Pearl Millet Aquaporin Gene PgPIP2;6 Improves Abiotic Stress Tolerance in Transgenic Tobacco

Abstract

Pearl millet [Pennisetum glaucum (L) R. Br.] is an important cereal crop of the semiarid tropics, which can withstand prolonged drought and heat stress. Considering an active involvement of the aquaporin (AQP) genes in water transport and desiccation tolerance besides several basic functions, their potential role in abiotic stress tolerance was systematically characterized and functionally validated. A total of 34 AQP genes from P. glaucum were identified and categorized into four subfamilies, viz., plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26-like intrinsic proteins (NIPs), and small basic intrinsic proteins (SIPs). Sequence analysis revealed that PgAQPs have conserved characters of AQP genes with a closer relationship to sorghum. The PgAQPs were expressed differentially under high vapor pressure deficit (VPD) and progressive drought stresses where the PgPIP2;6 gene showed significant expression under high VPD and drought stress. Transgenic tobacco plants were developed by heterologous expression of the PgPIP2;6 gene and functionally characterized under different abiotic stresses to further unravel their role. Transgenic tobacco plants in the T₂ generations displayed restricted transpiration and low root exudation rates in low- and high-VPD conditions. Under progressive drought stress, wild-type (WT) plants showed a quick or faster decline of soil moisture than transgenics. While under heat stress, PgPIP2;6 transgenics showed better adaptation to heat (40°C) with high canopy temperature depression (CTD) and low transpiration; under low-temperature stress, they displayed lower transpiration than their non-transgenic counterparts. Cumulatively, lower transpiration rate (Tr), low root exudation rate, declined transpiration, elevated CTD, and lower transpiration indicate that PgPIP2;6 plays a role under abiotic stress tolerance. Since the PgPIP2;6 transgenic plants exhibited better adaptation against major abiotic stresses such as drought, high VPD, heat, and cold stresses by virtue of enhanced transpiration efficiency, it has the potential to engineer abiotic stress tolerance for sustained growth and productivity of crops.

Keywords: Pennisetum glaucum; PgPIP2;6; canopy temperature depression (CTD); exudation rate; progressive drought stress; transpiration efficiency; transpiration rate; vapor pressure deficit.

FIGURE 1

Characterization of pearl millet AQP proteins. (A) The phylogenetic tree was constructed using MacVector software by the NJ method. (B) Exon and intron structure of pearl millet AQP genes is indicated by blue rectangles and thin dotted lines, respectively. The scale bar indicates nucleotide size length in the base pairs.

FIGURE 2

An expression profile of the 34 PgAQP genes in leaves and roots of VPD-sensitive and VPD-insensitive genotypes under (A) drought stress and (B) high VPD. The color bar represents normalized log2 values.

FIGURE 3

Characterization of the pearl millet PgPIP2;6 gene. (A) The 3D model of PgPIP2;6 protein built by using homology based in silico modeling. Aquaporin PgPIP2;6 is incorporated into the membranes in a tetrameric arrangement, comprising four individual pores. The protein structure has classical aquaporin structure with conserved NPA motif regions, which are highlighted in blue. The ligand of the protein binds to the protein near the NPA motifs. Ligand (Cadmium ion) is highlighted in gray spheres. (B) Schematic representation of the T-DNA region of the plant transformation vector (pMDC100), showing the two expression cassettes for nptII and PgPIP2;6 genes. RB, right border; 35SP, CaMV35s promoter; PgPIP2;6, pearl millet plasma intrinsic protein 2;6 gene; nptII, neomycin phosphotransferase gene; NosT, Nos terminator; LB, left border.

