Towards doubling fibre yield for cotton in the semiarid agricultural area by increasing tolerance to drought, heat and salinity simultaneously

Abstract

Abiotic stresses such as extreme temperatures, water-deficit and salinity negatively affect plant growth and development, and cause significant yield losses. It was previously shown that co-overexpression of the Arabidopsis vacuolar pyrophosphatase gene AVP1 and the rice SUMO E3 ligase gene OsSIZ1 in Arabidopsis significantly increased tolerance to multiple abiotic stresses and led to increased seed yield for plants grown under single or multiple abiotic stress conditions. It was hypothesized that there might be synergistic effects between AVP1 overexpression and OsSIZ1 overexpression, which could lead to substantially increased yields if these two genes are co-overexpressed in real crops. To test this hypothesis, AVP1 and OsSIZ1 were co-overexpressed in cotton, and the impact of OsSIZ1/AVP1 co-overexpression on cotton's performance under normal growth and multiple stress conditions were analysed. It was found that OsSIZ1/AVP1 co-overexpressing plants performed significantly better than AVP1-overexpressing, OsSIZ1-overexpressing and wild-type cotton plants under single, as well as under multiple stress conditions in laboratory and field conditions. Two field studies showed that OsSIZ1/AVP1 co-overexpressing plants produced 133% and 81% more fibre than wild-type cotton in the dryland conditions of West Texas. This research illustrates that co-overexpression of AVP1 and OsSIZ1 is a viable strategy for engineering abiotic stress-tolerant crops and could substantially improve crop yields in low input or marginal environments, providing a solution for food security for countries in arid and semiarid regions of the world.

Keywords: AVP1; OsSIZ1; co-overexpression; drought stress; heat stress; salinity; transgenic cotton.

Figure 1

Molecular analysis of OsSIZ1/AVP1 co‐overexpressing cotton plants. (a) PCR analysis of control and OsSIZ1/AVP1 co‐overexpressing cotton plants. The gene fragments amplified are labelled on the right. (b) RNA blot analysis of control and OsSIZ1/AVP1 co‐overexpressing cotton plants. The genes used as probes are listed on the right. (c) DNA blot analysis of control and OsSIZ1/AVP1 co‐overexpressing cotton plants. Molecular weight markers are on the right. WT, wild‐type plant; SNT, segregated non‐transgenic plant; A, AVP1‐overexpressing plant; O, OsSIZ1‐overexpressing plant; OA1 to OA4, four independent OsSIZ1/AVP1 co‐overexpressing plants.

Figure 2

The effects of combined drought and salt stresses on control and OsSIZ1/AVP1 co‐overexpressing cotton plants. (a) Phenotypes of cotton plants six weeks after combined drought and salt treatment. (b) Photosynthetic rates of cotton plants under normal irrigation (black bar) and combined drought and salt stresses (grey bar). (c) Total seed fibre yields under normal irrigation (black bar) and combined drought and salt stresses (grey bar). Results are the means ± SE (n = 9). (d) Root phenotypes of cotton plants under combined drought and salt stresses. (e) Root biomass analysis of cotton plants under combined drought and salt stresses. Results are the means ± SE (n = 5). WT, wild‐type plant; SNT, segregated non‐transgenic plant; A, AVP1‐overexpressing plant; O, OsSIZ1‐overexpressing plant; OA1 to OA4, four independent OsSIZ1/AVP1 co‐overexpressing plants. Samples denoted by different letters are significantly different (P < 0.05, Tukey correction).

Figure 3

The effects of combined drought and heat stresses on control and OsSIZ1/AVP1 co‐overexpressing cotton plants. (a) The phenotype of cotton plants six weeks after combined drought and heat treatment. (b) Photosynthetic rates of cotton plants two hours before heat treatment (black bar), during heat treatment at 37 °C (light grey bar), and 3 h after heat treatment (dark grey bar). (c) Total seed fibre yields under normal irrigation (black bar) and combined drought and heat stresses (grey bar). Results are the means ± SE (n = 6). (d) Root phenotypes of cotton plants under combined drought and heat stresses. (e) Root biomass analysis of cotton plants under combined drought and heat stresses. Results are the means ± SE (n = 6). WT, wild‐type plant; SNT, segregated non‐transgenic plant; A, AVP1‐overexpressing plant; O, OsSIZ1‐overexpressing plant; OA1 to OA4, four independent OsSIZ1/AVP1 co‐overexpressing plants. Samples denoted by different letters are significantly different (P < 0.05, Tukey correction).

