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Article

Identification, Characterization, and Stress Responsiveness of Glucose-6-phosphate Dehydrogenase Genes in Highland Barley

by
Ruijun Feng
†,
Xiaomin Wang
†,
Li He
,
Shengwang Wang
,
Junjie Li
,
Jie Jin
and
Yurong Bi
*
Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2020, 9(12), 1800; https://doi.org/10.3390/plants9121800
Submission received: 12 November 2020 / Revised: 8 December 2020 / Accepted: 13 December 2020 / Published: 18 December 2020
(This article belongs to the Special Issue Plant Physiology and Environmental Stresses)
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Abstract

:
G6PDH provides intermediate metabolites and reducing power (nicotinamide adenine dinucleotide phosphate, NADPH) for plant metabolism, and plays a pivotal role in the cellular redox homeostasis. In this study, we cloned five G6PDH genes (HvG6PDH1 to HvG6PDH5) from highland barley and characterized their encoded proteins. Functional analysis of HvG6PDHs in E. coli showed that HvG6PDH1 to HvG6PDH5 encode the functional G6PDH proteins. Subcellular localization and phylogenetic analysis indicated that HvG6PDH2 and HvG6PDH5 are localized in the cytoplasm, while HvG6PDH1, HvG6PDH3, and HvG6PDH4 are plastidic isoforms. Analysis of enzymatic activities and gene expression showed that HvG6PDH1 to HvG6PDH4 are involved in responses to salt and drought stresses. The cytosolic HvG6PDH2 is the major isoform against oxidative stress. HvG6PDH5 may be a house-keeping gene. In addition, HvG6PDH1 to HvG6PDH4 and their encoded enzymes responded to jasmonic acid (JA) and abscisic acid (ABA) treatments, implying that JA and ABA are probably critical regulators of HvG6PDHs (except for HvG6PDH5). Reactive oxygen species analysis showed that inhibition of cytosolic and plastidic G6PDH activities leads to increased H2O2 and O2− contents in highland barley under salt and drought stresses. These results suggest that G6PDH can maintain cellular redox homeostasis and that cytosolic HvG6PDH2 is an irreplaceable isoform against oxidative stress in highland barley.

1. Introduction

Drought and salinity are major abiotic stresses limiting plant growth and development, thus causing a significant reduction in crop yield [1,2]. Drought and salinity in plants impose severe water deficit, which causes the inhibition of essential enzymes, the decrease in photosynthesis, leaf transpiration rates, nutrient supply, as well as reactive oxygen species (ROS) accumulation [3,4,5]. Although ROS, as signaling molecules, can regulate diverse physiological and molecular processes [6,7,8], excess ROS can cause oxidative damage and affect the level and function of plant hormones, ultimately leading to cell death and protein modifications [9,10,11]. Therefore, the fine-tuned regulation of ROS homeostasis is the critical protective strategy against salt and drought stresses.
The oxidative pentose phosphate pathway (OPPP) is ubiquitous in eukaryotes. OPPP provides abundant intermediate metabolites and nicotinamide adenine dinucleotide phosphate (NADPH), which are necessary for the synthesis of nucleotide, amino acid, and fatty acid, carbon fixation as well as nitrogen assimilation [2,12,13,14,15,16,17,18]. Moreover, biosynthesis of the cellular redox buffer, reduced glutathione, depends on NADPH via glutathione reductase [6,19]. In plants, OPPP-associated NADPH synthesis is present in both cytoplasm and plastid through the rate-limiting glucose-6-phosphate dehydrogenase (G6PDH) [20,21]. Numerous literature have reported that G6PDH is involved in growth and development, such as oil and lipid accumulation, seed germination, and RNA biosynthesis [17,21,22]. Moreover, G6PDH responses to abiotic stresses such as drought [23,24], salt [25,26,27,28,29], metal [30,31], heat [32], cold [33,34], and so on; and in the dark, plastid G6PDH could provide NADPH instead of light reactions in the photosynthetic tissues [35,36]. Recently, studies of G6PDH mainly focus on its activity and gene expression, as well as the regulatory mechanism under various stresses, emphasizing the role of G6PDH in maintaining cellular redox homeostasis to enhance the tolerance to environmental stresses in plants.
Highland barley, also named “Qingke” in Chinese, is an important food crop and livestock feed and occupies 70% of crop lands in the Tibetan Plateau [37,38]. Compared to barley, which has covered caryopsis, highland barley has naked caryopsis and can adapt to extreme environmental conditions in Tibet with altitudes ranging from 2700 to 4000 m [37,38,39]. The majority of highland barley-growing areas expose to several environmental stresses, such as drought, low temperature, enhanced ultraviolet radiation, and salinity [39,40,41,42]. Our previous study has confirmed that G6PDH is a key regulator in the cross adaptation to salt and UV-B stresses by maintaining NADPH production in highland barley [40]. Moreover, G6PDH can regulate cellular redox balance and the response to oxidative stress by affecting the synthesis of antioxidant substances and activities of the plasma membrane (PM) H+-ATPase and Na+/H+ antiporter (SOS1) under salt and drought conditions [23,27,43,44]. However, the regulatory mechanism of the G6PDH gene family remains elusive in highland barley.
In the present study, we characterized five G6PDH genes in highland barley and analyzed their expression patterns and enzymatic activities. We found that HvG6PDH1 to HvG6PDH4 participate in response to salt and drought stresses and that the cytosolic HvG6PDH2 is the major isoform regulating cellular redox homeostasis. Moreover, jasmonic acid (JA) and abscisic acid (ABA) can positively regulate the expression of HvG6PDHs (except HvG6PDH5). Our data not only expand the information of the G6PDH gene family in plants but also offer new insights for metabolic adaptive mechanisms to abiotic stresses in highland barley.

