A β-ketoacyl carrier protein reductase confers heat tolerance via the regulation of fatty acid biosynthesis and stress signaling in rice

Abstract

Heat stress is a major environmental threat affecting crop growth and productivity. However, the molecular mechanisms associated with plant responses to heat stress are poorly understood. Here, we identified a heat stress-sensitive mutant, hts1, in rice. HTS1 encodes a thylakoid membrane-localized β-ketoacyl carrier protein reductase (KAR) involved in de novo fatty acid biosynthesis. Phylogenetic and bioinformatic analysis showed that HTS1 probably originated from streptophyte algae and is evolutionarily conserved in land plants. Thermostable HTS1 is predominantly expressed in green tissues and strongly induced by heat stress, but is less responsive to salinity, cold and drought treatments. An amino acid substitution at A254T in HTS1 causes a significant decrease in KAR enzymatic activity and, consequently, impairs fatty acid synthesis and lipid metabolism in the hts1 mutant, especially under heat stress. Compared to the wild-type, the hts1 mutant exhibited heat-induced higher H₂ O₂ accumulation, a larger Ca²⁺ influx to mesophyll cells, and more damage to membranes and chloroplasts. Also, disrupted heat stress signaling in the hts1 mutant depresses the transcriptional activation of HsfA2s and the downstream target genes. We suggest that HTS1 is critical for underpinning membrane stability, chloroplast integrity and stress signaling for heat tolerance in rice.

Keywords: Oryza sativa; heat; hydrogen peroxide; lipids; membrane; signaling.

Fig. 1

hts1 is a heat‐sensitive mutant. (a) Phenotypes of 2‐wk‐old rice seedlings before and after heat treatments. Two‐week‐old wild‐type (WT) and hts1 mutant seedlings grown at 28°C were transferred to 45°C for 3 d and then photographed after recovering at 28°C for 2 d. Bars, 20 mm. (b) Survival rates of WT and hts1 mutant seedlings after recovery from heat treatment. Data are means ± SD (n = 3). **, P < 0.01. (c) Heat‐sensitive phenotype in detached leaves of the hts1 mutant in comparison with the WT. (d, e) Visualization of programmed cell death (PCD) (d) and H₂O₂ accumulation (e) using DAB and trypan blue staining of leaves from 2‐wk‐old seedlings grown at 28°C and treated with heat (45°C) for 0 and 48 h.

Fig. 2

Cloning of the HTS1 gene and verification of HTS1 function. (a) Map‐based cloning of the HTS1 gene and a schematic representation of the HTS1 gene structure (bottom). The HTS1 locus was mapped primarily to the long arm of rice (Oryza sativa) chromosome 4 (Chr. 4) between markers yp398 and yp300. It was subsequently narrowed to an 80.2 kb region between yp430 and yp698 within the bacterial artificial chromosome clone AL607003 using 1370 homozygous mutant plants. The predicted ORFs in this region are highlighted with arrows. The HTS1 gene structure, including exons (black boxes), introns (black lines), the UTR (open boxes), ATG start and TGA stop codons, and a single base substitution (G to A) in hts1 are indicated. (b) The presence of normal transcripts of HTS1 in transgenic line hts1‐Com (hts1 with complemented expression of HTS1) was confirmed by PCR followed by restriction enzyme digestion. A 1290‐bp DNA fragment around the mutation is amplified with the specific primers (yp2243‐F and R) and then digested with the restriction enzyme MfeI. An MfeI restriction site (underlined) was abolished by the G‐to‐A substitution (red box) in the mutant sequence. (c) Iamges of 2‐wk‐old plants of the wild‐type, hts1 and hts1‐Com lines (Com‐1 and Com‐2) grown at 28°C and after 72 h of treatment at 45°C and 2 d recovery at 28°C. (d) Survival rates of the wild‐type, hts1 and the hts1‐Com lines after exposure to heat treatment (45°C, 72 h). (e) Images of the heat‐challenged detached leaves (left panel) from four different genotypes described in (c). DAB staining of these detached leaves is shown in the right panel. (f) MDA contents of the wild‐type, hts1 and hts1‐Com plants before and after exposure to heat treatment (45°C, 48 h). Data are means ± SD (n = 3). *, P < 0.05; **, P < 0.01.

Fig. 3

Phylogenetic trees and conserved domain of HTS1 proteins in plants and algae. (a) Conserved domain of HTS1 was predicted in the NCBI database (https://www.ncbi.nlm.nih.gov/cdd). (b) Phylogenetic trees of HTS1 proteins in representative species of major lineage of plants and algae. The maximum‐likelihood method was used to construct the trees. Clades are indicated by different colors. (c) Conserved domain alignment of HTS1s. KR, ketoreductase. Os, Oryza sativa; Pe, Picea engelmanii; Ac, Azolla caroliniana; Sm, Selaginella moellendorffii; Pp, Physcomitrella patens; Mp, Marchantia polymorpha; Kf, Klebsormidium flaccidum; Vc, Volvox carteri; Cm, Cyanidioschyzon merolae.

