A plastid-targeted heat shock cognate 70-kDa protein confers osmotic stress tolerance by enhancing ROS scavenging capability

Osmotic stress severely affects plant growth and development, resulting in massive loss of crop quality and quantity worldwide. The 70-kDa heat shock proteins (HSP70s) are highly conserved molecular chaperones that play essential roles in cellular processes including abiotic stress responses. However, whether and how plastid-targeted heat shock cognate 70 kDa protein (cpHSC70-1) participates in plant osmotic stress response remain elusive. Here, we report that the expression of cpHSC70-1 is significantly induced upon osmotic stress treatment. Phenotypic analyses reveal that the plants with cpHSC70-1 deficiency are sensitive to osmotic stress and the plants overexpressing cpHSC70-1 exhibit enhanced tolerance to osmotic stress. Consistently, the expression of the stress-responsive genes is lower in cphsc70-1 mutant but higher in 35S:: cpHSC70-1 lines than that in wild-type plants when challenged with osmotic stress. Further, the cphsc70-1 plants have less APX and SOD activity, and thus more ROS accumulation than the wild type when treated with mannitol, but the opposite is observed in the overexpression lines. Overall, our data reveal that cpHSC70-1 is induced and functions positively in plant response to osmotic stress by promoting the expression of the stress-responsive genes and reducing ROS accumulation.

Introduction

Environmental stresses such as drought, cold, and salinity can alter water availability by changes in solute concentrations (i.e., inorganic cations, sugars, anions, and salts) and impose osmotic stress on plants, which affects cell membrane integrity, photosynthetic capacity, and osmotic regulation, leading to severe restrictions on plant growth and development (Zhao et al., 2020). Osmotic stress reduces the water uptake of plants, bringing about not only ionic stress but also oxidative damages caused by overaccumulated reactive oxygen species (ROS) including hydroperoxide (H₂O₂), superoxide anion $(O_2^{•-})$ , and hydroxyl radicals (•OH) (Wenjing et al., 2020; Zhao et al., 2020; Wang et al., 2021b; Zhang et al., 2021). These ROS can affect proteins, lipids, and nucleic acids if accumulated over a certain threshold, leading to cell damage and death (Yuan et al., 2017; Nadarajah, 2020). To overcome this issue, plants have employed complex antioxidant defense mechanisms to protect plants from osmotic stress-induced oxidative damage by scavenging ROS and maintaining the balance of ROS production (Hasanuzzaman et al., 2021).

The antioxidant defense system includes a series of enzymes such as catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and non-enzymatic antioxidants (α-tocopherol, glutathione, β-carotene, ascorbate). They work in coordination to maintain ROS homeostasis by scavenging stress-induced excess ROS in plant cells (Waszczak et al., 2018; Sánchez-McSweeney et al., 2021). For instance, SOD can disproportionate O^2- to H₂O₂, and then H₂O₂ is further detoxified into H₂O by APX with the assistance of ascorbate in the chloroplast (Uzilday et al., 2014) and thus overexpressing SOD and APX can improve the plant’s tolerance under salt stress (Shafi et al., 2019). CATs, the enzymes directly degrading H₂O₂ without assistance of any reducing equivalent, are also necessary for scavenging ROS in plants under various stressed conditions (Hasanuzzaman et al., 2020). It is reported that the plants with CAT3 mutation accumulate higher H₂O₂ and are hypersensitive to water deprivation, but the plants overexpressing CAT3 have less H₂O₂ and are more tolerant to drought stress compared with the wild type (Zou et al., 2015). Similarly, the disruption of CAT2 causes excess H₂O₂ accumulation and reduces plant tolerance to high salinity (Bueso et al., 2007). Further, several factors have been indicated to regulate antioxidant enzymes in the stressed plants. For example, the zinc finger protein Zat12 involved in plant response to oxidative stress by promoting APX1 expression and Zat12-deficient plants are more sensitive to H₂O₂ application than wild-type plants (Rizhsky et al., 2004). Overexpression of AtbHLH112 increases salt and drought tolerance by promoting SOD activity to improve ROS scavenging ability (Liu et al., 2015). A recent report documented that leucine aminopeptidase 2 (LAP2), as a collaborator of CAT2, confers Arabidopsis plants increased osmotic stress tolerance possibly through maintaining CAT2 protein stability (Zhang et al., 2021). In addition, peroxisome-localized small heat shock protein Hsp17.6CII activates catalase by interacting with CAT2 and thus confers alkaline and salt stress tolerance in plants (Li et al., 2017). MeHSP90.9-silenced plants have repressed CAT1 expression, reduced CAT activity, and higher H₂O₂, resulting in more sensitivity to drought stress (Wei et al., 2020). These reports reveal that modulations of antioxidant enzymes play a vital role in regulating plant abiotic stress tolerance.

