HS usually causes assimilation branches of H. ammodendron seedlings to turn yellow and stop growing, resulting in the death of seedlings that are not deeply rooted when exposed to HS-induced drought stress (Yu et al. 2012). Therefore, acquiring the adaptability to HS through heat priming is of great significance for the survival of H. ammodendron seedlings and the natural regeneration of the H. ammodendron population.
14-3-3 proteins, a family of conserved molecules that target several protein clients through their ability to recognize well-defined phosphorylated motifs and regulate activities of a wide array of target proteins via protein-protein interactions, are involved in many biologically important processes, including cell cycle regulation, metabolism control, apoptosis, protein trafficking, stress response, and control of gene transcription (Yang et al. 2006; Chen et al. 2013; Mikhaylova et al. 2021). At present, the function and regulation mechanism of H. ammodendron 14-3-3 gene HaFT-1 are ambiguous. The study on H. ammodendron 14-3-3 gene HaFT-9 and heat shock factor gene HaHsfA5 showed that their expression has transcriptional memory to HS in H. ammodendron (Liu 2019b; Wang et al. 2020). In this study, we further discovered that the expression patterns of HaFT-1 were similar to that of HaFT-9 and HaHsfA5 during HS priming-and-triggering treatment (Fig. 1), indicating that HaFT-1 can regulate acquired thermotolerance. Unlike HaFT-9 or HaHsfA5, HaFT-1 is obviously up-regulated during the second HS and subsequent normal temperature recovery stage but repressed at the HS priming stage and unresponsive during the whole control treatment. However, it should be noted that stress memory regulating factors, including DNA methylation, chromatin remodeling, microRNA and histone modification, can affect and modulate HaFT-1 expression, which needs to be further explored. Transient expression of HaFT-1::YFP fusion protein in Arabidopsis protoplasts showed HaFT-1 localized in the cytoplasm (Fig. 2) indicating that as a molecular chaperon, HaFT-1 may interact with its target proteins, many of which act as acquired thermotolerance regulators in the cytoplasm. However, to date, there has been no report about target proteins of HaFT-1 or other 14-3-3 proteins in H. ammodendron.
Growth observation and cell death staining showed that HaFT-1 overexpression enhances HS priming and thermotolerance after priming-and-triggering treatment and non-primed control treatment in transgenic Arabidopsis (Fig. 3 and Fig. 4). These results confirmed the important role of HaFT-1 in thermotolerance. Furthermore, the phenotype difference between HaFT-1 transgenic lines and the WT Arabidopsis after the second HS was more obvious than that after control treatment, indicating that there might be additional regulation on HaFT-1 protein and its interaction with target proteins after HS priming. These results further suggest that the major role of HaFT-1 is to maintain the stability of acquired thermotolerance regulators. qRT-PCR further showed that at the HS priming stage, genes including HsfA2, HSP21, and HSP101 in P1 and P8 were highly expressed compared to those in WT Arabidopsis, implying that HaFT-1 may interact with certain transcriptional regulators, which promote the expression of heat shock factors and heat shock proteins at HS priming stage.
In this study, the transcriptomic analysis revealed more hints for understanding the function of HaFT-1. Venn diagrams showed genes that are up- and down expressed in HaFT-1 transgenic plants at the S2 stage (Fig. 5). Furthermore, KEGG enrichment analysis showed that energy generation and protein metabolism pathways were promoted among those genes, while signal transduction and biosynthesis pathways were repressed after second HS in HaFT-1 transgenic plants (Fig. 6). HaFT-1 might bind to its target proteins to facilitate the expression of genes in pathways specifically enriched in transgenic plants after second HS. This data suggested that upregulated genes in energy generation and protein metabolism pathways were more likely to promote HS-resistant activities after the second HS, while downregulated genes in signal transduction and biosynthesis may inhibit some metabolic activities from saving energy for promoting HS tolerance. Bioinformatics prediction further suggested that HaFT-1 may interact with HSFs, NACs, and other transcription factor family members (Liu 2019). Therefore, target proteins of HaFT-1 need to be further explored.
KEGG enrichment showed that genes specifically expressed in HaFT-1 overexpression lines and related to cell death inhibition and stress resistance belonged to proline metabolism, autophagy, chlorophyll metabolism, and ROS scavenging pathways. There is a close relationship between stress-related genes expression, ROS scavenging, and proline synthesis (Altuntaş et al. 2020; Medina et al. 2021; Li et al. 2022; Wang et al. 2022). A study on desiccation-tolerant resurrection plant Craterostigma plantagineum showed that after being primed by drought, expression of four representative stress-related genes and ROS pathway-related genes gradually increased, accompanied by increased SOD activity, proline content, and sucrose content, and a decrease in H2O2 content and electrolyte leakage (EL) (Liu et al. 2019c). Research on wheat and tall fescue confirmed that drought-priming leads to HS cross adaptation with higher leaf water potential, chlorophyll content, and photochemical efficiency. The improved protection results in higher grain yield and increased tolerance compared to the non-primed plants (Liu et al. 2017; Zhang et al. 2019). These examples suggest a positive role of HS priming in elevating chlorophyll content.
In this study, DAB staining showed higher staining in WT compared to P1 and P8 after both S2 and FS2 treatment (Fig. 7), indicating the accumulation of ROS in P1 and P8 plants was lower than that in WT at S2 and FS2 stages. These results suggested that the ROS scavenging ability of HaFT-1 transgenic plants was stronger than that of WT at S1 and R1 stages, resulting in less peroxide accumulation in HaFT-1 transgenic plants compared to that of WT at the S2 stage. Furthermore, DAB staining of WT and HaFT-1 transgenic plants at the FS2 stage was significantly higher than that at the S2 stage, which further showed that HS priming might reduce the accumulation of peroxide at the S2 stage. Meanwhile, expression of P5CS2 increased at the HS priming stage, accompanied by increased SOD, POD, and proline contents at S1, R1, and S2 stage, and chlorophyll content at the S2 stage in HaFT-1 transgenic Arabidopsis (Fig. 7, 8). Therefore, enhanced peroxide scavenging ability and proline biosynthesis during HS priming might contributed to maintaining chlorophyll stability and reduce cell death at the second HS stage in HaFT-1 transgenic Arabidopsis, leading to a gain of HS tolerance and HS memory.
Taken together, this study characterized HaFT-1 for its role in thermotolerance. HaFT-1 has the transcriptional memory to HS in H. ammodendron. Overexpression of HaFT-1 enhances HS priming and thermotolerance of transgenic Arabidopsis. Priming-and-triggering treatment in Arabidopsis overexpressing HaFT-1 promoted energy generation and protein metabolism, depressed signal transduction and biosynthesis, and strengthened physiological regulation of proline content and peroxide scavenging activity. This study showed that HaFT-1 is involved in the regulation of acquired thermotolerance rather than basic thermotolerance and its upregulated expression depends on HS priming in H. ammodendron.