Pyramiding of transcription factor, PgHSF4, and stress-responsive genes of p68, Pg47, and PsAKR1 impart multiple abiotic stress tolerance in rice (Oryza sativa L.)

Abstract

Abiotic stresses such as drought, salinity, and heat stress significantly affect rice crop growth and production. Under uncertain climatic conditions, the concurrent multiple abiotic stresses at different stages of rice production became a major challenge for agriculture. Hence, improving rice's multiple abiotic stress tolerance is essential to overcome unprecedented challenges under adverse environmental conditions. A significant challenge for rice breeding programs in improving abiotic stress tolerance involves multiple traits and their complexity. Multiple traits must be targeted to improve multiple stress tolerance in rice and uncover the mechanisms. With this hypothesis, in the present study gene stacking approach is used to integrate multiple traits involved in stress tolerance. The multigene transgenics co-expressing Pennisetum glaucum 47 (Pg47), Pea 68 (p68), Pennisetum glaucum Heat Shock Factor 4(PgHSF4), and Pseudomonas Aldo Keto Reductase 1 (PsAKR1) genes in the rice genotype (AC39020) were developed using the in-planta transformation method. The promising transgenic lines maintained higher yields under semi-irrigated aerobic cultivation (moisture stress). These 15 promising transgenic rice seedlings showed improved shoot and root growth traits under salinity, accelerating aging, temperature, and oxidative stress. They showed better physiological characteristics, such as chlorophyll content, membrane stability, and lower accumulation of reactive oxygen species, under multiple abiotic stresses than wild-type. Enhanced expression of transgenes and other stress-responsive downstream genes such as HSP70, SOD, APX, SOS, PP2C, and P5CS in transgenic lines suggest the possible molecular mechanism for imparting the abiotic stress tolerance. This study proved that multiple genes stacking as a novel strategy induce several mechanisms and responsible traits to overcome multiple abiotic stresses. This multigene combination can potentially improve tolerance to multiple abiotic stress conditions and pave the way for developing climate-resilient crops.

Keywords: abiotic stress; gene stacking; mechanism; multiple genes; stress tolerance; trait; transgenics.

Figure 1

Screening of T₃ putative transformants against different abiotic stresses. (A) Frequency distribution of rice transgenics based on root and shoot length for salinity stress response. (B) Photograph showing the salinity stress response of transgenic rice line (T-50). (C) Frequency distribution of rice transgenics based on root and shoot length for accelerated aging stress response. (D) Growth response of transgenic lines under accelerated aging stress. WT-wildtype, T-transgenic, C-Control, t- treated.

Figure 2

Rice transgenic seedlings’ growth response under NaCl-induced stress. (A) Germinated seeds were placed on 50, 100, and 200 mM NaCl (Induction treatment) for three h and transferred into the 350 mM NaCl (lethal concentration). After three days of stress treatment, seedlings were placed for recovery and shoot, and root lengths were recorded. (B) MDA estimation from control and NaCl-stressed samples (induction treatment sample). (C) Correlation between the seedling’s growth and percent increase in MDA content over control. Bars indicate standard error of replication, an asterisk (*) indicates the significance at P=0.05.

Figure 3

Response of rice transgenics to NaCl stress upon excised leaf disc assays. Leaf discs from transgenic and wild-type plants were placed on 350 mM NaCl,. (A) Photograph was taken 72 h after exposure to NaCl treatment. (B) Percent reduction in chlorophyll content (C) Percent increase in electrolyte leakage. Bars indicate a standard error of three replications, and an asterisk (*) indicates the significance at P=0.05, WT-wild-type, 1 to 252- different transgenic lines.

Figure 4

Multigene rice transgenics response to methyl induced viologen-induced oxidative stress. Two days germinated seedlings were placed on 0.6 µm methyl viologen for eight days. (A) shows the rice transgenic seedlings growth in response to methyl viologen stress. (B) Percent reduction in seedling growth of transgenics compared to wild type. WT- wild-type, 1 to 252-Transgenic lines. Error bars indicate the standard error of three replication.

Figure 5

Response of multigene rice transgenics to heat stress. Two days-old germinated seedlings were subjected to heat stress for three h and then kept at room temperature for eight days. (A) Photograph showing the rice transgenics seedling growth response upon exposure to the heat stress. (B) Percent reduction in seedling growth over control, (C) Percent reduction in protein content over control. Error bars indicate the standard error of three replication, and the asterisk (*) indicates the significance at P=0.05.

Figure 6

Quantification of reactive oxygen radicle using the histochemical assay. (A) One-week-old seedlings of wildtype and transgenic was incubated in 300 mM NaCl for 24 hours, seedlings were stained with NBT solution for quantification of superoxide radicles and (B) DAB solution staining for H₂O₂ quantification. WT-Wild-type, T50 and T210 – Transgenic lines.

Figure 7

Expression analyses of multigene-expressing rice transgenic plants. (A) Semi-quantitative expression of transgenes under heat stress, (B) Gene expression profiles of transgenes under salinity stress, (C) Semi-quantitative expression analysis of downstream genes in multigene-expressing transgenics under heat stress conditions, and (D) Semi-quantitative expression analysis of downstream genes in multigene-expressing transgenics under salinity stress conditions. The cDNA was amplified with gene-specific primers, and actin was used as a housekeeping gene. WT – Wild-type, T-25 to T-234 – Transgenic lines.

Figure 8

Overview of the stress tolerance mechanisms in multigene-expressing plants. The proposed model of multiple abiotic stress tolerance in multigene rice transgenics expressing PgHSF4, p68, and Pg47 genes along with PsAKR1. WT-Wild-type, T-Transgenics, ROS- Reactive oxygen species, RCC- Reactive carbonyl compounds. Pictures were adopted from Google and Vector Stocks.