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S H O R T C O M M U N I C A T I O N Open Access
OsProDH Negatively Regulates
Thermotolerance in Rice by Modulating
Proline Metabolism and Reactive Oxygen
Species Scavenging
Mingxin Guo
1,2*
, Xiaotian Zhang
1
, Jiajia Liu
1
, Linlin Hou
1
, Hanxiao Liu
1
and Xusheng Zhao
1,2*
Abstract
Background: Global warming threatens rice growth and reduces yields. Proline plays important roles in plant
abiotic stress tolerance. Previous research demonstrated that engineering proline metabolism-related genes can
enhance tolerance to freezing and salinity in Arabidopsis.OsProDH encodes a putative proline dehydrogenase and is
a single copy gene in rice. However, whether OsProDH plays roles in abiotic stress in rice remains unknown.
Findings: Quantitative RT-PCR analysis revealed that OsProDH transcript contents were relatively higher in leaf blade
and root tissues and the high temperature treatment repressed expression of OsProDH. The predicted OsProDH
protein localized in mitochondria. Using the Oryza sativa ssp. japonica cultivar KY131, we generated OsProDH
overexpression (OE) lines and knockout mutant lines using the CRISPR/Cas9 (CRI) system. Overexpression of
OsProDH decreased proline content, while mutation of OsProDH increased proline content compared with that of
KY131. The CRI and OE lines were respectively more resistant and sensitive to heat stress than KY131. Heat stress
induced proline accumulation and mutation of OsProDH led to proline overproduction which reduced H
2
O
2
accumulation in the seedlings.
Conclusions: OsProDH negatively regulates thermotolerance in rice. Our study provides a strategy to improve heat
tolerance in rice via manipulating proline metabolism.
Keywords: Rice, OsProDH, Proline, Thermotolerance, Reactive oxygen species
Findings
High temperature stress reduces plant growth and crop
productivity, potentially resulting in widespread risk of
food insecurity (Battisti and Naylor 2009; Lobell et al.
2011). Declines in yield of crops, such as wheat, maize,
and barley, have likely resulted from increases in global
temperatures (Lobell and Field 2007). In particular, rice
yield has declined by 10% per 1 °C increase during the
dry season of crop growth (Peng et al. 2004). Therefore
to reduce risks of food insecurity due to rising global
temperatures, we must improve modern plant breeding
strategies to increase crop tolerance to heat stress by
expanding our understanding of the molecular mecha-
nisms underlying plant responses to heat stress and gen-
etic modifications of plants.
Proline is an essential proteinogenic amino acid and
plays important roles in plant abiotic-stress tolerance
(Nanjo et al. 1999; Székely et al. 2008; Zhang et al. 2017;
Liu et al. 2018). To date, much is known about proline
synthesis and metabolism in higher plants. Proline is syn-
thesized mainly from glutamate being converted into
glutamate-semialdehyde (GSA) by pyrroline-5-carboxylate
synthetase (P5CS). Then GSA is spontaneously converted
into pyrroline-5-carboxylate (P5C), which is reduced to
proline by P5C reductase (P5CR). Proline is degraded into
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* Correspondence: mxguolynu@126.com;xszhaolynu@126.com
1
College of Life Sciences, Luoyang Normal University, Luoyang 471934, China
Full list of author information is available at the end of the article
Guo et al. Rice (2020) 13:61
https://doi.org/10.1186/s12284-020-00422-3
glutamate by two key mitochondrial enzymes: proline de-
hydrogenase (ProDH) and pyrroline-5-carboxylate de-
hydrogenase (P5CDH). First, ProDH oxidizes proline into
delta
1
-pyrroline-5-carboxylate (P5C) which is subse-
quently converted into glutamate by P5CDH (Szabados
and Savouré 2010). In Arabidopsis,thep5cs1 mutant ex-
hibited a salt-hypersensitive phenotype that led to hyper-
accumulation of H
2
O
2
, increased chlorophyll damage and
lipid peroxidation (Székely et al. 2008). There are two
genes (AtProDH1 and AtProDH2) encoding proline de-
hydrogenase in Arabidopsis. The predicted pre-proteins
AtProDH1 and AtProDH2 share 75% identical amino
acids (Funck et al. 2010). Antisense suppression of
AtProDH1 led to greater tolerance to freezing and salinity
stress in A. thaliana (Nanjo et al. 1999). AtProDH2 was
specifically induced during salt stress and promoted pro-
line accumulation under the stress (Funck et al. 2010). So
far, much is known about the biological functions of core
enzymes involved in proline synthesis and metabolism in
Arabidopsis. However, little is known about the biological
functions of these key enzymes in rice.
