Overexpression of PavHIPP16 from Prunus avium enhances cold stress tolerance in transgenic tobacco

Abstract

Background: The heavy metal-associated isoprenylated plant protein (HIPP) is an important regulatory element in response to abiotic stresses, especially playing a key role in low-temperature response.

Results: This study investigated the potential function of PavHIPP16 up-regulated in sweet cherry under cold stress by heterologous overexpression in tobacco. The results showed that the overexpression (OE) lines' growth state was better than wild type (WT), and the germination rate, root length, and fresh weight of OE lines were significantly higher than those of WT. In addition, the relative conductivity and malondialdehyde (MDA) content of the OE of tobacco under low-temperature treatment were substantially lower than those of WT. In contrast, peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) activities, hydrogen peroxide (H₂O₂), proline, soluble protein, and soluble sugar contents were significantly higher than those of WT. Yeast two-hybrid assay (Y2H) and luciferase complementation assay verified the interactions between PavbHLH106 and PavHIPP16, suggesting that these two proteins co-regulated the cold tolerance mechanism in plants. The research results indicated that the transgenic lines could perform better under low-temperature stress by increasing the antioxidant enzyme activity and osmoregulatory substance content of the transgenic plants.

Conclusions: This study provides genetic resources for analyzing the biological functions of PavHIPPs, which is important for elucidating the mechanisms of cold resistance in sweet cherry.

Keywords: Genetic transformation; HIPP; Low-temperature stress; Protein interaction; Sweet cherry.

Fig. 1

Sequence analysis and phylogenetic relationships of PavHIPP16. (a) Multiple sequence comparison of PavHIPP16 with homologous proteins from other plant species. The red background indicates that the amino acids at this position are fully conserved. (b) Phylogenetic tree constructed in MEGA11 using the NJ method and setting Bootstrap to 1000. Species include sweet cherry, Chinese plum, peach, white pear, apple, Arabidopsis, rice, potato, barley, sweet orange, tobacco, sorghum, and Brachypodium distachyon

Fig. 2

PCR validation and expression analysis of PavHIPP16 genetically transformed tobacco. (a) Specific primers for PCR validation of the transgene and WT. M is the Marker for D2000 and P indicates recombinant plasmid. (b) Expression level of PavHIPP16 in transgenic plants (Red boxes indicate overexpression plants for subsequent experiments). Data are shown as mean ± SE of three independent experiments (n = 3, biological replicates), with different letters representing significant differences at various levels (P < 0.05)

Fig. 3

Analysis of germination rate of PavHIPP16 transgenes tobacco. (a) Germination of tobacco OE and WT lines after 10 d of growth; (b) Germination statistics of tobacco seeds grown in MS medium for 10 d. Different colors are used to distinguish different tobacco lines. Data are shown as mean ± SE of three independent experiments (n = 3, biological replicates)

Fig. 4

Root length and fresh weight of tobacco under low-temperature treatment. (a-c) Root length of OE2, OE3, OE5, and WT tobacco under low-temperature stress (a: 4 °C, b: 8 °C, and c: 25 °C); (d) Root length statistics after low-temperature treatment. (e) Comparison of fresh weight after low-temperature treatment. Data are shown as mean ± SE of three independent experiments (n = 3, biological replicates), with different letters representing significant differences at different levels (P < 0.05)

Fig. 5

Observation of stomata after low-temperature treatment. (a) The scale is 20 μm. (b) Stomatal opening in OE and WT lines under low-temperature stress. Data are shown as mean ± SE of three independent experiments (n = 3, biological replicates), with different letters representing significant differences at different levels (P < 0.05)

Fig. 6

Phenotypic observation and related gene expression analysis of OE and WT. (a) WT and OE lines tobacco flowering; (b) WT and OE tobacco flowering time statistics; (c-g) represent the expression levels of NtFT, NtSCO1, NtCO, NtLFY, and NtFUL respectively. Data are shown as mean ± SE of three independent experiments (n = 3, biological replicates), with different letters representing significant differences at different levels (P < 0.05)

Fig. 7

Enzyme activity tests after low-temperature treatment. (a) 4 °C treatment of OE and WT tobacco for 7 d; (b-j) Represent relative conductivity, MDA content, POD activity, SOD activity, CAT activity, H₂O₂ content, proline content, soluble protein content, and soluble sugar content respectively. Data are shown as mean ± SE of three independent experiments (n = 3, biological replicates), with different letters representing significant differences at different levels (P < 0.05)

Fig. 8

Expression analysis of stress-responsive genes in different OE and WT under low-temperature stress and control conditions. (a-d) represent NtCBF1, NtCBF2, NtCOR47 and NtCOR78 respectively. Note: WT: Wild tobacco line; OE2, OE3, OE5: Transgenic tobacco lines. Data are shown as mean ± SE of three independent experiments (n = 3, biological replicates), with different letters representing significant differences at different levels (P < 0.05)

Fig. 9

Heterodimerization of PavbHLH106 and PavHIPP16. (a) Schematic diagram of the vector construction.; (b) The results of the Y2H experiments indicate that PavbHLH106 and PavHIPP16 interact. Yeast containing AD and BD recombinant plasmids grow and turn blue in SD/-Trp/-Leu/-Ade/-His medium. (c) Schematic diagram of the vector construction. (d) The results of luciferase complementation experiments indicate that PavbHLH106 and PavHIPP16 interact. The injected regions containing nLUC and cLUC recombinant plasmids were luminescent and brighter than other controls