2. Materials and Methods

2.1. Plant Material and Treatments

Seeds of highland barley (Hordeum vulgare L. var. nudum Hook.f.) cv Kunlun-14 were kindly provided by Qinghai Academy of Agricultural Sciences, Qinghai Province, China. The seeds of concordant size were surface sterilized with 2% NaClO (v/v), and then germinated overnight at 25 °C. The germinated seeds were carefully sowed into plastic containers filled with 1/4-strength Hoagland media [40]. All seedlings were grown in the greenhouse, where the photoperiod was 16 h light /8 h dark, and the temperature was set at 25 ± 2 °C with the relative humidity of 60–70% and 100 µmol·m−2·s−1 photon flux density.
Five-day-old seedlings were treated with the Hoagland medium supplemented with 150 mM NaCl (salt stress) or 20% PEG6000 (drought stress) for different times (3, 6, 12, 24, 48, 72, 96, 120, 144 h). For investigating the role of G6PDH on plasma membrane damage and the accumulation of active oxygen radicals (ROS), seedlings were first pretreated for 24 h with the Hoagland medium containing 10 mM glucosamine (Glucm), a competitive G6PDH inhibitor; and then, the seedlings were exposed to the Hoagland medium containing 10 mM Glucm (Glucm treatment), 150 mM NaCl and 10 mM Glucm (NaCl + Glum treatment), or 20% PEG 6000 and 10 mM Glum (PEG + Glum treatment) for 48 h, respectively. To examine the expression level of HvG6PDHs under phytohormone treatments, five-day-old seedlings were treated with ABA (100 µM) or JA (100 µM) for different times (3, 6, 12, 24, 48, 72, 96, 120, 144 h). The treatment solutions were replaced every two days. In the experiment, all the samples were collected at 9–10 o’clock in the morning and used immediately.

2.2. Identification of HvG6PDH Genes and Bioinformatics

The G6PDH sequences of Arabidopsis, maize, rice, and barley were searched in public databases, including HarvEST (http://harvest.ucr.edu/), Ensembl plant (http://plants.ensembl.org/Hordeum_vulgare/Info/Index), NCBI (www.ncbi.nlm.nih.gov), and BarleyGene Index (http://compbio.dfci.harvard.edu/tgi/plant.html). Based on the reduplicated assembled contigs, we synthesized multiple primers, which were used for amplifying the cDNA of HvG6PDHs (Supplementary Table S1). Multiple alignments of DNA and protein sequences were conducted using CLUSTAL X. The transit peptide sequences of HvG6PDH proteins were evaluated using the ProtComp 9.0 software (Softberry, http://linux1.softberry.com/).
A phylogenetic tree was constructed through a multiple alignment of amino acid sequences from T. urartu (EMS47668, EMS45717, EMS62742), A. tauschii (XP_020168771, XP_020156901, EMT03892, EMT16963, EMT23941), B. distachyon (XP_003580004, XP_003575296, XP_003558039, XP_003581458), O. sativa (XP_015627059, AAL79959, ABF95637, BAC84352, ABF96582), Z. mays (AFW57831, DAA45780, ACG39996, XP_008658752), S. bicolor (XP_002446185, XP_021306709, XP_021313884, XP_021318732), A. thaliana (Q9FJI5, Q43727, Q9IK23, Q8I743, Q93ZW0, Q9FY99), P. trichocarpa (Q9FY99, Q9FY99, EEE79649, ERP53365), R. communis (EEF50009, EEF47431, EEF32168), and H. vulgare using the MEGA 7.0 software (http://www.megasoftware.net/) with the Neighbor-Joining method. The robustness of each node in the phylogenetic tree was evaluated with 1000 replicates.