Heat shock proteins (HSPs) are a diverse group of multifamily proteins, functioning in adverse stimuli by preventing protein misfolding, reducing the aggregation of denatured proteins, and maintaining protein structural stability (Sable et al., 2018; Qi et al., 2019). Based on their apparent molecular weight, amino acid sequence homology, and functions, HSPs have been classified into HSP100s, HSP90s, HSP70s, HSP60s, and small heat shock protein (sHSP) families (Mayer and Bukau, 2005). Of all HSPs, HSP70 superfamily members are the most abundant, highly conserved, and well-characterized group of molecular chaperones in all organisms from prokaryotes to eukaryotes (Xu et al., 2020; Li et al., 2021). Structurally, HSP70s are identified by three distinct domains: a 45-kDa N-terminal ATPase, a 15-kDa β-sandwich domain, and a 10-kDa C-terminal α-helical domain (Zhu et al., 1996; Tang et al., 2016). Functionally, while HSP70s assist cellular machinery in regulating protein degradation and verifying proteins quality under normal conditions (Bukau et al., 2006; Su and Li, 2008; Hartl et al., 2011), they facilitate denatured protein refold, prevent denatured proteins from aggregating, and dissolve or degrade protein aggregates during stress (Wang et al., 2004; Lee and Tsai, 2005). In Arabidopsis, 18 HSP70s have been identified, and they are divided into four subclasses based on their subcellular localization: cytosol/nucleus, mitochondria, endoplasmic reticulum (ER), and plastids (Wu et al., 1994; Lin et al., 2001). The cytosolic/nuclear HSP70s mainly function in plant development, signaling pathways, abiotic stresses including drought, salinity, and high temperature, and biotic stresses such as virus infection (Jungkunz et al., 2011; Leng et al., 2017). The mitochondria-localized mtHSC70-1 and mtHSC70-2 are required for the mitochondrial Fe–S cluster assembly and aid in the translocation of precursor proteins to mitochondria as part of the translocon (Zhang and Glaser, 2002; Leaden et al., 2014). Early reports showed that overexpression of the rice (Oryza sativa) mtHSC70 inhibited heat- and H₂O₂-induced cell death in protoplasts through reducing reactive oxygen species (ROS) generation and sustaining mitochondrial membrane potential (Qi et al., 2011). A recent study showed that mtHSC70-1 mutation resulted in severe embryo defects (Li et al., 2021). As ER-localized HSP70s, immunoglobulin-binding proteins (BiPs) play an important role in male and female gametophyte development and unfolded protein responses (Srivastava et al., 2013; Maruyama et al., 2014). For plastid-localized HSP70s in Arabidopsis, two cpHSC70s (cpHSC70-1 and cpHSC70-2) are identified to be essential for maintaining chloroplast structure and functions (Sung et al., 2001; Latijnhouwers et al., 2010). While cphsc70-1 mutant plants exhibit variegated cotyledons, slow growth, deformed leaves, and impaired root growth under normal growth conditions, the stressed cphsc70-1 mutant plants are more sensitive to high temperature and drought stress (Su and Li, 2008; Latijnhouwers et al., 2010). However, whether and how cpHSC70-1 participates in plant response to osmotic stress remains unknown.

In this study, we report that cpHSC70-1 plays important roles in plant tolerance to osmotic stress. When challenged with osmotic stress, the expression of cpHSC70-1 is promoted in plants. Moreover, the knockout of cpHSC70-1 has enhanced sensitivity to osmotic stress with lower APX and SOD activities and increased ROS accumulation. Overexpression of cpHSC70-1 in wild type improves tolerance of transgenic Arabidopsis to osmotic stress, with a higher expression of genes encoding antioxidant enzymes and decreased ROS accumulation. Taken together, these results show that osmotic stress-induced cpHSC70-1 functions necessarily in the stress tolerance by modulating ROS scavenging capacity in Arabidopsis.