Figure 4

The performance of control and OsSIZ1/AVP1 co‐overexpressing cotton plants under rain‐fed conditions in 2016 and 2017. (a) Photosynthetic rates of cotton plants under the rain‐fed condition in 2016. (b) Photosynthetic rates of cotton plants under the rain‐fed condition in 2017. Results are the means ± SE (n = 8). (c) Fibre yield produced per plant under the rain‐fed conditions in 2016 (black bar) and 2017 (grey bar). Results are the means ± SE. WT, wild‐type plant; SNT, segregated non‐transgenic plant; A, AVP1‐overexpressing plant; O, OsSIZ1‐overexpressing plant; OA1 to OA4, four independent OsSIZ1/AVP1 co‐overexpressing plants. Samples denoted by different letters are significantly different (P < 0.05, Tukey correction).

Figure 5

Analysis of water use efficiency and canopy temperature of control and OsSIZ1/AVP1 co‐overexpressing cotton plants. (a) Phenotypes of cotton plants twenty‐five days after irrigating plants with an initial equal volume of water. (b) Water use efficiency of cotton plants in biomass produced per gram of water consumed by plant. Data are the means ± SE (n = 10). (c) Canopy temperatures of control and OsSIZ1/AVP1 co‐overexpressing cotton plants grown in field under the rain‐fed condition in 2017. Data are the means ± SE (n = 65). WUE, water use efficiency; WT, wild‐type plant; SNT, segregated non‐transgenic plant; A, AVP1‐overexpressing plant; O, OsSIZ1‐overexpressing plant; OA1 to OA4, four independent OsSIZ1/AVP1 co‐overexpressing plants. Samples denoted by different letters are significantly different (P < 0.05, Tukey correction).

Figure 6

Phenotype and biomass analysis of control and OsSIZ1/AVP1 co‐overexpressing cotton plants grown in hydroponic condition under different concentrations of inorganic phosphorus. (a) Phenotype of control and OsSIZ1/AVP1 co‐overexpressing cotton plants under low phosphate concentration (5 µm KH₂PO₄). (b) Phenotype of control and OsSIZ1/AVP1 co‐overexpressing cotton plants under high phosphate concentration (1000 µm KH₂PO₄). (c) Dry‐root biomass of control and OsSIZ1/AVP1 co‐overexpressing cotton plants grown under low (black bar) and high phosphate (grey bar) concentrations. (d) Dry‐shoot biomass of control and OsSIZ1/AVP1 co‐overexpressing cotton plants grown under low (black bar) and high phosphate (grey bar) conditions. Results are the means ± SE (n = 6). WT, wild‐type plant; SNT, segregated non‐transgenic plant; A, AVP1‐overexpressing plant; O, OsSIZ1‐overexpressing plant; OA1 to OA4, four independent OsSIZ1/AVP1 co‐overexpressing plants. Samples denoted by different letters are significantly different (P < 0.05, Tukey correction).

Figure 7

RNA‐sequencing analysis of control and OsSIZ1/AVP1 co‐overexpressing cotton plants in the field before and after rain. (a) Numbers of differentially expressed genes before and after rain. Black bars represent down‐regulated genes, and grey bars represent up‐regulated genes in OsSIZ1/AVP1 co‐overexpressing plants vs. wild‐type plants before and after rainfall. (b) Venn diagram of transcripts showing differential expression in leaf tissues of OsSIZ1/AVP1 co‐overexpressing plants vs. wild‐type plants before and after rain. (c) Heatmap of 45 differentially expressed genes in OsSIZ1/AVP1 co‐overexpressing plants compared to wild‐type plants before and after rain. (d) Quantitative real‐time PCR analysis of ten stress‐related genes in OsSIZ1/AVP1 co‐overexpressing plants vs. wild‐type plants before rain (black bar) and after rain (grey bar). Data are means ± SE (n = 3). DEGs, differentially expressed genes; WT, wild‐type plant; OA1, OsSIZ1/AVP1 co‐overexpressing plant 1. Samples denoted by different letters are significantly different (P < 0.05, Tukey correction).

Figure 8

Proposed model to illustrate the potential molecular mechanisms behind the increased abiotic stress tolerance in OsSIZ1/AVP1 co‐overexpressing cotton plants under rain‐fed conditions. Under drought and heat stress conditions, co‐overexpression of OsSIZ1 and AVP1 in cotton leads to up‐regulation of genes involved in cell wall biosynthesis, cell wall remodelling, antioxidation metabolism, plant hormone signalling pathways, and protein homeostasis, which in turn results in increased tolerance to abiotic stresses and higher yields under dryland conditions.