Improvement of plant tolerance to drought stress by cotton tubby-like protein 30 through stomatal movement regulation

Abstract

Introduction: Cotton is a vital industrial crop that is gradually shifting to planting in arid areas. However, tubby-like proteins (TULPs) involved in plant response to various stresses are rarely reported in cotton. The present study exhibited that GhTULP30 transcription in cotton was induced by drought stress.

Objective: The present study demonstrated the improvement of plant tolerance to drought stress by GhTULP30 through regulation of stomatal movement.

Methods: GhTULP30 response to drought and salt stress was preliminarily confirmed by qRT-PCR and yeast stress experiments. Ectopic expression in Arabidopsis and endogenous gene silencing in cotton were used to determine stomatal movement. Yeast two-hybrid and spilt-luciferase were used to screen the interacting proteins.

Results: Ectopic expression of GhTULP30 in yeast markedly improved yeast cell tolerance to salt and drought. Overexpression of GhTULP30 made Arabidopsis seeds more resistant to drought and salt stress during seed germination and increased the stomata closing speed of the plant under drought stress conditions. Silencing of GhTULP30 in cotton by virus-induced gene silencing (VIGS) technology slowed down the closure speed of stomata under drought stress and decreased the length and width of the stomata. The trypan blue and diaminobenzidine staining exhibited the severity of leaf cell necrosis of GhTULP30-silenced plants. Additionally, the contents of proline, malondialdehyde, and catalase of GhTULP30-silenced plants exhibited significant variations, with obvious leaf wilting. Protein interaction experiments exhibited the interaction of GhTULP30 with GhSKP1B and GhXERICO.

Conclusion: GhTULP30 participates in plant response to drought stress. The present study provides a reference and direction for further exploration of TULP functions in cotton plants.

Keywords: Cotton; Drought; GhTULP30; Seed germination; Stomatal movement.

Graphical abstract

Fig. 1

Structural characteristics of GhTLUP30. (A) Alignment of GhTLUP30 and TULP sequences from other plants. F-box and Tub domains are marked with solid green and purple lines. Black asterisks represent conserved PIP₂ binding sites. (B) Three-dimensional GhTLUP30 model. (C) Phylogenetic tree and motif analysis of GhTLUP30, AtTLPs, SlTLFP8, CsTLP8, MdTLP7, and CaTLP1. (D) Cis-acting element analysis of GhTLUP30 promoter region.

Fig. 2

GhTULP30 responds to drought and salt stress in cotton and yeast. (A and B) qRT-PCR analysis of the GhTULP30 expression level after treatment of CCRI36 with NaCl and PEG6000. (C and D) Growth curves of yeast cells in liquid medium containing NaCl and mannitol after ectopic expression of GhTULP30. Values represent the mean ± SD from three biological replicates. UBQ7 gene was used as a control. (E, F and G) After ectopic expression of GhTULP30, the growth of yeast cells in a control, solid medium containing NaCl and mannitol. 1, 10^-1, 10^-2, 10^-3 and 10^-4 indicate the initial concentration of OD₆₀₀ ≈ 1 yeast diluted at 1:10 for 4 times, respectively. * and ** denote p ≤ 0.05 and 0.01, respectively.

Fig. 3

GhTULP30 affects seed germination and stomatal movement in Arabidopsis. (A, B and C) The germination status of 35S:GhTULP30 and WT Arabidopsis seeds on empty MS medium and MS medium containing NaCl or PEG6000. (D) The stomata of transgenic and WT Arabidopsis were opened and closed after drought stress. The red and blue circles indicate closed and open stomata, respectively. (E) The germination rate of 35S:GhTULP30 and WT Arabidopsis seeds on empty MS medium and MS medium containing NaCl or PEG6000. (F) The stomatal opening ratio of transgenic and WT Arabidopsis under drought stress condition. * and ** denote p ≤ 0.05 and 0.01, respectively.

Fig. 4

Silencing GhTULP30 affects the movement and size of cotton stomata. (A) Stomatal closure of leaves of control and VIGS plants under drought conditions after silencing GhTULP30. The red and blue circles indicate closed and open stomata, respectively. (B) Single stomatal closure in leaves of control and VIGS plants under drought conditions after silencing GhTULP30. (C) Stomatal opening rate of control and VIGS cotton under drought stress. (D) Statistics of stomatal size of control and VIGS cotton plants under drought stress. The expression levels of stress response genes PXG1 (F), RAB18 (G), CPN60B2 (H), and UBI3 (I) in VIGS and WT plants under drought stress. * and ** denote p ≤ 0.05 and 0.01, respectively.

Fig. 5

Drought tolerance of cotton decreases after GhTULP30 silencing. (A) Phenotypes of drought-stressed VIGS and control cotton plants. (B) GhTULP30 expression level in VIGS and control cotton plants under drought stress. (C) Trypan blue and DAB staining of VIGS and control leaves after drought stress. (D, E and F) Proline, CAT, and MDA content of VIGS and control cotton plants under drought stress. ** denotes p ≤ 0.01.

Fig. 6

Protein interaction and co-expression network analysis. (A) Yeast two-hybrid assay exhibiting auto-activation and interaction of GhTULP30 with GhSKB1B and GhXERICO. (B) Luciferase experiment confirmed the interaction of GhTULP30 with GhSKB1B and GhXERICO. (C) Co-expression network of GhTULP30. Nodes denote individual genes, and edges denote significant co-expression between genes. Pink lines denote positive co-expression relationship with the target protein; blue lines represent negative co-expression relationship with the target protein; and the orange line represents protein interaction with the target protein.

Fig. 7

Hypothetical GhTULP30 functional mechanism model. Plant cells receive external stress signals through various sensors. Several secondary messengers such as reactive oxygen species and Ca²⁺, plant hormones, and signal converters transmit these stress signals and act on related transcription factors. Transcription factors bind to the GhTULP30 promoter region to induce their expression. The GhTULP30-PIP₂ complex is reversible in the G-protein signaling pathway. Under drought and salt stress, GhTULP30-PIP₂ disintegrates, and GhTULP30 is transferred from the plasma membrane to the nucleus to transmit signals in response to the corresponding stress and affect plant development. The interaction between GhTULP30 and GhXERICO also affects plant response to stress and development. The SCF-type complex and ubiquitination of target proteins regulate drought and salt stress. The SCF-type complex functions within the ubiquitination reaction through combined action with the E1 and E2 enzymes. TULPs bind to SKP and targets through their F-box domain and C-terminal domain, respectively, thereby presenting the ubiquitination target. PIP₂, phosphatidylinositol 4, 5-bisphosphate; Tub, Tub domain; F-box, F-box domain; TFs, transcription factors. Ub, ubiquitin. Rbx1, ring-box protein.