PeCHYR1, a ubiquitin E3 ligase from Populus euphratica, enhances drought tolerance via ABA-induced stomatal closure by ROS production in Populus

Abstract

Drought, a primary abiotic stress, seriously affects plant growth and productivity. Stomata play a vital role in regulating gas exchange and drought adaptation. However, limited knowledge exists of the molecular mechanisms underlying stomatal movement in trees. Here, PeCHYR1, a ubiquitin E3 ligase, was isolated from Populus euphratica, a model of stress adaptation in forest trees. PeCHYR1 was preferentially expressed in young leaves and was significantly induced by ABA (abscisic acid) and dehydration treatments. To study the potential biological functions of PeCHYR1, transgenic poplar 84K (Populus alba × Populus glandulosa) plants overexpressing PeCHYR1 were generated. PeCHYR1 overexpression significantly enhanced H₂ O₂ production and reduced stomatal aperture. Transgenic lines exhibited increased sensitivity to exogenous ABA and greater drought tolerance than that of WT (wild-type) controls. Moreover, up-regulation of PeCHYR1 promoted stomatal closure and decreased transpiration, resulting in strongly elevated WUE (water use efficiency). When exposed to drought stress, transgenic poplar maintained higher photosynthetic activity and biomass accumulation. Taken together, these results suggest that PeCHYR1 plays a crucial role in enhancing drought tolerance via ABA-induced stomatal closure caused by hydrogen peroxide (H₂ O₂ ) production in transgenic poplar plants.

Keywords: PeCHYR1; Populus; abscisic acid; drought tolerance; stomatal closure.

Figure 1

Amino acid sequence alignment and phylogenetic tree of different CHYR1 protein family members. (a) Phylogenetic analysis of the PeCHYR1 homologs from Populus, Amborella, Eucalyptus, Malus, Prunus, Theobroma, Picea, Pinus, Salix, Arabidopsis, rice and maize. (b) Multiple alignment of the amino acid sequences of CHYR1 proteins from Populus, Salix and Arabidopsis.

Figure 2

PeCHYR1 expression patterns in different tissues and under different treatments. (a) Relative expression levels of the PeCHYR1 gene in different tissues of P. euphratica. Young leaf; Adult leaf; Old leaf; Phloem; Xylem; Root. (b) Transcript levels of PeCHYR1 were measured by RT‐qPCR in response to dehydration. (c) Transcript levels of PeCHYR1 were measured by RT‐qPCR in response to ABA. (d) DAB staining in WT seedling leaves during ABA treatment. Error bars are means ± SE (n = 20). Asterisks denote significant differences: *P < 0.05; **P < 0.01.

Figure 3

PeCHYR1 is targeted to the endoplasmic reticulum (ER) and nucleus. (a) Subcellular localization of 35S: GFP and 35S:PeCHYR1‐GFP in transiently expressed tobacco leaves. HDEL‐RFP was used as an ER localization marker fused with red fluorescent protein (RFP). The nuclear dye DAPI (blue) was applied to mark the nucleus. Bar = 10 μm. (b) Subcellular localization of 35S: GFP and 35S:PeCHYR1‐GFP in transiently expressed Arabidopsis leaf protoplasts. 35S: HDELRFP was cotransformed with 35S:PeCHYR1‐GFP to verify the ER localization of PeCHYR1. The nuclear dye DAPI (blue) was applied to mark the nucleus. Bars = 5 μm.

Figure 4

PeCHYR1 promotes ABA‐induced stomatal closure via ROS production. (a) DAB staining shows different levels of ABA‐induced ROS production in the leaves of WT and 35S:PeCHYR1. Scale bars = 1 cm (b) Representative confocal images of 200 μm ABA‐induced H₂O₂ production (10 min) in the guard cells of WT poplar and transgenic lines coloured with H2DCFDA. (c) Quantification of H₂O₂ production in the guard cells of WT poplar and transgenic lines. (d) Detection of ABA‐induced stomatal closure in the leaves of the WT and transgenic lines; leaves were separated and immersed under light in stomata‐opening solution (OS) for 2 h and then treated with 5 μm ABA for 2 h (OS → ABA). (e) Stomatal closure was observed at 0, 1 and 2 h of ABA treatment with scanning electron microscopy of stomatal aperture. Scale bars = 50 μm. Error bars are means ± SE (n = 50). Asterisks denote significant differences: **P < 0.01.

Figure 5

Light response curves were measured in WT, OXPeCHYR1‐1 and OXPeCHYR1‐8. Light response curves were measured in the same greenhouse conditions. (a) A–light curve. (b) Gs–light curve. (c) Transpiration–light curve. (d) Instantaneous WUE–light curve. (e) VPD–light curve. Data are means ± SE (n = 25). Asterisks denote significant differences: **P < 0.01.

Figure 6

35S:PeCHYR1 plants exhibited increased tolerance during short‐term drought treatment. (a) Morphological differences in short‐term drought assays. Bars = 10 cm. Quantitative measurement of leaf RWC (b), MDA content analysis (c), REC (relative electrical conductance) (d), H₂O₂ (e), activity of peroxidase (POD) (f), and activity of superoxide dismutase (SOD) (g) in the leaves of WT and 35S:PeCHYR1 plants under normal and drought stress conditions. Values are means ± SE (n = 40). All asterisks denote significant differences: **P < 0.01.

Figure 7

Variation in photosynthetic parameters of 35S:PeCHYR1 plants relative to those of WT plants during drought treatment and changes in the water loss rate of 35S:PeCHYR1 plants relative to that of WT plants during natural dehydration. (a) Transpiration–drought time curve. (b) Gs–drought time curve. (c) A–drought time curve. (d) Water loss from detached leaves is indicated as a percentage of initial fresh weight. Values are means ± SE (n = 25). All asterisks denote significant differences: *P < 0.05; **P < 0.01.

Figure 8

35S:PeCHYR1 poplars exhibited high drought tolerance during long‐term drought. (a) Morphological differences between WT and transgenic plants during long‐term drought assays. Bars = 10 cm. The contents of chlorophyll a and b (b), Maximal PSII quantum yield (Fv/Fm) (c), and leaf relative water content (RWC) (d) under 70% soil RWC and 45% soil RWC. Data are means ± SE (n = 40). Asterisks denote significant differences: **P < 0.01.

Figure 9

PeCHYR1 positively regulates plant height and biomass increase under long‐term drought. (a) Plant height under 70% soil RWC and 45% soil RWC. (b) Stem height growth rate under 70% soil RWC and 45% soil RWC. (c) Shoot biomass, (d) root biomass, (e) whole plant biomass, and (f) root–shoot ratio in WT and 35S:PeCHYR1 poplars under no water stress and mild water stress. Data are means ± SE (n = 25). Asterisks denote significant differences: *P < 0.05; **P < 0.01.

Figure 10

Stress‐responsive genes expressed in WT and 35S:PeCHYR1 poplar plants under nonstress conditions and instantaneous drought stress conditions. Relative expression levels of PeSnRK2.6 (a), PeLEA14 (b), PeRbohD (c) and PeRbohF (d) in WT and 35S:PeCHYR1 poplar plants under nonstress conditions and 2 h of drought stress. Total RNA was extracted from 2‐week‐old seedlings treated with drought stress for 2 h, and RT‐qPCR was performed. Data are means ± SE (n = 20). Asterisks denote significant differences **P < 0.01.