2.3. RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from highland barley leaves or roots using Trizol reagent (Sangon, Shanghai, China). The cDNA synthesis was carried out using the M-MLV Reverse Transcriptase Kit (Sangon, Shanghai, China). After that the cDNA was diluted with RNAase-free water, which was used to assess the expression of HvG6PDH genes by qRT-PCR. The primers for HvG6PDH and HvACTIN (accession number: MK034133) genes are listed in Supplementary Table S1.
qRT-PCR was performed in a PCR cycle system (ABI 7500, ThermoFisher Scientific, USA) using the SYBR Green PCR master mix (Takara, Dalian, China). The PCR reaction was performed using the program of 95 °C for 30 s, and 40 cycles of 95 °C for 5 s and 60 °C for 34 s. The gene expression level was calculated according to the 2−ΔΔCt method [45], where the ΔΔCt value was the Ct value of HvG6PDH genes minus the Ct value of the HvACTIN.

2.4. Subcellular Localization

The open reading frame (ORF) of HvG6PDH1 to HvG6PDH5 were cloned into a pENTRTM/D-TOPO® vector (Invitrogen, USA) to generate attL1 and attL2 fragments into 5′ and 3′ terminal of HvG6PDH1 to HvG6PDH5 ORFs, respectively. The entry vectors (pENTR-HvG6PDH1 to pENTR-HvG6PDH5) were recombined into the attR1 and attR2-containing expression vector PGWB-RFP to produce HvG6PDHs-RFP proteins, which were driven by 35S promoter using the Gateway technology. All primers in the experiment are listed in Supplementary Table S1. All plasmids of destination constructs were transformed into the A. tumefaciens GV3101, and the resulting positive clones were infiltrated into N. benthamiana leaf for 48 h for transient expression [46]. The transformed N. benthamiana leaves were visualized through a laser scanning confocal microscope (C2 Plus, Nikon, Japan). The excitation wavelength for RFP and chloroplast autofluorescence was 561 nm and 556 nm, respectively, and the emission wavelength was 595 nm and 650 nm, respectively.

2.5. SDS-PAGE of HvG6PDH Proteins from Escherichia coli

Five HvG6PDHs ORFs were cloned into the vector pET28a(+), and then were transformed into E. coli BL21. Positive clones were inoculated into fresh liquid LB medium with 50 µg mL−1 kanamycin and cultured until the OD600 reached 0.5–0.6. Then 1.0 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) was added and induced protein expression. After shaking for 2 h, 10 mL LB medium was boiled for 7 min at 95 °C, and then centrifuged at 10,000× g for 10 min. After centrifugation, 10 mL supernatant was collected and used for protein electrophoresis in 12% SDS-PAGE. For analysis of the HvG6PDHs enzymes, the induced strains were ground but not boiled. The empty vector PET28(a) was used as the control. The SDS-PAGE gel was stained with Coomassie Brilliant Blue dye solution.

2.6. Extraction and Determination of G6PDH Activity

The extraction and assay of G6PDH were determined according to Hauschild and von Schaewen [47]. In order to determine the cytosolic G6PDH activity, the samples were incubated with 62.5 mM DTT at 25 °C for 5 min before measuring the enzyme activity, which can result in the inactivation of plastidic G6PDH [48]. Plastidic G6PDH activity was the difference between total and cytosolic G6PDH. In the analysis of enzyme activity, the extraction buffer was added into the reaction solution instead of the crude enzyme extract as blank control. Protein contents were analyzed using the method of Bradford [49].

2.7. Determination of Hydrogen Peroxide and Superoxide Anion Contents

Hydrogen peroxide (H2O2) content was measured as described by Veljovic-Jovanovic et al. [50]. H2O2 content was estimated by measuring the absorbance at 590 nm and determined from a calibration curve using 10–50 μM H2O2 as the standard. Superoxide anion (O2−) content was determined according to Elstner and Heupel [51]. Briefly, O2− was estimated by measuring the absorbance at 530 nm and recorded in a UV-VIS spectrophotometer.

2.8. Statistical Analysis

Each experiment was repeated at least three times. Data were expressed as mean ± SE, and analyzed by one-way variance analysis (ANOVA).