Materials and methods

Plant materials and growth conditions

The Columbia-0 (Col-0) Arabidopsis thaliana ecotype was employed in the present study. The mutants cphsc70-1 (Salk_140810) and cphsc70-2 (Salk_095715) were previously reported (Su and Li, 2008). Arabidopsis seeds were surface sterilized with 5% (w/v) bleach for 5 min, rinsed three times with sterile water, stored to 4°C for 3 days, and then grown on 1/2 strength MS (Murashige and Skoog) medium (pH 5.8) containing 1% (w/v) agar and 1% (w/v) sucrose, and plants were grown at 23°C with 16-h light (100 μmol m^–2 s^–1 illumination)/8-h dark conditions. For osmotic stress treatment, the corresponding seedlings were planted on 1/2 MS medium supplemented with or without 300 mM mannitol (Beijing Dingguo Changsheng Biotechnology Co., Ltd., DH190-2) for 5 days, and then the fresh weight and root length were determined and analyzed.

Plasmid construction and plant transformation

The full-length coding sequences (CDS) of cpHSC70-1 (AT4G24280), cpHSC70-2 (AT5G49910), and the promoter (2 kb) of cpHSC70-1 were amplified using PCR. The resulting fragments (cpHSC70-1 and cpHSC70-2) were cloned into the pCAMBIA1300S vector and verified by sequencing. cpHSC70-1 was also cloned into pCAMBIA1300 driven by the cpHSC70-1 promoter. The resultant plasmids were transformed into Col-0 or the homozygous cphsc70-1 mutant using Agrobacterium tumefaciens strain pGV3101 and the floral dip method. The primer sequences are listed in Supplemental Table 1.

RNA extraction and quantitative reverse transcription PCR

The RNA was extracted as previously described (Ding et al., 2022). Briefly, plants were thoroughly ground with liquid nitrogen, and TRIzol reagent (Invitrogen) was used to extract the total RNA by following the manufacturer’s instructions, and RQ1 RNase-free DNase I (Promega) was employed to remove the contaminated DNA. RNA reverse transcription was performed with ReverTra Ace kit (TOYOBO) according to the manufacturer’s instructions. qPCR was performed using a Bio-Rad CFX96 and SYBR Green I dye (Invitrogen) with the program of 95°C for 3 min, 35 cycles of 95°C for 10 s, and 60°C for 45 s, followed by 5 min incubation at 95°C. ACTIN2 (AT3G18780) was used as the reference gene, and all experiments included three independent biological replicates and three technical repetitions. Primer sequences are shown in Supplemental Table 1.

Seed germination and cotyledon expansion statistics

Germination was determined in the same way as before (Ding et al., 2022). Briefly, the seeds were planted on 1/2 MS medium with or without 300 mM mannitol. The radicle’s appearance served as a gauge for seed germination. The percentages of germinated seeds were counted at the specified times. For each seed germination experiment, at least 60 seeds of each genotype were used, and experiments were conducted three times. On the fifth day, the percentages of cotyledon expansion per plant was scored.

3,3-Diaminobenzidine and nitroblue tetrazolium staining

As detailed earlier (Yu et al., 2019; Luo et al., 2021), the 7-day-old seedlings grown on 1/2 MS medium were transferred to mannitol (300 mM) containing 1/2 MS medium for 48 h, then the seedlings were stained with 3,3-diaminobenzidine (DAB) or nitroblue tetrazolium (NBT) to determine H₂O₂ or superoxide anion accumulation. For DAB staining, seedlings were incubated for 8 h in freshly prepared DAB staining solution [1 mg/ml DAB (Beijing Dingguo Changsheng Biotechnology Co., Ltd., JD091) which was dissolved in 10 mM Na₂HPO₄ supplemented with 0.1% (v/v) Tween-20] and then washed with 70% ethanol to remove chlorophyll. The leaves were analyzed and photographed using a Nikon microscope (SMZ25; Nikon). The relative levels of DAB staining were quantitatively analyzed by using Photoshop CS6 software (Adobe). For superoxide anion labeling, seedlings were vacuum infiltrated with 0.1 mg/ml NBT (Sigma-Aldrich, N6876) in 25 mM HEPES buffer (pH 7.6) for 2 h in the dark. Seventy percent ethanol was used to remove chlorophyll from the leaves, then these leaves were imaged using a differential interference contrast (DIC) optical system (BX64; Olympus) and a charge-coupled device (CCD) camera (DP72; Olympus).

