Co-overexpression of the Constitutively Active Form of OsbZIP46 and ABA-Activated Protein Kinase SAPK6 Improves Drought and Temperature Stress Resistance in Rice

Abstract

Drought is one of the major abiotic stresses threatening rice (Oryza sativa) production worldwide. Drought resistance is controlled by multiple genes, and therefore, a multi-gene genetic engineering strategy is theoretically useful for improving drought resistance. However, the experimental evidence for such a strategy is still lacking. In this study, a few drought-responsive genes from rice were assembled by a multiple-round site-specific assembly system, and the constructs were introduced into the rice cultivar KY131 via Agrobacterium-mediated transformation. The transgenic lines of the multi-gene and corresponding single-gene constructs were pre-evaluated for drought resistance. We found that the co-overexpression of two genes, encoding a constitutively active form of a bZIP transcription factor (OsbZIP46CA1) and a protein kinase (SAPK6) involved in the abscisic acid signaling pathway, showed significantly enhanced drought resistance compared with the single-gene transgenic lines and the negative transgenic plants. Single-copy lines of this bi-gene combination (named XL22) and the corresponding single-gene lines were further evaluated for drought resistance in the field using agronomical traits. The results showed that XL22 exhibited greater yield, biomass, spikelet number, and grain number under moderate drought stress conditions. The seedling survival rate of XL22 and the single-gene overexpressors after drought stress treatment also supported the drought resistance results. Furthermore, expression profiling by RNA-Seq revealed that many genes involved in the stress response were specifically up-regulated in the drought-treated XL22 lines and some of the stress-related genes activated in CA1-OE and SAPK6-OE were distinct, which could partially explain the different performances of these lines with respect to drought resistance. In addition, the XL22 seedlings showed improved tolerance to heat and cold stresses. Our results demonstrate that the multi-gene assembly in an appropriate combination may be a promising approach in the genetic improvement of drought resistance.

Keywords: SNF-1 related protein kinase; bZIP transcription factor; co-overexpression; drought stress; genetic transformation; transcriptome.

FIGURE 1

Construction and characterization of transgenic materials. (A) Schematic representation of the T-DNA region in XL22. The construct consists of three expression cassettes (35S::Hpt, 35S::OsbZIP46CA1, and 35S::SAPK6), each driven by a CaMV 35S promoter and terminated by a NOS terminator. (B) Enhanced drought resistance of the T₁ XL22 transgenic families during the pre-screening of drought resistance in the field. (C) Real-time quantitative PCR analysis of transcript levels in T₂ generation transgenic seedlings. The relative expression level was calculated via the 2^-ΔΔCT method with ubiquitin (LOC_Os03g13170) as an internal control. Error bars indicate the standard deviation (SD) based on three replicates.

FIGURE 2

Increased ABA sensitivity of the XL22 seedlings. (A) Performance of KY131-N and one of the three tested independent transgenic lines of XL22, CA1-OE, and SAPK6-OE seedlings planted in 1/2 strength MS medium containing 3 μM ABA or normal 1/2 strength MS medium. (B) Plant lengths of three independent lines for each construct and KY131-N grown on the normal or ABA-containing 1/2 strength MS medium. The plant length, not including the root, was measured on the 15th day after germination when the photos in (A) were taken. Columns represent the average plant length of three independent biological replicates (10 seedlings each) and error bars indicate the standard error (SE). Asterisks indicate the significant difference (^∗∗P < 0.01; ^∗P < 0.05) by Student’s t-test).

FIGURE 3

Enhanced drought resistance of the XL22 transgenic lines. (A) The plant performance of XL22, CA1-OE, and KY131-N at the beginning of the drought stress treatment. (B) The plant performance after one-month of recovery from the drought stress treatment. (C) Comparison of the agronomic traits of all the overexpressors measured after the recovery. The radar plot shows the relative average values of panicle number, yield, biomass, spikelet number, grain number, and filling rate, which are normalized to those of KY131-N (set as 1 in the plot).

FIGURE 4

XL22 plants were more resistant to drought treatment at the seedling stage. (A) Seedling performance of one of the three tested independent XL22, CA1-OE, and SAPK6-OE lines in pots before, during, and after drought stress treatment. (B) Survival rates of drought-treated seedlings after recovery. (C) Water loss rates of detached leaves from the seedlings of XL22, CA1-OE, and SAPK6-OE at the indicated time intervals. Error bars in (B) and (C) indicate the SE based on three independent replicates. Asterisks indicate the significant difference (^∗∗P < 0.01; ^∗P < 0.05) by Student’s t-test.

FIGURE 5

XL22 seedlings were more tolerant to extreme temperature. (A,B) The phenotypes of the XL22, CA1-OE, SAPK6-OE lines and KY131-N before, during, and after heat (A) and cold (B) stress treatment, respectively. (C,D) Survival rates were measured after recovery from heat (C) and cold (D) treatment, respectively. Error bars indicate the SE of three replicates. Asterisks indicate the significant difference (^∗∗P < 0.01; ^∗P < 0.05) by Student’s t-test).

FIGURE 6

Transcriptome profiling of the transgenic plants. (A) Heat map showing the expression patterns of drought-responsive genes in XL22, CA1-OE, and SAPK6-OE plants in normal growth conditions (–N) and drought stress conditions (–D). (B) Venn diagrams illustrating the overlap of drought up-regulated genes between XL22 and two single-gene overexpressors. Group I and II represent genes specifically up-regulated in XL22 compared to the up-regulated genes in CA1-OE and SAPK6-OE, respectively. Group III and IV genes were up-regulated in CA1-OE and SAPK6-OE, respectively, when both the single gene overexpressors were compared. (C) GO enrichment of the Group I-IV genes. Functionally related GO terms were combined in the pie chart for categorization (details in GO terms were presented in Supplementary Files S4–S7). (D) KEGG pathway enrichment of the Group I and II genes.

FIGURE 7

Predicted protein–protein interaction (PPI) network of Group I and II proteins. Six proteins with the greatest number of interactors within each group are highlighted. The histogram shows the relative expression levels of the highlighted genes in response to drought stress detected by qPCR using the same drought-treated samples for RNA-Seq. The relative expression levels were normalized to the corresponding lines grown under normal conditions (arbitrarily set as 1).