3. Results

3.1. Identification and Preliminary Characterization of Putative HvG6PDH Genes in Highland Barley

Highland barley, also named naked barley, is a relative species of barley (Hordeum vulgare L). In the study, five putative G6PDH genes in the genome of barley were selected as candidates to clone and identify HvG6PDH genes from the cDNA database of highland barley, which were finally named as HvG6PDH1, HvG6PDH2, HvG6PDH3, HvG6PDH4, HvG6PDH5. The cDNA sequences of HvG6PDH1-5 genes were submitted to GeneBank with accession numbers listed in Table 1. The number of G6PDH genes in highland barley is similar to the number reported in other species: six in A. thaliana [49], five in O. sativa, four in T. urartu [52], four in Z. mays [53], and five in A. tauschii [54].
G6PDH isozymes have various biochemical properties in plants [28,55,56]. In order to analyze the biochemical properties of HvG6PDH proteins, we predicted the isoelectric point (pI), molecular weight, and subcellular localization of each HvG6PDH protein. The open reading frame (ORF) length and deduced amino acid number of HvG6PDH1 to HvG6PDH5 range from 1527 to 1788 bp and 509 to 596 amino acids, respectively. The molecular weight and pI values range from 57.9 to 67.0 kD and 5.4 to 8.3, respectively (Table 1). Analysis of predicted transit peptides and subcellular localization of the HvG6PDH proteins indicated that HvG6PDH2 and 5 are localized in the cytosol, while HvG6PDH1, 3, and 4 are localized in the chloroplast/plastid (Table 1). A multiple comparison analysis of HvG6PDH proteins showed that the identity of amino acid sequences ranges from 44.8% to 81.6%, and the highest and lowest identity was found between HvG6PDH2 and 5 and between HvG6PDH1 and 3, respectively. Three highly conserved amino acid sequences were found in the substrate-binding site (IDHYLG), NADP-binding site (NEFVIRLQP), and Rossman fold (GASGDLAKKK) domain (Figure 1).
To confirm whether the encoded proteins of these putative HvG6PDH genes possess G6PDH activity, we expressed HvG6PDH genes in the pET28(a) vector driven by a T7 promoter. The results suggested that the five HvG6PDH genes are highly expressed with the expected molecular mass (Supplementary Figure S1). The expressed HvG6PDH1 to HvG6PDH5 proteins showed the remarkable G6PDH activity in comparison with that in the empty vector (the own G6PDH activity of E. coli.). These results indicated that the five HvG6PDH1-5 are members of the G6PDH gene family in highland barley, and encode the functional G6PDH proteins.

3.2. Phylogenetic Profiling and Subcellular Localization of HvG6PDH Proteins

To explore the similarity between HvG6PDHs and other determined G6PDH proteins, we constructed a phylogenetic tree based on a multiple alignment of HvG6PDHs and other 43 G6PDH proteins from monocots (Z. mays, T. urartu, S. bicolor, A. tauschii, O. sativa, T. aestivum, and B. distachyon) and eudicots (P. trichocarpa, R. communis, and A. thaliana; Figure 2). Amino acid alignment revealed that both NADP+ and glucose-6-phosphate binding motifs are highly conserved in these G6PDH proteins (Figure 1). Based on the classification of G6PDH proteins in A. thaliana, G6PDH1 to G6PDH4 has been verified to locate in plastids, and G6PDH5 and G6PDH6 are located in the cytoplasm [14,21]. Four clusters are formed in the phylogenetic cladogram. The early branch corresponding to plastidic G6PDH isoforms is further divided into three subclusters (P0, P1, and P2), in which HvG6PDH1, 3 and 4, are distributed. The cytosolic G6PDH (Cyt) branch includes HvG6PDH2 and HvG6PDH5, (Figure 2). It is worth noting that the HvG6PDH isoforms share high homology with those G6PDHs from T. aestivum, T. urartu, and A. tauschii in each cluster, with 85%-95% homology in amino acids among the G6PDH members (Figure 2). To further ascertain the subcellular localization of HvG6PDH1 to HvG6PDH5, we fused HvG6PDHs into the N-terminal of GFP and transiently expressed it in N. benthamiana leaves. As expected, HvG6PDH1, 3, 4 are unequivocally located in chloroplasts, and HvG6PDH2 and HvG6PDH5 proteins are clearly located in the cytoplasm (Figure 3), confirming the results from phylogenic analysis and previous online prediction for HvG6PDHs.

