Comprehensive Genomic Analysis and Expression Profiling of the NOX Gene Families under Abiotic Stresses and Hormones in Plants

Abstract

Plasma membrane NADPH oxidases (NOXs) are key producers of reactive oxygen species under both normal and stress conditions in plants and they form functional subfamilies. Studies of these subfamilies indicated that they show considerable evolutionary selection. We performed a comparative genomic analysis that identified 50 ferric reduction oxidases (FRO) and 77 NOX gene homologs from 20 species representing the eight major plant lineages within the supergroup Plantae: glaucophytes, rhodophytes, chlorophytes, bryophytes, lycophytes, gymnosperms, monocots, and eudicots. Phylogenetic and structural analysis classified these FRO and NOX genes into four well-conserved groups represented as NOX, FRO I, FRO II, and FRO III. Further analysis of NOXs of phylogenetic and exon/intron structures showed that single intron loss and gain had occurred, yielding the diversified gene structures during the evolution of NOXs family genes and which were classified into four conserved subfamilies which are represented as Sub.I, Sub.II, Sub.III, and Sub.IV. Additionally, both available global microarray data analysis and quantitative real-time PCR experiments revealed that the NOX genes in Arabidopsis and rice (Oryza sativa) have different expression patterns in different developmental stages, various abiotic stresses and hormone treatments. Finally, coexpression network analysis of NOX genes in Arabidopsis and rice revealed that NOXs have significantly correlated expression profiles with genes which are involved in plants metabolic and resistance progresses. All these results suggest that NOX family underscores the functional diversity and divergence in plants. This finding will facilitate further studies of the NOX family and provide valuable information for functional validation of this family in plants.

Keywords: NADPH oxidase (NOX); abiotic stress; coexpression; hormone; phylogenetic analysis.

Fig. 1.—

The FRO and NOX gene family in Plantae. (A) Systematic evolutionary relationships of 20 species among eight lineages within the supergroup Plantae. (B) The numbers of FRO and NOX homologs in each species. (C) The typical domain organization of FRO and NOX proteins. The transmembrane regions are shown with dotted pink box.

Fig. 2.—

Phylogenetic relationships and domain organization of FRO and NOX genes in plants. (A) The unrooted maximum-likelihood phylogenetic tree of FRO and NOX family members was inferred from the amino acid sequence alignment of the Ferric_reduct, FAD_binding_8 and NAD_binding_6 domains. The four conserved groups are marked by different colors and represented as NOX, FRO I, FRO II and FRO III. Scale bar represents 0.2 amino acid substitution per site. (B) Domain organizations of the four conserved groups of FRO and NOX proteins. The transmembrane regions are shown with dotted pink box.

Fig. 3.—

Phylogenetic relationships and exon/intron structure of NOX family genes in land plants. (A) The unrooted maximum-likelihood phylogenetic tree of FRO and NOX family members was inferred from the amino acid sequence alignment of the NADPH_Ox, Ferric_reduct, FAD_binding_8 and NAD_binding_6 domains. Numbers above the nodes represent bootstrap values from 1,000 replications. (B) Exon/intron structure of NOX family genes in land plants. Black boxes represent exons; black lines represent introns; The length of the boxes and lines are scaled relative to the length of the gene, and longer introns are denoted by a double slash; numbers 0, 1, and 2 are intron phases, which indicates the positions of introns between or within codons. Phase 0 introns are located between two consecutive codons, phase 1 introns are located between the first and second nucleotides of a codon, and phase 2 introns are located between the second and third nucleotides of a codon. The introns labeled with red mumber, that is, “1” or “2” were gained introns. The four conserved subfamilies of NOXs are represented as Sub. I, Sub. II, Sub.III and Sub.IV at the left side of the gene structures.

Fig. 4.—

The expansion and evolution of the FRO and NOX gene family in plants. (A) Schematic comparison of intron distribution in NOX orthologs of land plants generated with the CIWOG software. Black horizontal lines are aligned sequences; gray horizontal lines are gaps in the alignment; black vertical bars are conserved common introns and represented as I₁～I₁₄; red vertical bars are gained introns. The numbers 0, 1, and 2 are intron phases. The intron numbers of each NOX genes were showed on the right side and the gray numbers, that is, “+1,” “+2,” or “+3” are represented the number of gained introns. (B) A model for the expansion and evolution of the NOX gene family in Plantae. The evolutionary relationships among eight lineages (left) and a model for the expansion and evolution of the FRO and NOX gene family (right) within the supergroup Plantae.

Fig. 5.—

Developmental expression patterns of NOX family genes in Arabidopsis and rice. Expression profiles of (A) AtNOXs (i.e., AtRBOHA–J) and (B) OsNOXs (i.e., OsNOX1–9) in different developmental stages obtained from microarray data reported in Genevestigator. Results are shown as heat maps in white/gray/red (low to high) that reflect the percent of expression.

Fig. 6.—

Inducible expression patterns of NOX family genes in Arabidopsis under abiotic-stresses and hormone treatments. Expression levels of AtRBOHA–I assayed by qRT-PCR under (A) cold (4 °C), (B) heat (30 °C), (C) drought (20% PEG6000), (D) salt (200 mM NaCl), (E) oxidative (30 µM MV) stresses and (F) ABA (100 µM), (G) MeJA (100 µM) hormone treatments. Data are means ±SD (n = 3) and are representative of similar results from three independent experiments.

Fig. 7.—

Inducible expression patterns of NOX family genes in rice under abiotic-stresses and hormone treatments. Expression levels of OsNOX1–9 assayed by qRT-PCR under (A) cold (4 °C), (B) heat (40 °C), (C) drought (20% PEG6000), (D) salt (200 mM NaCl), (E) oxidative (30 µM MV) stresses and (F) ABA (100 µM), (G) MeJA (100 µM) hormone treatments. Data are means ± SD (n = 3) and are representative of similar results from three independent experiments.

Fig. 8.—

Expression profiles analysis of coexpression genes with OsNOXs by qRT-PCR under MeJA hormone treatment on the shoot of rice seedling. Data are means ± SD (n = 3) and are representative of similar results from three independent experiments. Abbreviations and locus names of genes used are available in supplementary table S2, Supplementary Material online.