Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field

Abstract

Drought stress has often caused significant decreases in crop production which could be associated with global warming. Enhancing drought tolerance without a grain yield penalty has been a great challenge in crop improvement. Here, we report the Arabidopsis thaliana galactinol synthase 2 gene (AtGolS2) was able to confer drought tolerance and increase grain yield in two different rice (Oryza sativa) genotypes under dry field conditions. The developed transgenic lines expressing AtGolS2 under the control of the constitutive maize ubiquitin promoter (Ubi:AtGolS2) also had higher levels of galactinol than the non-transgenic control. The increased grain yield of the transgenic rice under drought conditions was related to a higher number of panicles, grain fertility and biomass. Extensive confined field trials using Ubi:AtGolS2 transgenic lines in Curinga, tropical japonica and NERICA4, interspecific hybrid across two different seasons and environments revealed the verified lines have the proven field drought tolerance of the Ubi:AtGolS2 transgenic rice. The amended drought tolerance was associated with higher relative water content of leaves, higher photosynthesis activity, lesser reduction in plant growth and faster recovering ability. Collectively, our results provide strong evidence that AtGolS2 is a useful biotechnological tool to reduce grain yield losses in rice beyond genetic differences under field drought stress.

Keywords: confined field trial; drought; galactinol synthase; grain yield; transgenic rice.

Figure 1

Ectopic overexpression of AtGolS2 gene confers higher galactinol accumulation on transgenic Curinga and NERICA4 rice. (a) Expression of AtGolS2 in Ubi:AtGolS2 transgenic rice was analysed by quantitative real‐time PCR. Expression of OsUbi1 was analysed as an internal control to normalize the expression of AtGolS2. (b) Accumulation of galactinol in Ubi:AtGolS2 transgenic rice was analysed using GC‐TOF‐MS. The highest average value of the samples was set as 100, and the relative values were shown in (a) and (b). Five plants were combined into one sample for each line, and the relative values are mean ± SD of three technical replicates. Nontransgenic (NT) Curinga and NERICA4 were used as the control.

Figure 2

Ubi:AtGolS2 improves vegetative drought tolerance in transgenic Curinga. (a) Soil moisture profile during the vegetative drought stress rainout shelter experiment. Fragmented line and dotted line indicate upper (0–20 cm) and average lower (20–60 cm) soil moisture, respectively. Arrowheads indicate drought stress scheduling and sampling time. (b) Variation in leaf rolling score among the transgenic lines during peak stress (three weeks after stress). (c) Variation in plant dry biomass among the transgenic lines at the end of the stress. (d) Variation in plant dry biomass among the transgenic lines at the end of the harvest after stress recovery. (e) Variation plant height among the transgenic lines at the end of the stress. Each dry biomass and plant height value represents the mean ± SE (n = 3), in each replication data point derived from three individual uniform plants; leaf rolling score based on whole plot performance and represents the mean ± SE (n = 3) from three replications. Different letters in each column denote significant differences at P < 0.05 by Tukey–Kramer method.

Figure 3

Ubi:AtGolS2 improves rice grain yield in Managed Drought Stress Environment (MDSE) over the growing seasons—rainout shelter experiments, CIAT, Palmira. Single plant grain yield performance of Curinga transgenic lines in 2012‐rainy season‐MDSE‐Trial‐1 (a), 2012‐dry season‐MDSE‐Trial‐2 (b) and 2014‐rainy season‐MDSE‐Trial (c). (d) Field performance of NT Curinga and promising transgenic lines in 2014‐rainy‐MDSE‐Trial. Photographs were taken next day after rewatering for recovery after drought stress at the flowering stage. Each single plant yield value represents the mean ± SE (n = 9–24). Different letters in each column denote significant differences at P < 0.05 by Tukey–Kramer method.

Figure 4

Variation in physiological parameters among the Curinga Ubi:AtGolS2 transgenic lines in 2014‐rainy‐MDSE‐Trial. (a) Percentage of relative water content (RWC) values of transgenic and NT lines at before, first, second and third weeks after stress (WAS). (b) Changes in chlorophyll fluorescence ( F _V / F _M ) of transgenic and NT lines at before (BS), first, second and third weeks after stress (WAS). (c) SPAD chlorophyll values of transgenic and NT lines at peak drought stress. (d) RT‐PCR analysis of Ubi:AtGolS2 transgenic and NT lines evaluated at different timing points: before stress (BS), during peak stress (DS) and after stress (AS). RT‐PCR analyses were performed using RNAs from leaf tissue at different points of drought development using Ubi:AtGolS2 gene‐specific primers. Each physiological parameter value represents the mean ± SE (n = 9), three individual plants from three replications. Different letters in each figure denote significant differences at P < 0.05 by Tukey–Kramer method.

Figure 5

Rainfall and temperature pattern during crop period in upland confined rainfed field trial, CIAT, Santa Rosa upland station. (a) Climatic profile of TE‐2012‐13‐Trial. (b) Climatic profile of TE‐2013‐14‐Trial. (c) Climatic profile of TE‐2014‐15‐Trial. Black bar shows amount of rainfall received during crop period, and dotted line graph shows the maximum daily temperature during trial period. Temperature data are daily averages and rainfall is daily total.

Figure 6

Ubi:AtGolS2 improves Curinga grain yield in Target Environment (TE)—Santa Rosa rainfed Trial, CIAT upland rainfed station, Villavicencio. (a) Grain yield performance of Curinga transgenic lines in TE‐2012‐13‐Trial. (b) Grain yield performance of Curinga transgenic lines in TE‐2013‐14‐Trial. (c) Grain yield performance of Curinga transgenic lines in TE‐2014‐15‐Trial. (d) Field performance of NT Curinga and promising transgenic event 2580 at TE‐2013‐14‐Trial. Photographs were taken during stress at the grain‐filling stage. Estimated grain yield (kg/ha) was derived from plot yield from three replications, and value represents the mean ± SE (n = 3). Different letters in each column denote significant differences at P < 0.05 by Tukey–Kramer method.

Figure 7

Ubi:AtGolS2 improves NERICA4 grain yield in rainfed upland trials. (a) Grain yield performance of NERICA4 lines in Target Environment (TE)‐2013‐14‐Trial at Santa Rosa rainfed trial. (b) Grain yield performance of NERICA4 lines in TE‐2014‐15‐Trial at Santa Rosa rainfed trial. Estimated grain yield (kg/ha) derived from plot yield. Estimated grain yield (kg/ha) was derived from plot yield from three replications, and value represents the mean ± SE (n = 3). Different letters in each column denote significant differences at P < 0.05 by Tukey–Kramer method.