Homologous Drought-Induced 19 Proteins, PtDi19-2 and PtDi19-7, Enhance Drought Tolerance in Transgenic Plants

Abstract

Drought-induced 19 (Di19) proteins play important roles in abiotic stress responses. Thus far, there are no reports about Di19 family in woody plants. Here, eight Di19 genes were identified in poplar. We analyzed phylogenetic tree, conserved protein domain, and gene structure of Di19 gene members in seven species. The results showed the Di19 gene family was very conservative in both dicotyledonous and monocotyledonous forms. On the basis of transcriptome data, the expression patterns of Di19s in poplar under abiotic stress and ABA treatment were further studied. Subsequently, homologous genes PtDi19-2 and PtDi19-7 with strong response to drought stress were identified. PtDi19-2 functions as a nuclear transcriptional activator with a transactivation domain at the C-terminus. PtDi19-7 is a nuclear and membrane localization protein. Additionally, PtDi19-2 and PtDi19-7 were able to interact with each other in yeast two-hybrid system. Overexpression of PtDi19-2 and PtDi19-7 in Arabidopsis was found. Phenotype identification and physiological parameter analysis showed that transgenic Arabidopsis increased ABA sensitivity and drought tolerance. PtDi19-7 was overexpressed in hybrid poplar 84K (Populus alba × Populus glandulosa). Under drought treatment, the phenotype and physiological parameters of transgenic poplar were consistent with those of transgenic Arabidopsis. In addition, exogenous ABA treatment induced lateral bud dormancy of transgenic poplar and stomatal closure of transgenic Arabidopsis. The expression of ABA/drought-related marker genes was upregulated under drought treatment. These results indicated that PtDi19-2 and PtDi19-7 might play a similar role in improving the drought tolerance of transgenic plants through ABA-dependent signaling pathways.

Keywords: ABA; PtDi19-2; PtDi19-7; drought stress; lateral bud dormancy; stomatal closure.

Figure 1

Phylogenetic tree and conserved protein structure of Di19s in seven species. (A) The phylogenetic tree constructed by the neighbor-joining method implemented by MEGA software. Different colors represent different subfamilies; Di19-S1/-S2/-S3 subfamilies use green, blue, and red, respectively. (B) Conserved protein domain analysis of Di19s. The amino acid length, zf-Di19 domain, and Di19_C domain are represented by black, olive green, and blue, respectively. The length of each pattern is displayed proportionally.

Figure 2

Gene structure of Di19s in seven species. Use GSDS online tool for gene structure analysis. Yellow boxes and black lines represent exons and introns, respectively. Blue box indicates the 5′ and 3′ non-coding regions. The length represents the size of exon and intron.

Figure 3

RNA-seq and qRT-PCR analysis of PtDi19 genes under stress. (A) Heat maps of the expression levels of eight PtDi19 genes in different tissues under drought, salt, and cold treatment. The legend was to show the relative high and low expressions, value = log₂ (fold change). (B,C) The induced expression pattern of PtDi19 with 20% PEG 6000 and 200 mmol/L NaCl root irrigation, respectively. The Y-axis indicates the relative expression levels, and 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h (X-axis) indicate hours of treatment.

Figure 4

qRT-PCR analysis of the expression profile of PtDi19 gene. (A,B) The induced expression pattern of PtDi19 with 100 µmol/L ABA root irrigation and 4 °C low temperature treatment, respectively. The Y-axis indicates the relative expression levels, and 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h (X-axis) indicate hours of treatment.

Figure 5

Subcellular localization of PtDi19–2 and PtDi19–7 in tobacco leaves. (A) The control and 35S::PtDi19–2::GFP fusion protein was separately expressed in tobacco leaves and observed by the use of a fluorescence microscope. The signal of the GFP channel exhibits a green color, and DAPI staining for the nucleus presents as a blue fluorescence; the bright field was jointly used for forming the merged channel. (B) The control and 35S::PtDi19–7::GFP fusion protein were separately expressed in tobacco leaves and observed by the use of a fluorescence microscope. The signal of the GFP channel exhibits a green color, and RFP channel exhibits red fluorescence; the bright field was jointly used for forming the merged channel. Scale bars = 20 µm.

Figure 6

PtDi19–2 and PtDi19–7 proteins both had transcriptional activity and interaction with each other in yeast. (A) A schematic diagram illustrating the PtDi19–7 and cDNA fragments encoding different portions of PtDi19–2, which were fused to DNA sequences encoding the GAL DNA binding domain in the yeast vector pGBKT7. (B) Transactivation activity of the PtDi19–2 and PtDi19–7 protein in yeast. (C) Recombinant plasmids of BD–PtDi19–2–N and AD–PtDi19–7 co-transformed into yeast strain AH109, and then plated on a selective medium.

Figure 7

The overexpression Arabidopsis of PtDi19–2 and PtDi19–7 were sensitive to ABA and showed strong drought tolerance. (A) We spotted the seeds of WT and overexpression lines evenly on 1/2MS, 1/2MS+0.7 µMABA, or 1/2MS+mannitol (300 mM, 350 mM, and 400 mM) to observe seed germination. (B) Counting of the germination rate of different lines. Asterisks indicate a significant difference compared to the corresponding controls (* p < 0.05 and ** p < 0.01). Error bars indicate SEs from three replicates.

Figure 8

The PtDi19–2 and PtDi19–7 improved drought resistance in Arabidopsis. (A) Three week WT and transgenic plants were withheld water for 10 days to induce dehydration. After dehydration for 10 days, the representative images taken. (B) Relative water content of the leaves. (C) Electrolyte leakage. (D) Proline contents. (E) POD activity. (F) MDA content. (G) DAB staining. A p-value of <0.01 was considered to be extremely significant (**).

Figure 9

PtDi19–7 improved drought resistance in poplar. (A) One-month-old wild-type 84K poplar and oxPtDi19–7 lines were withheld water for 8 days to induce dehydration. After dehydration for 8 days, the representative images were taken. (B) Relative water content of the leaves. (C) Proline contents. (D) Chlorophyll content. (E) MDA content. (F) POD activity. (G) Electrolyte leakage. A p-value of <0.01 was considered to be extremely significant (**).

Figure 10

PtDi19–7 affects root development of transgenic poplar and responds to exogenous ABA. (A) Phenotypic analysis of root length after 8 days of drought treatment. (B) The root length measurement. (C) The root fresh weight. (D) The root dry weight. (E) Lateral bud outgrowth of short shoot segments grown for 3 weeks on 1/2 MS medium supplemented with ABA (5/10 µM) or without ABA of WT and oxPtDi19–7 plants. A p-value of <0.05 was considered to be significant (*), and a p-value of <0.01 was considered to be extremely significant (**).