AtHSPR may function in salt-induced cell death and ER stress in Arabidopsis

ABSTRACT

Salt stress is a harmful and global abiotic stress to plants and has an adverse effect on all physiological processes of plants. Recently, we cloned and identified a novel AtHSPR (Arabidopsis thaliana Heat Shock Protein Related), which encodes a nuclear-localized protein with ATPase activity, participates in salt and drought tolerance in Arabidopsis. Transcript profiling analysis revealed a differential expression of genes involved in accumulation of reactive oxygen species (ROS), abscisic acid (ABA) signaling, stress response and photosynthesis between athspr mutant and WT under salt stress. Here, we provide further analysis of the data showing the regulation of salt-induced cell death and endoplasmic reticulum (ER) stress response in Arabidopsis and propose a hypothetical model for the role of AtHSPR in the regulation of the salt tolerance in Arabidopsis.

Salt stress limits crop growth since high sodium ions in soil or irrigation water are toxic to plants. Under salt stress, the plant can adapt itself to high salinity via toxic ion exclusion, compartmentalization, long-distance transport, the synthesis of molecular chaperones like Heat Shock Proteins (HSPs) and the expression of stress response genes. Previous studies have revealed that the evolutionarily conserved Salt Overly Sensitive (SOS) pathway plays a key role in regulating Na⁺/K⁺ homeostasis during salt stress.^1,2 Two calcium sensors SOS3 and SCaBP8 (SOS3-like Calcium Binding Protein8) sense an increased cytosolic calcium signal induced by salt stress and interact with and activate SOS2, a serine/threonine protein kinase. Subsequently, the SOS3/SCaBP8-SOS2 protein kinase complex then phosphorylates and activates the SOS1, a plasma membrane-localized Na⁺/H⁺ antiporter which transport the Na⁺ out of plant cells.^1,3,4 Salt stress induces unfolded protein response (UPR) in plants.^5,6 Then, excessively accumulated unfolded proteins may cause ER stress.⁷ Many previous studies have demonstrated that ER stress and UPR are involved in plant responses to environmental stresses.^8-11 AtHSPR (Arabidopsis thaliana Heat Shock Protein Related) is a member of a new SMXL(SUPPRESSOR OF MORE AXILLARY GROWTH2-LIKE) family consisting of eight 8 genes that are closely related to HSP101, also called SMXL4 in the Arabidopsis Columbia background.^12,13 Recently, SMAX1 [SUPPRESSOR OF MORE AXILLARY GROWTH2(MAX2) 1]¹³ has been demonstrated to suppress the seed germination and seedling growth phenotypes of max2. MAX2 has been reported to play a role in Strigolactone (SL) responses.^14,15 Previous data showing that MAX2 plays a role in regulating the drought/salt stress response, but it is not yet clear if SLs regulate drought/ salt tolerance. Ha et al. showed that SL-deficient mutants max3-11 and max4-7, as well as SL-signaling max2-3 mutant are sensitive to drought and salt stress, and exogenous SLs application rescues drought sensitivity of SL-deficient max3-11 and max4-7 mutants and enhances the drought tolerance of wild-type, suggesting that SLs positively regulates drought and high salinity responses in Arabidopsis.^15,16 However, Bu et al. reported contradictory data that SL-biosynthetic max1, max3 and max4 mutants did not display sensitive phenotype to drought stress, suggesting that SLs are not involved in the drought response.¹⁷ Recent studies have demonstrated that three rice D53 (DWARF53)-like proteins, SMXL6, SMXL7 and SMXL8 regulate shoot branching and leaf development through distinct MAX2-dependent responses to SLs in Arabidopsis.^18,19 However, the roles of AtHSPR/SMXL4 and other SMXL in plant stress tolerance are still unknown. More recently, we report that AtHSPR/SMXL4 as a positive regulator enhanced salt tolerance and drought resistance in transgenic Arabidopsis through modulation of ROS levels, ABA-dependent stomatal closure, photosynthesis and K⁺/Na⁺ homeostasis.¹² In this study, we further show a potential role of AtHSPR in salt-induced cell death and ER stress signaling in Arabidopsis.

T-DNA-inserted athspr mutant not only exhibit reduced organs in size, darker green rosette leaves and delayed flowering time,¹² but also show slight cell death in leaf apex under normal soil growth condition compared with WT (Fig. 1A). DAB staining (for assessing the level of H₂O₂ accumulation) and Evan's Blue staining (for assessing the extent of cell death) were also observed in the leaf tip of the athspr mutant but not in the WT, AtHSPR complementation (COM) and overexpression (OE) lines (Fig. 1A). Meanwhile, the transcriptome data from our recent publication¹² indicated several genes involved in both regulations of cell death and aging were highly expressed in the athspr mutant under both normal and salt conditions (Fig. 1B). For example, the senescence-related gene SRG2 (SENESCENCE-RELATED GENE 2) and senescence-activated genes, including SAG13 and SAG29 (SENESCENCE-ASSOCIATED GENE 13/29), and seven MAPKs genes, were up-regulated in salt-treated athspr plants than those in salt-treated WT plants. qRT-PCR assays also indicated that the expression level of senescence-associated genes SAG13 is largely increased in athspr mutant compared with wild-type plants during both 3 and 6 h after 200 mM NaCl treatment (Fig. 1D). These results were consistent with the evidence that cell death was dramatically higher in the athspr mutant after salt treatment compared with the salt-treated WT plants.¹²

Figure 1.