FIGURE 4

Performance of the transgenic and non-transgenic WT plants under stable and increasing VPD conditions. (A) Tr response to increasing VPD conditions in PgPIP2;6 transgenic and WT tobacco plants. A plot of transpiration rates against VPD for three transgenic tobacco lines [E 1–20 (the blue-color line); E 11–10 (the green-color line); E 28–9 (the pink-color line)] and WT (the red-color line) plants. Compared to WT, transgenic events (except E 1–20) showed limited Tr under high-VPD conditions. Each data point represents means (± SE) six replicates. (B) Mean Tr of PgPIP2;6 transgenic and WT plants under low-VPD and high-VPD conditions. The bars with blue color represent low-VPD (1.2 kPa) Tr, and bars with red color represent high-VPD (3.8 kPa) Tr. Data represent means (± SE) six replicates, and means were analyzed by the Tukey–Kramer test. The “P” indicates the probability of a difference among the transgenic events and WT plants in low and high VPD. Bars with different capital letters and small letters indicate significantly different at P ≤ 0.05 in high-VPD and low-VPD conditions. The asterisks indicate the significant difference at P ≤ 0.05 between low- and high-VPD treatments. “Bars with asterisk ** and *** symbols are significantly different at P < 0.01, P < 0.001 between low VPD and high VPD stress treatment. (C) The root exudation rate of transgenic and WT tobacco plants subjected to low (1.2 kPa) and high-(3.8 kPa) VPD stress. The exudation rate was calculated by sampling the root sap. Data were analyzed by the Tukey-Kramer test. Values represent means ± SE (n = 6). Bars with different capital letters and small letters indicate significantly different at P ≤ 0.05 in high VPD and low VPD conditions. Bars with asterisk * symbol is significantly different at P < 0.05, between low and high VPD exudation. Bars with NS indicate that both low and high VPD root exudation are statistically non-significant.

FIGURE 5

Progressive drought stress. (A) Relationship between the normalized transpiration rate (NTR) and fraction of transpirable soil water (FTSW) of transgenic [E 1–20 (blue color line); E 11–10 (pink color line); E 28–9 (green color line)] and WT (red color line) during progressive drought stress treatment. The FTSW thresholds where the transpiration initiated its decline were calculated with segmental regression procedure from Graphpad prism. Then, the regression lines of the relationships between the NTR and FTSW were drawn by fitting NTR to FTSW data above and below the respective thresholds for transpiration decline in each genotype. The dotted lines represent the NTR of well-watered control plants. Each point is the mean of NTR (n = 9), and the error bars are the SE of the mean. (B) Total transpiration content in PgPIP2;6 transgenic tobacco lines under well-watered (WW) and water stress (WS) treatment. Bars with different capital letters and small letters indicate significantly different at P ≤ 0.05 in both low and high VPD conditions. Bars with asterisk *** symbol are significantly different at P < 0.001 between WW and WS treatment.

FIGURE 6

Physiological performance of PgPIP2;6 transgenic tobacco lines under heat stress at 40°C. (A) Tr in transgenic lines and WT plants subjected to heat stress treatment (40°C for 4 h). (B) Thermal infrared images of leaf from WT and transgenic tobacco plants. The images were taken after 4 h of heat stress treatment. The color scale to the right shows the variation in leaf temperature produced by heat. (C) Canopy temperature of the transgenic lines E 1–20, E 11–10, and E 28–9 and WT tobacco plants under 40°C heat stress and control (28°C) conditions. Measurements were taken after 4 h of stress treatment on three replicates of each line. Data are means ± SE. (D) Canopy temperature depression in transgenic tobacco lines and WT plants under heat stress. Data are means ± SE. Bars with different capital letters and small letters indicate significantly different at P≤0.05 in both control and heat stress conditions. Bars with asterisk *** symbols from (A,C,D) are significantly different at P < 0.001, between control and heat stress treatment.

FIGURE 7

The transpiration rate in transgenic and WT plants subjected to cold stress treatment (4°C for 4 h). Bars with different capital letters and small letters indicate significantly different at P≤0.05 in both control and cold stress conditions. Bars with asterisk *, **, and *** symbols are significantly different at P < 0.05, P < 0.01, P < 0.001 between control and cold stress treatment.

FIGURE 8

The relative expression of PgPIP2;6 gene in transgenic tobacco plants under high VPD (A), progressive drought (B), heat (C), and cold (D) stresses. Y-axis indicates the relative gene expression values, and names on the X-axis represent the transgenic events. Dark black-color bars indicate the leaf expression, and gray-color bars indicate root expression. Data points represent the PgPIP2;6 transgene expression values, obtained after normalization against the reference gene (UBC) and corresponding control samples by 2^–ΔΔCt method. Each data point represents mean of three biological replications with standard error (±SE). Each biological replication represents mean of three technical replications. The “P” indicates the probability of a difference among the transgenic events expression in leaf and root tissues. Bars with different capital letters and small letters are indicated significantly different at P ≤ 0.05 in root and leaf tissues.