Transcript Profiling Reveals the Presence of Abiotic Stress and Developmental Stage Specific Ascorbate Oxidase Genes in Plants

Abstract

Abiotic stress and climate change is the major concern for plant growth and crop yield. Abiotic stresses lead to enhanced accumulation of reactive oxygen species (ROS) consequently resulting in cellular damage and major losses in crop yield. One of the major scavengers of ROS is ascorbate (AA) which acts as first line of defense against external oxidants. An enzyme named ascorbate oxidase (AAO) is known to oxidize AA and deleteriously affect the plant system in response to stress. Genome-wide analysis of AAO gene family has led to the identification of five, three, seven, four, and six AAO genes in Oryza sativa, Arabidopsis, Glycine max, Zea mays, and Sorghum bicolor genomes, respectively. Expression profiling of these genes was carried out in response to various abiotic stresses and during various stages of vegetative and reproductive development using publicly available microarray database. Expression analysis in Oryza sativa revealed tissue specific expression of AAO genes wherein few members were exclusively expressed in either root or shoot. These genes were found to be regulated by both developmental cues as well as diverse stress conditions. The qRT-PCR analysis in response to salinity and drought stress in rice shoots revealed OsAAO2 to be the most stress responsive gene. On the other hand, OsAAO3 and OsAAO4 genes showed enhanced expression in roots under salinity/drought stresses. This study provides lead about important stress responsive AAO genes in various crop plants, which could be used to engineer climate resilient crop plants.

Keywords: abiotic stress; ascorbate oxidase; genome-wide analysis; qRT-PCR; reactive oxygen species (ROS).

FIGURE 1

Chromosomal distribution of AAO genes in diverse genera. Chromosomal distribution of AAO genes in (A) rice, (B) Arabidopsis, (C) Zea mays, (D) Glycine max, and (E) Sorghum bicolor. Only the chromosomes having ascorbate oxidase (AAO) genes are shown. The scale is in (300 Mb). The chromosomal position (Mb) depicted in parenthesis along with gene name for each AAO is also marked as horizontal bar on the chromosome. Pairs of segmentally duplicated genes are shown by a dotted line.

FIGURE 2

Phylogenetic analysis of AAO from diverse genera. An unrooted parsimonious tree of AAO proteins showing different clades. Clade I is marked in orange color while, clade II and clade III are marked in purple and green color, respectively. The outer circle denotes monocot (blue color) and dicot (red color) plant species taken for phylogenetic study. The tree was plotted using Mega7 software.

FIGURE 3

Conserved motif identification in AAO proteins from diverse plant genera (A) Interpro analysis revealed three types of domains as a part of cupredoxin family in previously defined AAO proteins. (B) De novo motif identification of AAO proteins; motifs 1, 2, 3, 4, 5, and 6 show resemblance to cupredoxin family and motifs 7, 8, 9, and 10 are unidentified motifs.

FIGURE 4

Stress regulated expression of AAO genes. (A) Heatmap analysis of AAO genes OsAAO1, OsAAO2, OsAAO3, and OsAAO5 from rice in 7 days old seedlings. Heatmap is based on the microarray data depicting the expression profile of AAO genes under abiotic stress conditions such as, drought, salt, and cold (B) Heatmap analysis of AAO genes AtAAO1, AtAAO2, and AtAAO3 from 18 days old Arabidopsis seedlings. Microarray data depicting expression of AAO gene under various abiotic stress such as osmotic, cold, salt, drought, and oxidative stress. The dataset obtained correspond to shoot and root tissue at different time point of stress such as 0.5, 1, 3, 6, 12, and 24 h with respect to control. (C) Heatmap analysis of AAO genes ZmAAO1, ZmAAO2, ZmAAO3, and ZmAAO4 in response to abiotic stress. Heatmap is based on the microarray data depicting expression of AAO genes (i) under cold stress on different varieties of Zea mays (MO17, B73, and OH43), (ii) under drought stress of 6 and 24 h on different anatomical parts of germination stage with water potential ranging from -0.2 M. Pa to -0.8 M. Pa, (iii) in response to drought stress on different anatomical parts harvested from various developmental stages such as seedling stage, stem elongation, inflorescence and anthesis stage. The color bar in all figures represents the expression values, green color representing down regulation, black no change in expression and red signifies highest level of expression.

FIGURE 5

Developmental stage specific expression of AAO genes. Scatter plot displays the expression of AAO genes across various stages of development in (A) Oryza sativa, (B) Arabidopsis thaliana, (C) Glycine max, (D) Zea mays, and (E) Sorghum bicolor. Expression of different genes are represented by different color dots.

FIGURE 6

Expression pattern of AAO genes in rice based on RT-PCR and qRT-PCR analysis. (A) EtBr stained agarose gel depicting the expression of AAO genes in root and shoot tissue of rice seedling. Individual lanes show amplicons corresponding to OsAAO1–OsAAO5 amplified from rice root and shoot tissue using gene specific real-time PCR primers. M corresponds to 50 bp ladder. (B) Histogram representing fold change of OsAAO1, OsAAO2, OsAAO3, and OsAAO4 in 1 and 24 h stress treated shoot and root tissue of rice seedling based on qRT-PCR analysis. OsAAO5 could not be amplified hence it was not included in real-time analysis. Real-time PCR was done with cDNA template synthesized from shoot and root tissue of 10 days old control or stressed (salinity 200 mM NaCl and drought) rice seedlings.

FIGURE 7

Mechanistic details of possible pathway of AAO action in regulating stress phenotype of plants. (A) Reduced AA availability due to the oxidizing activity of AAO leads to stress sensitive phenotype in WT plants. (B) Enhanced AA availability due to the knock down of AAO gene leads to stress tolerant phenotype in AAO knock down plants.