Detection of H₂O₂ and $O_2^{•-}$ contents

The POD-coupled assay was utilized to quantify the H₂O₂ levels as previously reported (Yuan et al., 2017). First, 2 ml of HClO₄ (1 M) containing insoluble polyvinylpyrrolidone (5%) was used to extract 0.2 g of the Arabidopsis seedlings. The homogenate was centrifuged at 12,000 g for 10 min, and the supernatant was neutralized to pH 5.6 with 5 M K₂CO₃ to pH 5.6 in the presence of 100 μl of 0.3 M phosphate buffer, pH 5.6. The solution was centrifuged for 1 min at 12,000 g, and the supernatant was then mixed with 1 unit of ascorbate oxidase and left to stand for 10 min at 25°C. 3-(Dimethylamino)benzoic acid (3.3 mM), 0.07 mM 3-methyl-2-benzothiazoline hydrazone, and 0.1 M phosphate buffer (pH 6.5) were added to 500 μl of the reaction mixture to start the final reaction. After standing for 30 min at 25°C, the 590-nm absorbance change in the solution was determined. A superoxide anion content detection kit (Beijing Solarbio Science and Technology Co., Ltd., BC1290) was used to measure the $O_2^{•-}$ contents according to the provided instructions.

Determination of catalase, ascorbate peroxidase, and superoxide dismutase activities

Total CAT, APX, and SOD activities were analyzed using a Catalase Assay Kit (Beyotime Biotechnology), Ascorbate Peroxidase Assay Kit (Gelatins), and a Total Superoxide Dismutase Assay Kit with NBT (Beyotime Biotechnology), respectively, following supplied protocols. The CAT activity was assayed based on decreases in H₂O₂ accumulation. The APX activity was measured by calculating the AsA oxidation rate. The SOD activity was detected by NBT photoreduction inhibition.

Western blot analysis

Total proteins were extracted using the plant protein extracting buffer containing 375 mM NaCl, 2.5 mM EDTA, 1% β-mercaptoethanol, 125 mM Tris–HCl (pH8.0), and 1% SDS. Protein concentrations were assayed using a bicinchoninic acid assay (BCA) protein assay kit (Beijing Dingguo Changsheng Biotechnology Co., Ltd.). Afterward, the proteins were separated by electrophoresis using 12% SDS-PAGE gel. Immunoblotting was performed on PVDF membranes with anti-HSC70 (Agrisera, AS08348) and anti-ACTIN (Abmart, M20009M). The intensity of each immunodetection band was determined using an image-processing and analysis software package (ImageJ, version 1.52v). Relative protein levels were normalized against those in control, which were set to 1.

Results

Osmotic stress promotes the expression of cpHSC70-1

HSP70s play key roles in ensuring cellular homeostasis, whether cells are in normal or stressful environments (Leng et al., 2017). Although their family members, cpHSC70s, have been reported to be involved in some biotic stresses such as heat and drought (Su and Li, 2008; Latijnhouwers et al., 2010), the function of cpHSC70s in osmotic stress remains unknown. To investigate whether cpHSC70s are involved in plants osmotic stress response, we examined whether mannitol treatment affects the expression of cpHSC70-1 and cpHSC70-2. Our reverse transcription PCR (RT-qPCR) analyses showed that osmotic stress significantly induced the expression of cpHSC70-1, but not cpHSC70-2 (Figure 1A). Moreover, we measured cpHSC70 proteins in mannitol-treated wild-type plants; our Western blot analyses indicated that the mannitol-treated wild-type seedlings had higher cpHSC70 accumulation than untreated control (Figure 1B). Thus, our data suggest that osmotic stress promotes cpHSC70-1 expression in the stressed plant.

FIGURE 1

Figure 1 Osmotic stress induces cpHSC70-1 expression. (A) The cpHSC70-1 and cpHSC70-2 expression levels of 5-day-old wild-type seedlings subjected to osmotic stress (300 mM mannitol for 6, 12 h) and its control. The data are means ( ± SEM) from at least three independent experiments. Asterisks indicate significant differences revealed using a Student’s t-test (***p < 0.001). (B) cpHSC70 protein level of 5-day-old wild-type seedlings treated with 300 mM mannitol for 0, 12, and 24 h. The intensity of each immunodetection band was measured by using an image-processing and analysis software package (ImageJ). The protein levels of 0 h were set to 1. Actin was used as control.