In this study, we focused on OsProDH (Os10g0550900),
a single copy gene encoding the putative proline dehydro-
genase in rice. We cloned the coding sequence (CDS) and
genomic DNA sequence of OsProDH by PCR method
using the japonica variety Kongyu131 (KY131). We com-
pared the CDS with genomic DNA and found four exons
in OsProDH genomic DNA. The predicted pre-proteins of
OsProDH has 454 amino acids and with the proline de-
hydrogenase domain located at residues 133–436 by
searching the NCBI Conserved Domain Database (Add-
itional file 2: Figure S1). Furthermore, alignment of the
predicted protein sequences of OsProDH with AtProDH1,
AtProDH2, ZmProDH1, ZmProDH2 and SbProDH,
showed that these proteins all had proline dehydrogenase
domain and high similarity (Additional file 2: Figure S2).
Quantitative RT-PCR (qPCR) analysis revealed that
OsProDH transcripts can be detected in various tissues:
root, stem, leaf blade, leaf sheath, and young panicle. Ex-
pression levels were relatively higher in leaf blade and root
than other tissues (Fig. 1a). To examine the transcriptional
response of OsProDH to heat stress, two-leaf stage seed-
lings of KY131 were subjected to 45 °C treatment and the
shoots were sampled at 0, 0.5, 1, 2, 6, 12 h after treatment.
The results showed that heat stress clearly repressed the
expression level of OsProDH (Fig. 1b).
To determine the subcellular localization of OsProDH,
a35S::OsProDH-GFP vector was introduced into rice
protoplasts. As AtProDH1 and AtProDH2 were all local-
ized in mitochondria (Funck et al. 2010), we inferred
that the OsProDH might also be located in mitochon-
dria. Using a mitochondrial tracker, we observed that
OsProDH-GFP localized in mitochondria, whereas GFP
alone localized in the cytoplasm (Fig. 2). Thus results
show that OsProDH is a mitochondria-localized protein.
To investigate the biological function of OsProDH in
abiotic stress, we generated OsProDH overexpression
(OE) lines and knockout mutant lines using CRISPR/
Cas9 (CRI) system under KY131 background. Two OE
and CRI lines were chosen and characterized in detail
for this study (Fig. 3a, b). In CRI-1 and CRI-2 mutants,
there was a G and T insertion in the second exon, re-
spectively, resulting in frameshift, which led to a trun-
cated protein (453 aa) and mutant protein that is
Fig. 1 Expresson pattern of OsProDH.aTissue-specific expression of OsProDH detected by qPCR. R: root; S: stem; LB: leaf blade; LS: leaf sheath; YP:
young panicle. Roots were sampled from two-leaf stage seedlings. Stem, leaf blade, leaf sheath were sampled from two-month old plants. Panicle
in 5 cm length. The data shown are the mean values of three technical repeats with the SD. bTranscriptional response of OsProDH to high
temperature stress. Two-leaf stage KY131 seedlings were subjected to 45 °C treatment and OsProDH expression was determined in the shoots at
the indicated time points by qPCR analysis. OsActin was used as internal control. The data shown are the mean values of three technical repeats
with the SD
Guo et al. Rice (2020) 13:61 Page 2 of 5
disrupted at Leu202 and lacks the proline dehydrogenase
domain (Fig. 3b and Additional file 2: Figure S3).
Based on gene annotation and domain analyses, we
conclude that OsProDH encodes proline dehydrogenase.
To validate this conclusion, we determined the proline
contents of OE and CRI lines and KY131. Results re-
vealed that proline contents of the two CRI lines were
highest, followed by that of KY131, and then those of
the two OE lines (Fig. 3c). These results were consistent
with the annotation results.
We then investigated the biological function of OsProDH
in seedlings exposed to drought, salt and heat stresses.
Under drought and salt conditions, no obvious phenotypic
difference was detected between transgenic lines and
KY131 (data not shown). However, under high temperature
stress conditions, CRI and OE lines were respectively more
resistant and sensitive to heat stress than KY131 (Fig. 3d).
Specifically, the survival rates were higher in CRI lines and
much lower in OE lines than compared with that in KY131
(Fig. 3e). These results indicate that OsProDH negatively
regulate thermotolerance in rice seedlings.
Previous studies have reported that environmental
stresses such as drought (Choudhary et al. 2005), salinity
(Yoshiba et al. 1995), high light and UV irradiation (Sar-
adhi et al. 1995), heavy metals (Schat et al. 1997), and
oxidative stress (Yang et al. 2009) can induce proline ac-
cumulation in higher plants. Moreover, proline
accumulation in plants has a protective function under
stress conditions (Kishor et al. 2005; Verbruggen and
Hermans 2008). Therefore, we compared the proline
contents of wild type KY131 and transgenic seedlings
(OE and CRI lines) under 45 °C treatment for 48 h. The
results show that proline contents of all seedlings under
heat stress were greater than those of the seedlings
under the normal condition (Fig. 3c, f). Further, similar
to normal conditions, proline contents of CRI and OE
lines were significantly higher and lower than proline
content of KY131, respectively (Fig. 3f).
