GmTDN1 improves wheat yields by inducing dual tolerance to both drought and low-N stress

Abstract

Genetically enhancing drought tolerance and nutrient use efficacy enables sustainable and stable wheat production in drought-prone areas exposed to water shortages and low soil fertility, due to global warming and declining natural resources. In this study, wheat plants, exhibiting improved drought tolerance and N-use efficacy, were developed by introducing GmTDN1, a gene encoding a DREB-like transcription factor, into two modern winter wheat varieties, cv Shi4185 and Jimai22. Overexpressing GmTDN1 in wheat resulted in significantly improved drought and low-N tolerance under drought and N-deficient conditions in the greenhouse. Field trials conducted at three different locations over a period of 2-3 consecutive years showed that both Shi4185 and Jimai22 GmTDN1 transgenic lines were agronomically superior to wild-type plants, and produced significantly higher yields under both drought and N-deficient conditions. No yield penalties were observed in these transgenic lines under normal well irrigation conditions. Overexpressing GmTDN1 enhanced photosynthetic and osmotic adjustment capacity, antioxidant metabolism, and root mass of wheat plants, compared to those of wild-type plants, by orchestrating the expression of a set of drought stress-related genes as well as the nitrate transporter, NRT2.5. Furthermore, transgenic wheat with overexpressed NRT2.5 can improve drought tolerance and nitrogen (N) absorption, suggesting that improving N absorption in GmTDN1 transgenic wheat may contribute to drought tolerance. These findings may lead to the development of new methodologies with the capacity to simultaneously improve drought tolerance and N-use efficacy in cereal crops to ensure sustainable agriculture and global food security.

Keywords: DREB-like transcription factor; drought; field trial; nitrogen deficiency; transgenic wheat (Triticum aestivum L.).

Figure 1

GmTDN1 overexpression confers drought and low nitrogen (LN) stress tolerance. (a) Photographs of Shi4185 and S‐OE lines under normal (non‐stressed conditions), drought (7 and 21 days), drought (21 or 28 days) and then rehydration for 7 days. Three‐leaf stage plants were subjected to drought stress (stop watering for 7 or 21 days, and then allowed to recover for 7 days) prior to capturing these images. (b) Results of a water loss assay for Shi4185 and S‐OE lines. Leaves of three‐leaf stage plants were detached for 5 h (n = 4 biologically independent samples; error bars represent means ± SD). (c) Survival rates of wheat seedlings. (d) Seedlings of Shi4185 and the S‐OE plants grown under LN and normal conditions. Three days after germination, plants were grown hydroponically for 21 days under LN and normal nutrient solution conditions. (e) Shoot fresh weight. (f) Root fresh weight. Data in e and f are presented as means ± SD from three independent experiments (at least 20 plants in each experiment). (g) ${\text{NO}}_3^{‐}$ flux rates; NO₃ ^‐ flux rate was measured via NMT with 0.2 mm ${\text{NO}}_3^{‐}$ in the measuring solution. Here, seedlings were pretreated with 0.2 mm ${\text{NO}}_3^{‐}$ and then used to measure ${\text{NO}}_3^{‐}$ flux rates at the root surface in a measuring solution that contained 0.2 mm ${\text{NO}}_3^{‐}$. Positive and negative flux values indicate efflux and influx, respectively. Data represent means ± SD of at least six replicates. *P < 0.05 and **P < 0.01 between the transgenic line and the WT control according to Student’s t‐test (two‐tailed). Values are represented by the mean ± SD.

Figure 2

Drought tolerance of the Shi4185 transgenic lines assessed by improved nitrate acquisition and utilization. (a) Photographs of Shi4185 and S‐OE lines under normal, drought (28 and 21 days), and rehydration for 7 days in normal and nitrogen‐deficient field soil. Three‐leaf stage plants were subjected to drought stress (stop watering for 28 or 21 days and then allowed to recover for 7 days) before capturing photographs. (b) Survival rates of wheat seedlings. Data represent means ± SD of at least three replicates (at least 20 plants in each experiment). *P < 0.05 and **P < 0.01 between the transgenic line and WT control according to Student’s t‐test (two‐tailed). Values are represented by the mean ± SD.

Figure 3

The effect of GmTDN1 on grain yields in field experiments, and physiological characterization under drought stress field conditions. (a) Images of OE and Shi4185 plants during the wheat‐growing season at the Shijiazhuang site under limited‐irrigated conditions (LIR). (b) All year averages for grain yields of GmTDN1 OE in the Shi4185 wheat background at all three locations. (c) All year averages for the grain yield of GmTDN1 OE in the Jm22 wheat background at all three locations. (d) and (e) Water use efficiency (WUE) of the Shi4185 (d) and Jm22 (e) OE plants at the Shijiazhuang site. (f–g) Flag and secondary leaves were collected and examined for physiological characteristics under the LIR condition, Net photosynthesis rate (Pn) (f), SOD activity (g). *P < 0.05 and **P < 0.01 between OE and WT plants assessed via Student’s t‐test (two‐tailed). Values are represented by the mean ± SD.

Figure 4

Grain yield and N use‐related traits of the wild type and the GmTDN1 OE lines in low nitrogen condition field experiments. (a) The images represent OE and Jm22 WT plants under low N conditions. (b) Grain yields for two consecutive years (2016–2017). (c) Spike number per m². (d) Grain number per spike. (e) 1000‐grain weight. (f) NUE of lines and WT in field plots. (g) Grain N concentration. (g) Grain N distribution. NUE, grain yield/applied N fertilizer. *P < 0.05 and **P < 0.01 between the transgenic line and the WT control assessed via Student’s t‐test (two‐tailed). Values are represented by the mean ± SD.

Figure 5

Identification of direct targets of transgenic GmTDN1 by ChIP?seq assay. (a) Binding GmTDN1 to promoters containing DRE cis‐acting elements in GmTDN1 target genes. (b) The enrichment for occupancy of GmTDN1 at target gene loci detected in the ChIP‐seq data was paired with qPCR‐based analysis of potential increased transcription of these genes. (c) GmTDN1‐GFP specifically activated the expression of the Luc gene as driven by the native promoters of NRT2.5 and LEA.

Figure 6

NRT2.5 overexpression improved drought stress and nitrogen uptake in wheat. (a) Representative images of WT and overexpression line seedlings grown hydroponically with PEG stress. WT, G3, and G4 seedlings grown hydroponically for 3 days followed by treatment with or without 25% PEG6000 for an additional 10 days, and then recovered for 8 days. (b) Representative images showing the chlorate tolerance of WT and overexpression lines. The wheat plants were grown hydroponically with water for 3 days followed by transfer to Hoagland’s liquid medium for 3 days, and then moved to the same hydroponic system supplied with 2 mm ${\text{KClO}}_3^{‐}$ for another 3 days. Wheat plants were cultured in Hoagland’s liquid medium without ${\text{KClO}}_3^{‐}$ for 6 days were used as controls. The data represent the mean ± SD from three independent experiments (at least 20 plants in each experiment). *P < 0.05, **P < 0.01 (two‐tailed Student’s t‐test).

Figure 7

Proposed model for GmTDN1 action in wheat. GmTDN1 binds to and promotes the transcription of target genes (in blue), with downstream impacts on biochemical and physiological processes including N uptake, plant morphology, GA synthesis, photosynthesis, and water stress (in red), leading to enhancement of drought tolerance, N utilization efficiency, and increased grain yield.