The cpHSC70-1 functions positively in plant response to osmotic stress

The induction of cpHSC70-1 expression by osmotic stress as shown in our above results implies a possible role of this gene in plant response to osmotic stress. To verify this possibility, we obtained the mutant cphsc70-1 (SALK_140810), in which T-DNA was inserted in the second intron of cpHSC70-1 and cpHSC70-1 expression was significantly reduced in the mutant (Supplementary Figures 1A, B) and generated the complementation lines (cpHSC70-1::cpHSC70-1 cphsc70-1) by expressing the full-length coding sequence of cpHSC70-1 driven under its native promoter in cphsc70-1 mutant (Supplementary Figure 1B). Then, we examined the phenotypes of the mutant and its complementation lines under osmotic stress. We found that when grown under normal conditions, cphsc70-1 exhibited variegated cotyledons, malformed leaves, small size, and impaired root growth compared with the wild type as previously reported (Su and Li, 2008; Chu et al., 2020), but cphsc70-1 plants showed a similar germination rate and cotyledon expansion rate as the wild type had (Figures 2A-C). When challenged with osmotic stress, the cphsc70-1 seedlings had a reduced germination rate and cotyledon expansion rate than wild-type seedlings (Figures 2B, C). In addition, the fresh weight and primary root elongation were further repressed by osmotic stress in the mutant compared with the wild type (Figures 2D, H). Regarding the cpHSC70-1::cpHSC70-1 cphsc70-1 plants, they behaved as the wild type under either normal or stressed conditions (Figures 2A-H). Taken together, our results indicate that cpHSC70-1 functions positively in plant response to osmotic stress.

FIGURE 2

Figure 2 Loss-of-function mutation of cpHSC70-1 increases plant sensitivity to osmotic stress. (A) Images of 5-day-old wild-type, cphsc70-1, and cpHSC70-1::cpHSC70-1 cphsc70-1 seedlings grown on 1/2 MS medium with or without (Mock) 300 mM mannitol. Bars = 1 cm. (B) Germination rate of the wild-type, cphsc70-1, and cpHSC70-1::cpHSC70-1 cphsc70-1 plants in response to 300 mM mannitol. (C) Rate of cotyledon expansion, (D) fresh weight, and (E) relative fresh weight of the wild-type, cphsc70-1, and cpHSC70-1::cpHSC70-1 cphsc70-1 plants in panel (A). (F) Images of 5-day-old wild-type, cphsc70-1, and cpHSC70-1::cpHSC70-1 cphsc70-1 seedlings grown on 1/2 MS medium with or without (Mock) 300 mM mannitol. Bars = 1 cm. (G) Root length and (H) relative root length of the plants shown in (F). The data are means ( ± SD) from at least three independent experiments (n ≥60 for germination rate and n ≥30 for root length). Asterisks indicate significant differences revealed using a Student’s t-test (*p < 0.05; **p < 0.01; ***p< 0.001). Different letters indicate significant differences as determined using ANOVA followed by Tukey’s test (P < 0.05).

Overexpression of cpHSC70-1 enhances plant tolerance to osmotic stress

Further, we tested whether overexpression of this gene can confer plants more tolerance to osmotic stress. For this end, we generated transgenic lines 35S::cpHSC70-1 by overexpressing cpHSC70-1 under the control of the 35S promoter in the Col-0 background Arabidopsis. RT-qPCR results showed that the cpHSC70-1 expression in the overexpression lines was higher than that in the wild type (Supplementary Figure 1B). While 35S::cpHSC70-1 plants showed no aberrant phenotype compared to the wild type under normal growth conditions (Figure 3), these seedlings had a higher germination rate, promoted the cotyledon expansion rate, and increased fresh weight and longer primary root length than the wild-type seedlings (Figures 3A-F). These findings reveal that overexpression of cpHSC70-1 enhances osmotic stress tolerance in the transgenic plants.

FIGURE 3

Figure 3 Overexpression of cpHSC70-1 enhanced osmotic stress tolerance. (A) Images of 5-day-old wild-type and 35S::cpHSC70-1 seedlings grown on 1/2 MS medium with or without (Mock) 300 mM mannitol. Bars = 1 cm. (B) Germination rate of the wild-type and 35S::cpHSC70-1 plants in response to 300 mM mannitol. (C) Rate of cotyledon expansion and (D) fresh weight of wild-type and 35S::cpHSC70-1 plants in panel (A). (E) Images of 5-day-old wild-type and 35S::cpHSC70-1 seedlings grown on 1/2 MS medium with or without (Mock) 300 mM mannitol. Bars = 1 cm. (F) Root length of the plants shown in (E). The data are means ( ± SD) from at least three independent experiments (n ≥60 for germination rate and n ≥ 30 for root length). Asterisks indicate significant differences revealed using a Student’s t-test (* p < 0.05; **p < 0.01; ***p < 0.001). Different letters indicate significant differences as determined using ANOVA followed by Tukey’s test (p < 0.05).