Fig. 4

HTS1 is a thylakoid membrane‐localized protein and is in response to heat stress in rice (Oryza sativa). (a) Confocal imaging showing chloroplast targeting of HTS1. 35S::GFP (upper panel) and 35S::HTS1‐GFP (lower panel) transiently expressed in rice protoplast cells. Left to right, GFP fluorescence, chloroplast autofluorescence, merged images and bright field images. Bars, 5 μm. (b) Suborganelle localization of the HTS1 protein in the chloroplast. Intact chloroplasts were isolated from WT leaves then separated into thylakoid membrane, envelope and stroma fractions. Polyclonal antibodies against D1 (thylakoid marker), RbcL (stroma marker) and Tic110 (envelope marker) were used as markers. (c, d) The mRNA (c) and protein (d) levels of HTS1 in wild‐type leaves after 45°C heat treatment. The protein levels of HTS1 were measured by western blotting. Equal protein loading was confirmed with antiserum against actin.

Fig. 5

The predicted protein structures and enzymatic activity of 3‐oxoacyl‐acyl‐carrier‐protein reductase (HTS1) and HTS1^A254T. (a) Three‐dimensional models of HTS1 and HTS1^A254T predicted by Swiss‐Model. (b) The purification of GST‐tagged HTS1 and mutant proteins from Escherichia coli. (c) Enzyme activity assay of HTS1 in vitro. (d) Western blot of purified HTS1 protein incubated at 25°C (control) and 45°C for the indicated times. Purified Rubisco activase (RCA) protein was used as the control (Ristic et al., 2007). (e) Enzyme activity of purified HTS1 protein incubated at 25°C (control) and 45°C. Data represent means ± SD (n = 3). **, P < 0.01.

Fig. 6

Lipidomic analysis of leaves of wild‐type and hts1 mutant rice (Oryza sativa). (a) Lipid compositions in leaves from 2‐wk‐old wild‐type and hts1 seedlings. Lipid classes are presented as the percentage of the total amount of all detected lipid classes. (b) Relative abundance of different molecular species of glycerolipids (DG and MGDG) in the wild‐type and hts1 mutant. Data represent means ± SD (n = 3). **, P < 0.01; *, P < 0.05. Cer, ceramides; DG, diglyceride; GlcCer, glucosylceramide; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; SQDG, sulfoquinovosyldiacylglycerol; TG, triglyceride.

Fig. 7

Disturbance of HTS1 expression accelerates plasma membrane disruption and chloroplast degradation under heat treatment. (a) Change of cellular ion leakage of the fully expanded leaves from WT, hts1 and hts1‐Com1 plants challenged with heat treatment (45°C) for the indicated time. Data are means ± SD (n = 3). **, P < 0.01. (b) TEM subcellular observation of the plasma membrane of the 2‐wk‐old WT, hts1 and hts1‐Com1 plants treated at 45°C for 0, 24 and 48 h. Red arrows indicate the plasma membrane blebbing in the hts1 mutant. PM, plasma membrane; CW, cell wall; V, vacuole. Bar, 0.2 μm. (c) Confocal images of mesophyll cells of the 2‐wk‐old WT, hts1 and hts1‐Com1 plants treated at 45°C for 0 and 2 h. White arrows indicate disrupted chloroplasts. Bars, 10 μm (×3.0) and 5 μm (×8.0). (d) Quantification of the occurrence of chloroplast disruption (swelling and collapsing) in mesophyll cells after 2 h of heat treatment. Data are means ± SD (n = 5 with 30–50 cells). **, P < 0.01. (e) TEM observation of chloroplast collapse of the 2‐wk‐old WT, hts1 and hts1‐Com1 plants treated at 45°C for 0 and 48 h. Bars, 1 μm.

Fig. 8

Intracellular Ca²⁺ influxes and H₂O₂ production are increased in the hts1 mutant after heat treatment. (a) Transient net Ca²⁺ influxes measured from mesophyll cells of leaves of WT and the hts1 mutant in response to heat treatment. Data are means ± SD (n = 5–8). (b) Heat‐challenged phenotypes of WT and the hts1 mutant as examined with detached leaves with or without 100 μM EGTA supplement. (c) DAB staining of the heat‐challenged detached leaves of WT and hts1 as described in (b). (d) Representative images of H2DCFDA fluorescence from mesophyll cells from leaves of WT and the hts1 mutant measured in the control (28°C) and after 30 and 60 min of heat treatment (45°C). Bars, 15 μm. (e) Corrected total cell fluorescence of H2DCFDA for heat‐induced H₂O₂ production. Data are means ± SD (n = 5 with 30–50 cells). **, P < 0.01.

Fig. 9

Mutation of HTS1 inhibits transcriptional activation of HsfA2 and its target genes in response to heat stress. qRT‐PCR analysis of mRNA levels of HsfA2 (a), its representative target genes, including APX2, GolS1 (b) and HSP genes (c) in leaves from 2‐wk‐old WT and mutant seedlings challenged with heat treatment (45°C) for the indicated times. The actin gene (LOC_Os03g50885) was used as the internal standard. Data represent means ± SD (n = 3).

Fig. 10

A proposed working model of HTS1 in modulating heat tolerance in rice (Oryza sativa). A deficiency of HTS1 directly impairs the biosynthesis of fatty acids in the hts1 mutant. The reduced fatty acid content (mainly C16 : 0 and C18 : 3) disrupts the integrity and stability of cell membrane systems under heat stress, which may cause abnormal heat‐induced reactive oxygen species (ROS) and Ca²⁺ signaling. The mutation in HTS1 also causes a repression of the transcriptional activity of heat stress transcription factor (HSF) and heat stress protein (HSP) networks, leading to an impaired heat stress response (HSR) regulation and uncontrolled heat damage. Red arrows represent increased/decreased expression or content. Dashed arrow indicates multiple steps. Blunt‐ended arrow indicates inhibition.