Plant resistance inducer AMHA enhances antioxidant capacities to promote cold tolerance by regulating the upgrade of glutathione S-transferase in tea plant

Abstract

Plant resistance inducers represent an alternative strategy that mitigate stress-induced damage in plants. Previously, 2-amino-3-methylhexanoic acid (AMHA), a novel natural plant resistance inducer, was shown to significantly bolster cold tolerance, thermotolerance, and pathogen resistance in plants. However, the intricate mechanisms underlying AMHA's response to cold stress remain elusive. Thus, we investigated the physiological and transcriptomic analyses of AMHA pretreatment on tea plant to determine its substantial role of AMHA under cold stress. The results showed that pretreatment with 100 nM AMHA effectively mitigated the detrimental effects of cold stress on photosynthesis and growth. Furthermore, differentially expressed genes were identified through RNA-seq during pretreatment, cold stress, and 2 days of recovery. These genes were mainly enriched in pathways related to flavonoid/anthocyanin, carotenoid, and ascorbic acid-glutathione (AsA-GSH) cycle, including GST (encoding glutathione S-transferase). Potential regulatory relationships between the identified genes and transcription factors were also established. Antisense oligodeoxynucleotide-silencing and overexpression experiments revealed that CsGSTU7 enhances cold resistance by maintaining redox homeostasis. In conclusion, our study suggests that antioxidant-related signaling molecules play a critical role in the signaling cascades and transcriptional regulation mediating AMHA-induced cold-stress resistance in tea plant.

Figure 1

Effects of AMHA treatment on the phenotypes and photosynthetic performance of tea plant at different times under cold stress conditions. (a) Phenotypic changes and pseudo-color images of 1-year-old tea plant treated with AMHA under cold stress. Scale bars: 2 cm. (b) Statistical analysis of F_V/F_M values in AMHA or mock-treated plants. The pseudo-color gradient indicates damage levels, with 1 indicating no damage and 0 indicating severe damage. (c) Chlorophyll content. (d–f) Raw fluorescence rise kinetics of AMHA-treated plants at 12 and 24 hours of post-cold stress and after 2 days of recovery. Untreated plants were healthy tea plants. (g) Maximal fluorescence at the peak P of OJIP (F_M). (h) Performance index (PI_ABS) representing overall photosynthetic efficiency. (i) The amount of PSII active RCs (Q_A-reducing centers). Values indicate means ± SD (n = 3, ^*P < 0.05, and ^**P < 0.01, Student’s t-test), and this method was utilized for significance analyses below.

Figure 2

Dynamic effects of AMHA on osmotic adjustment substances contents, ROS levels, and antioxidant accumulation in tea plant during pretreatment, low temperature (LT), and recovery stages. (a–d) Contents of proline (a), soluble sugar (b), soluble protein (c), and MDA (d). (e) and (f) Accumulation of H₂O₂ and O₂^·− detected by NBT and DAB staining, respectively. Scale bars: 2 cm. (g) and (h) Contents of H₂O₂ and O₂^·−. (i-l) Enzyme activities of SOD (i), POD (j), CAT (k), APX (l). Values indicate means ± SD (n = 3, ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001, Student’s t-test)

Figure 3

Identification and functional enrichment analysis of DEGs in AMHA-treated plants compared to mock-treated plants. (a–e) Venn diagrams showing common and unique DEGs in AMHA- or mock-treated plants during the pretreatment, cold stress, and 2 days of recovery stages. (f) Bar plot indicating the total number of DEGs identified in the comparisons outlined. (g) Volcano plots displaying DEGs. (h, i) Lollipop plots displaying KEGG enrichment scores based on enrichment score (ES) ranking derived from differential expression analysis.

Figure 4

Effects of AMHA on flavonoid/anthocyanin and AsA metabolic pathways in tea plant. (a) and (b) Metabolism pathway and transcriptional levels of DEGs in the flavonoid/anthocyanin. (c) and (d) Total contents of flavonoid and anthocyanin, respectively. (e) and (f) metabolism pathway and transcriptional levels of DEGs in the AsA metabolism pathway. (g) Total contents of GSH and AsA, respectively. Values indicate means ± SD (n = 3, ^*P < 0.05, and ^**P < 0.01, Student’s t-test), and this method was utilized for significance analyses below.

Figure 5

Analysis of transcription factors (TFs) potentially correlated with flavonoid/anthocyanin, carotenoid, and AsA biosynthesis, as well as GST expression in AMHA-treated plants. (a) Diagram depicting the transcriptional regulatory network of flavonoid/anthocyanin, carotenoid, and AsA biosynthesis pathways, as well as GSTs. Non-TFs are represented by hexagons; TFs are depicted by different circles, with interconnecting lines showing potential co-expression associations between TFs and non-TF genes. (b) Heatmap displaying differentially expressed TFs following AMHA exposure. (c) RT-qPCR validation results of key TFs. Values indicate means ± SD (n = 3, ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001, Student’s t-test)

Figure 6

Effects of AMHA on ROS homeostasis in tea plant of AsODN_GSTs under cold stress conditions. (a) Phenotype and photosynthetic pigments of sODN and AsODN_GSTs of AMHA or mock-treated plants. Scale bars: 1 cm. (b) F_V/F_M value differences between sODN and AsODN_GSTs of AMHA or mock-treated plants. (c) and (d) H₂O₂ and O₂^·− contents of sODN and AsODN_GST of AMHA or mock-treated plants. (e-g) Changes in GST, SOD, and POD activities in sODN and AsODN_GSTs of AMHA or mock-treated plants. Data are presented as means ± SEs (15 biological replicates). Different lowercase letters indicate statistically significant differences by the LSD multiple range test (P < 0.05).

Figure 7

Overexpression of CsGSTU7 promoted stress resistance in transgenic tea plant under cold stress. (a) Phenotypes under room- and low-temperature conditions with AMHA or mock-treated plants. Scale bars: 1 cm. (b) Expression pattern analysis of the CsGSTU7 under room- and low-temperature conditions. (c-d) Measurement of H₂O₂ and O₂^·− contents under room- and low-temperature conditions. (f-h) Measurement of GST, SOD, and POD activities under room- and low-temperature conditions. The values are presented as the means ± SDs of three biological experiments. Different lowercase letters indicate statistically significant differences by the LSD multiple range test (P < 0.05).

Figure 8

A putative regulatory work model showing how AMHA is involved in mitigating cold damage in tea plant. AMHA pretreatment elicits physiological responses, including enhanced antioxidant enzymes, chloroplast protection, and osmotic adjustment, while also affecting the expression levels of glutathione S-transferase (GST). AMHA significantly increases the levels of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), anthocyanins, β-carotenes, ascorbic acid (AsA), glutathione (GSH), and soluble sugars, which are related to cold stress resistance. This alleviates physiological and metabolic damages, such as impaired photosynthetic efficiency, increased reactive oxygen species (ROS), and disrupted osmotic stability, caused by cold stress. AMHA mitigates the damage of cold stress by inducing the expression levels of CsGSTU7 and other key genes, as well as by stimulating the synthesis of the AsA-GSH system. AMHA maintains normal tea plant growth under cold-stress exposure by inducing genes related to cold resistance and osmolyte production.