cpHSC70-1 regulates the expression of the genes involved in plant stress response

Previous studies reported that various abiotic stresses including high salinity and osmotic stress modulate the expression of stress-responsive genes (Wang et al., 2021a; Ding et al., 2022). To explore the impact of cpHSC70-1 on the molecular basis of osmotic stress response, we detected the transcripts of several key genes involved in osmotic stress (RD29A, KIN1, COR15A, and P5CS1) (Wang et al., 2021b). We found that the transcripts of all tested genes were similar in cphsc70-1, 35S::cpHSC70-1, cpHSC70-1::cpHSC70-1 cphsc70-1, and wild-type plants under normal conditions (Figures 4A-D). However, upon osmotic stress, phsc70-1 plants suppressed but 35S::cpHSC70-1 plants promoted the expression of these genes compared with both wild-type and cpHSC70-1::cpHSC70-1 cphsc70-1 plants (Figures 4A-D). These results suggest that the expression of the stress-responsive genes could be involved in cpHSC70-1-mediated plant osmotic stress response.

FIGURE 4

Figure 4 Effects of cpHSC70-1 on expression of genes involved in stress response and ROS-scavenging system. (A–G) The expression levels of (A) RD29A, (B) COR15A, (C) P5CS1, (D) KIN1, (E) sAPX, (F) tAPX, and (G) CSD2 of the 5-day-old wild-type, cphsc70-1, cpHSC70-1::cpHSC70-1 cphsc70-1, and 35S::cpHSC70-1 plants treated with or without (Mock) 300 mM mannitol for 12 h. The expression of these genes was determined by RT-qPCR and correlated with that of the WT, the value of which was set as 1. ACTIN2 was used as the reference gene. The data are means ( ± SEM) from at least three independent experiments. Different letters indicate significant differences as determined using ANOVA followed by Tukey’s test (p < 0.05).

The cpHSC70-1 regulates ROS homeostasis of plants under osmotic stress

It is reported that ROS is involved in plant response to osmotic stress (Foyer and Noctor, 2005); (Koussevitzky et al., 2008; Vishwakarma et al., 2015; Li et al., 2019; Wang et al., 2021a). Thus, we also tested the expression of genes involved in the ROS-scavenging system (CAT1, CAT2, CAT3; APX1, sAPX, tAPX; CSD1, CSD2, CSD3) in cphsc70-1 and 35S::cpHSC70-1 plants under osmotic stress. Our results showed that while cphsc70-1, 35S::cpHSC70-1, cpHSC70-1::cpHSC70-1 cphsc70-1, and wild-type plants had similar expressions of CAT1, CAT2, CAT3, APX1, CSD1, and CSD3, the cphsc70-1 mutant reduced but cpHSC70-1 overexpression lines increased the expression of sAPX, tAPX, and CSD2 compared with cpHSC70-1::cpHSC70-1 cphsc70-1 and wild-type plants when subjected with mannitol treatment (Figures 4E-G; Supplementary Figures 2A-F). Consistently, all tested plants had similar CAT activity when grown under both normal and stressful conditions; however, osmotic stress-promoted activities of APX and SOD in both cpHSC70-1::cpHSC70-1 cphsc70-1 and wild-type plants were repressed in cphsc70-1 mutant but enhanced in 35S::cpHSC70-1 plants (Figures 5A-C; Supplementary Figures 3A-C). These results suggest the possible involvement of ROS homeostasis in cpHSC70-1-mediated plant osmotic stress response. Therefore, we examined the contents of H₂O₂ in plant response to osmotic stress by using both 3,3-diaminobenzidine (DAB) staining experiments and the POD-coupled assay. Our results showed that while the stress elevated H₂O₂ levels in the stressed cphsc70-1, 35S::cpHSC70-1, cpHSC70-1::cpHSC70-1 cphsc70-1 and wild-type plants compared with their untreated control, respectively, H₂O₂ accumulation was higher in cphsc70-1 but lower in 35S::cpHSC70-1 than that in cpHSC70-1::cpHSC70-1 cphsc70-1 and the wild type upon osmotic stress treatment (Figures 5D, E; Supplementary Figures 3D, E). Similar results were found when we assayed the abundance of the superoxide anion $(O_2^{•-})$ using NBT staining and spectrophotometry (Figures 5F, G; Supplementary Figures 3F, G). These findings indicate that cpHSC70-1 regulates plant ROS homeostasis under osmotic stress.

