and HS. D, The change of W_k between F₀ and F₃₀₀μs: W_k = (F_t-F₀)/(F₃₀₀μs-F₀) and E, the differences of the stressed samples to the control samples (ΔW_k). To intuitively display the change, the data of W_k and ΔW_k were numerically fit with the nonlinear equation; Polynomial, Cubic f = y₀ + a*x + b*x²+c*x³. OE, overexpression.

Figure 6

Histochemical analysis for the generation of H₂O₂ and ${\mathrm{O}}_2^{-}$ stained with DAB and NBT in WT, fahsp17.8-CII, and FaHSP17.8-CII-OE leaves under ACC+HS and HS. Brown precipitations and blue spots represent the presence of H₂O₂ (A) and ${\mathrm{O}}_2^{-}$ (B), respectively. Mutant and OE represents fahsp17.8-CII and FaHSP17.8-CII-OE, respectively. Three independent replicates were performed. Scale bars: 500 μm. OE, overexpression.

Figure 7

Quantification analysis of H₂O₂ and O⁻₂. and antioxidant enzyme activities in WT, fahsp17.8-CII and FaHSP17.8-CII-OE leaves under ACC+HS and HS. The accumulation level of H₂O₂ (A) and O⁻₂. (B). The activities of APX (C), CAT (D), SOD (E) and POD (F). Vertical bars indicate means ± s.e. (n = 4). Different letters above the bars indicate significant difference based on the LSD test at P < 0.05. ACC, heat acclimation or priming. HS, heat stress. OE, overexpression. WT, wild type. FM, fresh weight. APX, ascorbate peroxidase. CAT, catalase. SOD, superoxide dismutase. POD, peroxidase. The activities of APX, CAT, SOD and POD were present relative to the soluble protein content (Pro.) in each sample. DAB, 3,3′-diaminobenzidine. NBT, nitroblue tetrazolium.

Figure 8

Transcript levels of ROS/PSII signaling-related genes in WT, fahsp17.8-CII, and FaHSP17.8-CII-OE during thermomemory phase. The sample point was shown in Figure 1, A. Vertical bars indicate means ± se (n = 4). Different letters above the bars indicate significant difference based on the LSD test at P < 0.05.

Figure 9

A proposed model depicting the thermomemory mediated through FaHSP17.8-CII–PSII-ROS work module in tall fescue. When grasses were exposed to heat ACC, the stress memory was acquired. During the temporal profile of stress memory, FaHSP17.8-CII-OE (red) obtained greater PSII function and lower ROS level than WT (blue), but fahsp17.8-CII (golden) showed exactly the reverse effect (A and B), leading to stronger thermotolerance in FaHSP17.8-CII-OE relative to WT (C). OE, overexpression.