GhLCYε-3 characterized as a lycopene cyclase gene responding to drought stress in cotton

Abstract

Drought stress significantly affects crop productivity. Carotenoids are essential photosynthetic pigment for plants, bacteria, and algae, with signaling and antioxidant functions. Lutein is a crucial branch product in the carotenoid synthesis pathway, which effectively improves the stress tolerance of higher plants. lycopene cyclase, a central enzyme for lutein synthesis, holds great significance in regulating lutein production. This research establishes a correlation between lutein content and stress resistance by measuring the drought resistance and lutein content of various cotton materials. To identify which crucial genes are associated with lutein, the lycopene cyclase family (LCYs) was analyzed. The research found that LCYs form a highly conserved family divided into two subfamilies, LCY-ε (lycopene ε-cyclase) and LCY-β (lycopene β-cyclase). Most members of the LCY family contain photoresponsive elements and abscisic acid elements. qRT-PCR demonstrates showed that most genes responded positively to drought stress, and GhLCYε-3 was expressed significantly differently under drought stress. Virus-induced gene silencing (VIGS) assay showed that the content of GhLCYε-3 was significantly increased with MDA and PRO, and the contents of chlorophyll and lutein were significantly decreased in pYL156 plants. The decrease in GhLCYε-3 expression is speculated to lead to reduced lutein content in vivo, resulting in the accumulation of reactive oxygen species (ROS) and decreased drought tolerance. This research enriched the understanding of LCY gene family and lutein function, and provided a new reference for cotton planting in arid areas.

Synopsis: Lycopene cyclase plays an important role in enhancing the ability of scavenging ROS and drought resistance of plants.

Keywords: Carotenoids; Cotton; Functional verification; Lutein; Lycopene cyclase; Reactive oxygen species (ROS).

Graphical abstract

Fig. 1

Correlation between germination, root length, fresh weight and lutein content of 12 cotton materials. Red shading indicate a positive correlation. Blue shading represents the strength of the negative correlation coefficient, with a closer absolute value to 1 indicating a stronger correlation.

Fig. 2

Conservative motifs analysis of LCYs sequences. Annotation：(A) and (B) representconserved basal sequences of four cotton species in the LCY-β group and LCY-ε group, respectively.

Fig. 3

A: Evolutionary analysis of LCY family members in four cotton species; B: Phylogenetic relationships of 49 LCYs proteins form G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, A. thaliana, O. sativa, V. vinifera, P. trichocarpa, T. cacao, O. sativa, and Z. mays.

Fig. 4

Chromosome distribution of LCYs gene family in four cotton species. Annotation (A) (B) (C) (D) Chromosomal location of LCYs on chromosomes in G. hirsutum、 G. arboreum、 G. barbadense.and G. raimondii.

Fig. 5

Conservative motifs andcis-actingelements of LCY gene family from G. hirsutum, G. barbadense, G. raimondii, and G. arboreum.

Fig. 6

Analysis of expression patterns for LCYs genes in response to abiotic stresses. Annotation：.

Fig. 7

Protein–protein interaction analysis of LCYs proteins. Annotation: 10 protein in terrestrial cotton interact with the GhLCYε-3.

Fig. 8

3D structure of 8 GhLCYs proteins. α-helices are indicated by red, β-strands are indicated by yellow, and random coils are indicated by blue. Annotation:α-helices are indicated by red, β-strands are indicated by yellow, and random coils are indicated by blue.

Fig. 9

Analysis of GhLCY gene family expression under two treatments (CK and PEG) Annotation:* *P ≤ 0.01,*P ≤ 0.05.

Fig. 10

(A) Phenotype of cotton after silence of GhLCYε-3 gene under stress. PDS: positive control; CK: control plant; pYL156 plants: negative control; VIGS: pYL156 GhLCY ε- 3 plants; VIGS+ PEG: pYL156 GhLCY ε- 3 plants+ PEG (B) Silencing efficiency of GhLCYε-3 (C) Chlorophyll content of pYL156 +PEG and VIGS + PEG leaves under drought stress (D) Lutein content of pYL156 +PEG and VIGS + PEG leaves under drought stress (E) (F) MDA and Pro content in pYL156 + PEG and VIGS + PEG leaves. (G) Water loss rates of CK, pYL156 +PEG and VIGS+PEG cotton leaves under drought (H) DAB staining (I) Expression of 10 genes downstream of lutein. Annotation:* 0.01 <p < 0.05, P * * < 0.01.

Fig. 11

Mechanism of GhLCYε-3 regulating cotton response to drought stress. Annotation：lycopene (Synthetic substrates).GhLCYε-3 (Lycopene ε-cyclase) lutein (Clear ROS) MDA (Malondialdehyde) PRO (Proline).