FIGURE 5

Figure 5 The cphsc70-1 mutant has higher ROS accumulation and reduced APX and SOD activity. (A) Catalase activity, (B) APX activity, and (C) SOD activity of the 5-day-old wild-type, cphsc70-1, and cpHSC70-1::cpHSC70-1 cphsc70-1 plants treated with or without (Mock) 300 mM mannitol for 2 days. (D) The DAB-staining images of leaves from the 5-day-old wild-type, cphsc70-1, and cpHSC70-1::cpHSC70-1 cphsc70-1 plants treated with or without (Mock) 300 mM mannitol for 2 days. (E) Measurements of H₂O₂ in the 5-day-old wild-type, cphsc70-1, and cpHSC70-1::cpHSC70-1 cphsc70-1 plants treated with or without (Mock) 300 mM mannitol for 2 days. (F) The NBT-staining images of leaves from the 5-day-old wild-type, cphsc70-1, and cpHSC70-1::cpHSC70-1 cphsc70-1 plants treated with or without (Mock) 300 mM mannitol for 2 days. (G) Measurements of $O_2^{•-}$ in the 5-day-old wild-type, cphsc70-1, and cpHSC70-1::cpHSC70-1 cphsc70-1 plants treated with or without (Mock) 300 mM mannitol for 2 days. The data are means ( ± SD) from at least three independent experiments. Different letters indicate significant differences as determined using ANOVA followed by Tukey’s test (p < 0.05).

Discussion

HSP70 proteins are widespread and play key roles in organisms ranging from prokaryotes to land plants. They have been mainly reported in heat shock responses, protein folding and translocation, and prevention of protein aggregation (Miemyk, 1997). However, whether the chloroplast-located cpHSC70-1 participates in plant response to osmotic stress remains unknown. Here, we provide evidence that cpHSC70-1 is required for plant tolerance to osmotic stress because cphsc70-1 plants have higher sensitivity to osmotic stress and 35S::cpHSC70-1 plants exhibit higher osmotic stress tolerance.

It has been documented that there are two chloroplast-localized HSPs (cpHSC70-1 and cpHSC70-2) in Arabidopsis. These two proteins are structurally homologous based on their protein sequence alignment and play important roles in chloroplast development and functional integrity (Sung et al., 2001; Latijnhouwers et al., 2010). In this study, we found that osmotic stress induces the expression of cpHSC70-1, but the cpHSC70-2 expression is not affected by the stress (Figure 1). Thus, we also examined whether cpHSC70-2 is involved in plant response to osmotic stress. We obtained the mutant cphsc70-2 (SALK_095715), in which T-DNA was inserted in the second intron of cpHSC70-2 and cpHSC70-2 expression was significantly reduced in the mutant (Supplementary Figures 1C, D), and we assayed the stress response of this mutant under osmotic stress. We found that cphsc70-2 and wild-type plants had no substantial distinction in terms of germination rate, cotyledon expansion rate, fresh weight, and primary root elongation under either normal or osmotic-stressed conditions (Supplementary Figure 4). Further, the transgenic 35S::cpHSC70-2 lines, in which pHSC70-2 is overexpressed under the control of the 35S promoter (Supplementary Figure 1D), does not show higher stress tolerance compared with the wild type (Supplementary Figure 4). Thus, our data do not support the involvement of cpHSC70-2 in plant response to osmotic stress. Similar observations were also reported when the role of cpHSC70-1/2 was examined in plant response to drought and heat stresses (Su and Li, 2008; Latijnhouwers et al., 2010). These studies indicate that cpHSC70-1 acts positively in plant response to these stresses, but cpHSC70-2 is not required for plant tolerance to these stresses.