Abiotic stress induces ROS accumulation and exces-
sive ROS leads to programmed cell death (Gill and
Tuteja 2010). Several studies have revealed that proline
exhibits scavenging activity for reactive oxygen species
(ROS) and acts as a singlet oxygen quencher (Smirnoff
and Cumbes 1989; Matysik et al. 2002). These previous
discoveries led us to compare the H
2
O
2
levels of KY131
and transgenic seedlings under heat stress. We used 3,
3′-diaminobenzidine (DAB) staining to visually evaluate
H
2
O
2
accumulation in leaves. The brown precipitate in-
dicative of H
2
O
2
accumulation was generally not distrib-
uted in the leaves of both KY131 and transgenic lines
prior to the heat treatment (Fig. 3g). However, after
treatment, we observed more precipitate present in OE
leaves than in those of KY131, and more precipitate
present in KY131 leaves than those in CRI (Fig. 3g).
Fig. 2 Subcellular localization of OsProDH. GFP and the OsProDH-GFP fusion were transiently expressed in rice protoplasts. Using mitochondrial
tracker, it indicated the OsProDH -GFP fusion protein was specifically expressed in the mitochondria
Guo et al. Rice (2020) 13:61 Page 3 of 5
Taken together, these data suggest that mutation of
OsProDH led to greater proline accumulation which re-
duced H
2
O
2
accumulation and oxidative stress, ultim-
ately conferring higher survival rates despite the heat
treatment. Our study provides robust evidence support-
ing potential genetic approaches to improve crop ther-
motolerance by engineering proline metabolism.
Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s12284-020-00422-3.
Additional file 1. Materials and Methods.
Additional file 2: Figure S1. Gene structure and domain annotation of
OsProDH. Figure S2. Multiple sequence alignment of OsProDH,
Fig. 3 OsProDH negatively regulates thermotolerance in rice aTranscript levels of OsProDH detected by qPCR in KY131 and transgenic two
overexpression (OE) lines in shoots at the two-leaf stage rice seedlings. The data shown are the mean values of three technical repeats with the
SD. bGene structure of OsProDH and sequencing results at target sites in T1 plants produced by CRISPR/Cas9. cProline contents of KY131, OE
and CRI lines in shoots at the two-leaf stage rice seedlings under normal conditions. Values are the means ± SE, n= 3. Differences between the
KY131 and transgenic lines were analyzed with Student’st-test. (*P< 0.05). dPhenotypes of KY131, OE and CRI lines under 45 °C treatment. The
two-leaf stage seedlings were subjected to high temperature treatment for 48 h and then recovered at normal conditions. eSurvival rates of
KY131, OE and CRI lines after recovering 15 days at normal conditions. (n= 3 × 20). fProline contents of KY131, OE and CRI lines in shoots at the
two-leaf stage rice seedlings after 45 °C treatment for 48 h. Values are the means± SE, n= 3. Differences between the KY131 and transgenic lines
were analyzed with Student’st-test. (*P< 0.05; **P< 0.01). gDAB staining of KY131, OE and CRI lines leaves from plants under normal (left) and
stressed (right, 45 °C) conditions, respectively
Guo et al. Rice (2020) 13:61 Page 4 of 5
AtProDH1, AtProDH2, ZmProDH1, ZmProDH2 and SbProDH. The blue
lines indicated the conserved proline dehydrogenase domain. Figure S3.
Characterization of mutation in OsProDH. Protein sequences of OsProDH
in KY131 and mutants (CRI-1 and CRI-2) derived from the CRISPR-Cas9
system.
Additional file 3: Table S1. Primers used in this study were listed.
Abbreviations
KY131: Kongyu131; CDS: Coding sequence; OE: Overexpression; CRI: CRISPR/
Cas; GSA: Glutamate-semialdehyde; P5CS: Pyrroline-5-carboxylate synthetase;
P5CR: Pyrroline-5-carboxylate reductase; ProDH: Proline dehydrogenase;
P5CDH: Pyrroline-5-carboxylate dehydrogenase
Acknowledgements
Not applicable.
Authors’Contributions
MXG and XSZ conceived and designed the experiments. MXG, XTZ, JJL, LLH,
HXL performed the experiments. MXG analyzed the data. MXG wrote the
manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by the Key Science and Technology Program of
Henan Province (192102110058).
Availability of Data and Materials
The datasets supporting the conclusions of this article are included within
the article and its additional files.
Ethics Approval and Consent to Participate
Not applicable.
Consent for Publication
Not applicable.
Competing Interests
The authors declare that they have no competing interests.
Author details
1
College of Life Sciences, Luoyang Normal University, Luoyang 471934,
China.
2
Jujube Research Center, Luoyang Normal University, Luoyang 471934,
China.
Received: 8 May 2020 Accepted: 20 August 2020
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