Vol.:(0123456789) 1 3
Plant Cell Reports
https://doi.org/10.1007/s00299-021-02726-0
ORIGINAL ARTICLE
Characterization of APX and APX-R gene family in Brassica juncea
and B. rapa for tolerance against abiotic stresses
Deepika Verma
1
· Santosh Kumar Upadhyay
2
· Kashmir Singh
1
Received: 30 March 2021 / Accepted: 31 May 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021
Abstract
Key Message APX and APX-R gene families were identified and characterized in two important oilseed species of
Brassica. Gene expression under abiotic stress conditions, recombinant protein expression, and analysis further
divulged their drought, heat, and salt-responsive behavior.
Abstract Ascorbate peroxidases (APX) are heme-dependent enzymes that rid the cells of H
2
O
2
and regulate diverse biological
processes. In the present study, we performed APX gene family characterization in two Brassica sp. (B. juncea and B. rapa)
as these are commercially important oilseed crops and afected severely by abiotic stresses. We identifed 16 BjuAPX and 9
BraAPX genes and 2 APX-R genes each in B. juncea and B. rapa genomes, respectively. Phylogenetic analysis divided the
APX genes into fve distinct clades, which exhibited conservation in the gene structure, motif organization, and sub-cellular
location within the clade. Structural analysis of APX and APX-R proteins revealed the amino acid substitutions in conserved
domains of APX-R proteins. The expression profling of BjuAPX and BraAPX genes showed that 3 BjuAPX, 7BraAPX, and 2
BraAPX-R genes were drought and heat responsive. Notably, BjuAAPX1a, BjuAPX1d, BjuAAPX6, BraAAPX1a, BraAAPX2,
and BraAAPX3b showed high expression levels in RT-qPCR. Cis-regulatory elements in APX and APX-R gene promoters
supported the diferential behavior of these genes. Further, two stress-responsive genes BjuAPX1d and BraAAPX2 were
cloned, characterized, and their roles were validated under heat, drought, salt, and cold stress in bacterial expression system.
This study for the frst time reports the presence of APX activity in dimeric and LMW form of purifed BraAAPX2 protein.
The study may help pave way for developing abiotic stress-tolerant Brassica crops.
Keywords Ascorbate peroxidase · Gene expression · Cloning and characterization · APX activity · Overexpression ·
Brassica
Introduction
Exposure of plants to abiotic and biotic stresses produce
reactive-oxygen species (ROS) like superoxide anion (O
2
−
),
hydrogen peroxide (H
2
O
2
), singlet oxygen (
1
O
2
), and the
hydroxyl radical (HO·) in diferent parts of cell (Chen et al.
2017; Zhou et al. 2018). Defense mechanisms to cope
with serious threats posed by ROS includes enzymatic and
non-enzymatic components. The non-enzymatic components
are ascorbate (AsA), carotenoids, tocopherol, glutathione
(GSH), and phenolic compounds. Major defensive enzymes
are Superoxide Dismutase (SOD), Catalase (CAT), and
enzymes of ascorbate–glutathione cycle [Ascorbate Peroxi-
dase (APX), Glutathione Peroxidase (GPX), Mono Dehy-
dro Ascorbate Reductase (MDHAR), Dehydro Ascorbate
Reductase (DHAR), Glutathione Reductase (GR)]. Func-
tional existence of ROS scavenging enzymes is reported
in various cellular compartments like cytosol, chloroplast
(stroma and thylakoid), mitochondrion, glyoxisome, peroxi-
some, etc. (Mittler 2002; Mittler et al. 2004).
APX (E.C.1.1.11.1), a Class I type heme-peroxidase is an
important component of the Ascorbate–Glutathione cycle
“Foyer-Halliwell-Asada pathway”, which uses ascorbate as
an electron donor to convert H
2
O
2
into H
2
O and O
2
(Caver-
zan et al. 2012; Das and Roychoudhury 2014). Three classes
Communicated by Vijay Pratap Singh.
* Kashmir Singh
kashmirbio@pu.ac.in; kashmir123@gmail.com
1
Department of Biotechnology, BMS Block I, Panjab
University, Sector 25, Chandigarh 160014, India
2
Department of Botany, Panjab University, Sector 14,
Chandigarh 160014, India