Many studies indicate that various abiotic stresses induce the expression of stress-responsive genes (Zhang et al., 2019; Fu et al., 2021; Ding et al., 2022). There have been various reports that hypersensitive mutants have increased the induction of stress-inducible genes. For example, the salt stress-induced expression of COR15A and ABCG6 is enhanced in salt stress-sensitive glyI2 mutants (Fu et al., 2021). Similarly, dpg1 mutants have higher expressions of RD29A and RD29B than the wild type under high salinity (Yi et al., 2019), and gcn20 mutants are more sensitive to salt stress with higher expressions of KIN1, KIN2, and COR15A (Ding et al., 2022). However, it is widely documented that hypersensitive mutants have a decreased induction of stress-inducible genes. For instance, cand2-1 mutants are hypersensitive to osmotic stress with decreased induction of RD29A, KIN1, P5CS1, and COR15A (Wang et al., 2021b). Also, mutation of DCD results in the inhibition of cadmium stress-induced PCR1 and PDR8 (Zhang et al., 2020). A recent report documents that expression levels of COR15A, COR47, and RD29A are significantly lower in rboh-D, rboh-F, and rboh-DF mutants than in the WT when treated with cold, and these mutants are less tolerant to cold stress (Liu et al., 2022a). In addition, salt and drought stress-induced RD29A and RD22 are significantly higher in the amiR-TSB1 mutants than in the wild type when the mutants show higher tolerances to drought and salt stress than the wild type (Liu et al., 2022b). Our experimental results indicate that osmotic stress promoted the expression of stress-responsive genes such as RD29A, COR15A, P5CS1, and KIN1 in wild-type plants which are repressed in cphsc70-1 mutant but enhanced in 35S::cpHSC70-1 plants. However, how cpHSC70-1 modulates the expression of these genes is unclear. ABA is a central phytohormone regulating plant responses to osmotic stress (Tivendale et al., 2014; Julkowska and Testerink, 2015). Many ABA-inducible genes such as RD29A, KIN1, and COR15A function in various abiotic stresses including osmotic stress (Ding et al., 2022). We speculate that cpHSC70-1 may be involved in regulating the expression of these stress-responsive genes by affecting ABA accumulation or ABA signaling, which is worthy of further exploration. Additionally, it has been documented that cpHSC70-1 can interact with GENOMES UNCOUPLED1 (GUN1), a protein that participates in multiple retrograde signaling pathways, regulating the expression of many nuclear-encoded genes (Wu et al., 2019). Thus, investigating whether GUN1 functions in cpHSC70-1-mediated plant osmotic stress response by modulating the expression of nuclear-encoded stress-responsive genes could be a future research direction.

Various environmental stresses including osmotic stress, result in oxidative damages caused by overaccumulated reactive oxygen species (ROS) (Wenjing et al., 2020; Zhao et al., 2020; Wang et al., 2021b; Zhang et al., 2021). These ROS can affect proteins, lipids, and nucleic acids if accumulated over a certain threshold, leading to cell damage and death (Yuan et al., 2017; Nadarajah, 2020). Thus, reducing stress-induced ROS overaccumulation is one of the most important and common protective mechanisms for plants under an adverse environment. Our study shows that the knockout of cpHSC70-1 has decreased APX and SOD activities and increased ROS accumulation, and overexpression of cpHSC70-1 in wild-type lines has higher ROS detoxification capacity and less ROS. It is known that a system of posttranslational protein transport into the chloroplast is absolutely essential for its functions such as photosynthesis, and cpHSC70-1 is a motor for protein import into the chloroplast and its mutation significantly reduces the efficiency of protein import (Su and Li, 2010; Li et al., 2020). In animals, HSP70 modulates SOD2 activity by promoting the import of SOD2 into the mitochondria (Zemanovic et al., 2018). We also notice that the mutation of cpHSC70-1 reduces activities of APX and SOD, but not CAT, under osmotic stress. It is possible that cpHSC70-1 may regulate the import of sAPX, tAPX, and CSD2 into the chloroplast, but not CAT1, CAT2, and CAT3, into the peroxisome, resulting in changes in the activities of APX and SOD under osmotic stress. Chloroplasts are thought to be the main source and target of ROS (Waszczak et al., 2018); thus, we speculate that when plants are subjected to osmotic stress, this impaired import of sAPX, tAPX, and CSD2 into the chloroplast in cphsc70-1 mutants, resulting in reduced activities of APX and SOD, which in turn causes ROS accumulation. Further experiments are needed to verify this hypothesis.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Author contributions

FD and BZ conceived and designed the project. FD and FL performed the experiments. FD and BZ analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.

Acknowledgments

We thank Prof. Ying-Tang Lu (Wuhan University, China) for providing guidance and valuable advice.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2022.1012145/full#supplementary-material

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