Disruption of AtHSPR induced cell death, H₂O₂ accumulation in the leaf tip, and up-regulated expression of genes involved in cell death and ER stress response under both normal and salt conditions. (A). Visible cell death in the leaf tip of athspr mutant. The DAB staining or Evan's Blue staining was used for assessing H₂O₂ accumulation or cell death in WT, athspr mutant, complementation (COM) and overexpression (OE) lines, respectively. (B-C). Hierarchical clustering of the cell death genes or ER stress genes in four different pair-wise comparisons, which include: WT-s/WT (NaCl-treated WT relative to WT), athspr/WT (athspr relative to WT), athspr-s/athspr (NaCl-treated athspr relative to athspr) and athspr-s/WT-s (NaCl-treated athspr relative to NaCl-treated WT). The bottom bar shows the color scale for regulation ratio (log₂) relative to the control samples. (D-F). The relative expression of senescence-associated genes SAG13 and ER stress chaperone genes BIP1 and CNX1 in WT, athspr mutant and OE seedlings in response to NaCl treatment by qRT-PCR. Plants were grown for 2 weeks, and then treated with medium containing 200 mM NaCl for different time points. Error bars show standard deviations from three independent RNA extractions. Statistical significance was determined by a Duncan's multiple range test; significant differences (P = 0.05) are indicated by different lower-case letters.

Also upregulated genes that respond to unfolded / misfolded proteins or to ER stress were found in comparison of salt-treated athspr plants with salt-treated WT(athspr-s/WT-s, Fig. 1C). One example is ERO1 (ENDOPLASMIC RETICULUM OXIDOREDUCTINS 1), encoding an oxidoreductase that catalyzes the formation and isomerization of protein disulfide bonds in the ER,^20,21 which was significantly increased in athspr mutant under both normal and salt-stress condition. ERO1 can be induced by ER stress under saline condition and may contribute to H₂O₂ formation,²² furthering lipid peroxidation and protein oxidation. Molecular chaperones like HSPs are key factors for protein folding under various stresses especially in the heat, drought, salt and heavy metal stresses. HSPs can protect the newly synthetic proteins from denaturalizing to maintain their functions.^23,24 Genes such as HSF4 (HEAT SHOCK FACTOR 4), HSP17.4, HSP17.6A, HSP17.6II, HSP23.5, HSP20-like, HSP70-2, HSP70-4 and HSP90-1 showed higher expression levels in athspr mutant than those in WT under both normal and salt-stress condition. These well-known molecular chaperone genes are considered to be early markers of oxidative stress and ER stress. There are consistent data showing that the expression levels of ER stress marker genes BIP1 as well as CNX1⁶ are largely increased by NaCl treatment (Fig. 1E and F). These findings imply that salt-stressed athspr plants may have a greater accumulation of unfolded/misfolded proteins in the ER, which may initiate ER stress. A prolonged unfolded protein response would lead to inhibition of plant growth by affecting protein synthesis. These results are consistent with the salt-sensitive phenotype of athspr plants. However, direct involvement of the AtHSPR protein in salt-induced ER stress needs to be further investigated.

The present study, combining with our recent publication,¹², a possible model was presented for the involvement of AtHSPR in enhancing the tolerance to salt/drought stress (Fig. 2). AtHSPR enhance salt tolerance of transgenic plants through enhancing antioxidant system to reduce accumulation of salt-induced ROS and maintain Na⁺/K⁺ homeostasis, which in turn alleviates the impairment in photosynthesis and cell death. AtHSPR also involve in ABA-mediated signaling pathway by regulating the negative regulator of ABA under salt stress. Subsequently, elevated endogenous ABA activates a series of stress-related genes to enhance salt tolerance. Moreover, AtHSPR may involve in the salt-induced ER-stress response, and excess ER-stress exacerbated the accumulation of ROS and cell death. In summary, AtHSPR may protect cells from death upon salt stress through enhancing antioxidant defense, modulating ABA-dependent stomatal closure and signal transduction, and maintaining photosynthesis and Na⁺/K⁺ homeostasis, alleviating salt-induced ER-stress.

Figure 2.

Working model of AtHSPR in responses to salt stress in Arabidopsis. AtHSPR confers salt tolerance by enhancing antioxidant defense, modulating ABA-dependent stomatal closure and signal transduction, maintaining photosynthesis and Na⁺/K⁺ homeostasis, and alleviating salt-induced ER-stress.

Our recent work found that athspr showed deficient seed germination and increased shoot-branching phenotypes, which were similar to other SLs deficient mutants (data not shown), which implies that AtHSPR protein may be associated with SLs signal transduction. We now focus on whether AtHSPR does regulate SLs signaling and SLs does involve in abiotic stress response. Furthermore, functional study of AtHSPR/SMXL4 and SMXLs family genes in developmental processes and stress response will contribute to insights into SLs regulating development and stress response.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the National Natural Science Foundation of China (NSFC) (Grant No. 31270304) and the Fundamental Research Funds for the Central Universities of China (lzujbky-2013-bt05, lzujbky-2014-bt05).