Genome-wide investigation and expression profiling of APX gene family in Gossypium hirsutum provide new insights in redox homeostasis maintenance during different fiber development stages

Abstract

Ascorbate peroxidase (APX) is a member of heme-containing peroxidases which catalyze the H₂O₂-dependent oxidation of a wide range of substrates in plants and animals. As is known, H₂O₂ acts as a signaling molecule in the regulation of fiber development. Our previous work reported that ascorbate peroxidase 1 (GhAPX1) was important for cotton fiber elongation. However, knowledge about APX gene family members and their evolutionary and functional characteristics in cotton is limited. Here, we report 26 GhAPX genes by genome-wide investigation of tetraploid cotton Gossypium hirsutum. Phylogenetic and gene structure analyses classified these APX members into five clades and syntenic analysis suggested two duplication events. Expression profiling of the 26 APXs revealed that ten members are expressed in cotton fibers. Notably, GhAPX10A, GhAPX10D, GhAPX12A, and GhAPX12D showed high expression levels in 30-day fiber, while GhAPX1A/D, GhAPX3A/D, and GhAPX6A/D showed very low expression levels. The enzyme activity and H₂O₂ content assays revealed that cotton fiber kept high enzyme activity and the lowest H₂O₂ level in 30-day fibers, indicating that other than GhAPX1, the newly reported APX members are responsible for the reactive oxygen species homeostasis in the cotton fiber maturation stages. Expression profiling of ten fiber-expressed APXs after phytohormone treatments revealed their regulation patterns by different stimuli, suggesting that GhAPX1, GhAPX12A, and GhAPX12D are responsible to most phytohormone treatments. Our data provided evolutionary and functional information of GhAPX gene family members and revealed that different members are responsible to redox homeostasis during different cotton fiber development stages.

Keywords: Ascorbate peroxidase; Cotton fiber; Expression profiling; Gossypium hirsutum; Redox homeostasis.

Fig. 1

Chromosome distribution of GhAPX gene family. The 26 GhAPX genes were mapped to the chromosomes of A sub-genome (a) and un-assembled scaffolds (b), as well as that of D sub-genome (c) and un-assembled scaffold (d). Genes were referred as GhAPX1A–13A and GhAPX1D–13D, according to their organization order on the chromosomes, except for the previously reported GhAPX1A/D. Different scales were used for chromosomes and un-assembled scaffolds. Note that the chromosome information of scaffolds was known, although they could not be assembled to the corresponding chromosomes

Fig. 2

Phylogenetic and intron–exon structure analyses of APX family genes. a Protein sequences of APX gene families from Arabidopsis thaliana (AtAPX), G. hirsutum (GhAPX), Oryza sativa (OsAPX), Theobroma cacao (TcAPX), and Vitis vinifera (VvAPX) were used to construct an NJ phylogenetic tree. Bootstrap was set to 1000 replicates. Five clades were named as sub-family I to V, shadowed with different colours. b Intron–exon organization structure analysis of the 26 GhAPX genes was shown. Boxes and lines represented the exons and introns, and the genomic length was indicated at the bottom. (Color figure online)

Fig. 3

Tandem duplication and segmental duplication of GhAPXs and syntenic analysis between G. hirsutum and T. cacao APXs. Chromosomes and scaffolds from cotton (Gh) and cacao (Tc) are shown in yellow and blue segments, respectively. The positions of the APX genes are marked by black lines in the circus. Duplicated GhAPXs are linked by red lines and syntenic relationships between cotton and cacao are linked by purple lines. (Color figure online)

Fig. 4

Motif analysis of GhAPXs. Conserved motifs of GhAPX protein sequences were analyzed (a). Ten different motifs were recognized and indicated with different colours. The organization order of motifs for each member of GhAPXs was highlighted. The conservation of the sequences for each conserved domain was also presented (b). (Color figure online)

Fig. 5

Expression profiling of GhAPX genes in different cotton tissues and different development stage of cotton fibers. a Heat map of the qRT-PCR data for the 26 GhAPXs in eight different cotton tissues. Orthologs with very high identities that could not be distinguished by gene-specific primers were examined together use the same primers and indicated by slashes. b Semi-quantitative RT-PCR results of GhAPX genes corresponding to that of in a. c Heat map of the qRT-PCR data for ten fiber-preferentially accumulated GhAPXs in eight development stages of cotton fibers. Orthologs GhAPX1A/D, GhAPX3A/D, and GhAPX6A/D were detected use the same primers, respectively. d Semi-quantitative RT-PCR results of GhAPX genes corresponding to that of in c. Relative expression levels were normalized by the internal control UBQ gene

Fig. 6

Dynamic changes of the APX enzyme activity and H₂O₂ content in different stages of cotton fibers. a Dynamic changes of APX enzyme activity in different stages of cotton fibers (from 5 to 30 dpa). b Dynamic changes of H₂O₂ content in different stages of cotton fibers (from 5 to 30 dpa)

Fig. 7

Cis-regulatory elements analysis and expression profiling of GhAPXs response to plant hormone (a–e). The putative cis-regulatory elements of GhAPXs were labeled with capital letters in the figure according to their relative position. The green letters stand for stress response cis-elements, the blue letters stand for plant hormone response cis-elements, and the red letters stand for transcription initiation cis-elements. More details about cis-regulatory elements are shown in S4 Table. f Heat map of the qRT-PCR data for ten fiber-preferentially accumulated GhAPXs in 10-day fibers treated with different plant hormones at different hours. Orthologs GhAPX1A/D, GhAPX3A/D, and GhAPX6A/D were detected using the same primers, respectively. Relative expression levels were normalized by the internal control UBQ gene. (Color figure online)