3.3. NaCl and PEG Treatments Induce the G6PDH Activity in Highland Barley

To investigate the responses of G6PDH under NaCl and PEG conditions, we determined the activities of cytosolic and plastidic G6PDH in leaves and roots from highland barley seedlings. In the presence of 150 mM NaCl, the cytosolic G6PDH activity in leaves was significantly increased at 120 h and 144 h, but the plastidic G6PDH activity did not change (Figure 4A). The cytosolic and plastidic G6PDH activities in roots reached their maximum under NaCl treatment for 96 h, and were increased to 4.5-fold and 1.7-fold of control, respectively (Figure 4B). In contrast, PEG treatment rapidly induced the activity of cytosolic G6PDH and the highest value in leaves (2.0-fold) and roots (2.5-fold) appeared at 120 h and 96 h, respectively (Figure 4C,D). The activity of plastidic G6PDH in roots had a remarkable increase from 72 h to 120 h but had no obvious change in leaves (Figure 4C,D). Moreover, G6PDH activity was similar among the seedlings at different growth stages under the control conditions (Supplementary Figure S2) These results suggested that NaCl and PEG treatment can induce the activity of G6PDH in highland barley, and the cytosolic G6PDH is more responsive than plastidic G6PDH in salt and drought stresses.

3.4. NaCl and PEG Treatments Activate the Expression of HvG6PDH Genes in Highland Barley

To further understand the response of HvG6PDH genes under NaCl and PEG treatments, we examined the expression level of five HvG6PDH genes in the leaves and roots of highland barley seedlings. Under normal conditions, the five HvG6PDH genes exhibited different expression levels in roots and leaves (Figure 5). The transcript level of HvG6PDH5 in leaves upon 150 mM NaCl stress showed no change within 120 h, whereas the expression of HvG6PDH1, HvG6PDH2, HvG6PDH3, and HvG6PDH4 rapidly increased after 3 h and reached peak values at 12, 6, 12, and 12 h, respectively (Figure 5A). However, when highland barley roots were exposed to NaCl treatment, the transcript level of HvG6PDH1, HvG6PDH2, and HvG6PDH4 decreased sharply after 3 h, but gradually increased after 36 h. The expression of G6PDH3 and G6PDH5 had no obvious changes in this period (Figure 5B).
Figure 5C showed that under 20% PEG treatment, except for HvG6PDH3 and HvG6PDH4 expression, the HvG6PDH1, HvG6PDH2, HvG6PDH5 transcription gradually increased after 3 h and reached the highest values during 12–36 h compared to those in the leaves of the untreated seedlings (0 h). In roots, the expression of HvG6PDH2 peaked at 72 h under 20% PEG treatment; HvG6PDH1 and HvG6PDH4 gradually increased and decreased during the treatment, respectively; comparatively, HvG6PDH3 had no change (Figure 5D). These results suggest that the expression patterns of five HvG6PDH genes vary in response to salt and drought stresses, implying that they may play different roles in these processes.

3.5. Abscisic Acid and Jasmonic Acid Positively Regulate the Activity and Expression of G6PDH

Abscisic acid (ABA) and jasmonic acid (JA) are critical signaling molecules in regulating gene expression under salt and drought stresses [57]. To determine whether ABA and JA affect G6PDH in highland barley, we analyzed the activity of cytosolic and plastidic G6PDH and the transcript abundance of HvG6PDHs under ABA and JA treatments. In the presence of ABA, the activity of cytosolic and plastidic G6PDH in leaves was rapidly increased after 6 h and reached the highest value at 12 h, and which increased by 47% and 113% compared to the control, respectively (Figure 6A). Moreover in roots, the activity of cytosolic G6PDH showed a similar trend with that in leaves, but the plastidic G6PDH activity gradually increased during the treatment (Figure 6B). Under JA treatment, the activity of both cytosolic and plastidic G6PDH in leaves reached the maximum at 24 h and then displayed a decreasing trend (Figure 6C). However, in roots, they reached the highest value at 12 h. The activity of cytosolic and plastidic G6PDH increased by 22% and 66%, respectively (Figure 6D).
The CGTCA motif (JA-responsive element) was found in the promoters of HvG6PDH1, HvG6PDH2, HvG6PDH3, and HvG6PDH4, and the ABRE motif (ABA-responsive element) was also found in the promoters of HvG6PDH2, HvG6PDH3, and HvG6PDH4. Neither CGTCA nor ABRE motif was found in the promoter of HvG6PDH5 (Supplementary Figure S3). As expected, under ABA treatments, the expression levels of HvG6PDH2, HvG6PDH3, and HvG6PDH4 in leaves increased rapidly after 3 h and reached the highest values at 12 h. The expression levels of HvG6PDH1 and HvG6PDH5 had no changes within 120 h of ABA treatment (Figure 7A). In roots, these genes showed similar patterns of expression as in leaves under ABA treatment, but they peaked at 6 h (Figure 7B). JA treatment also can stimulate the expression of HvG6PDH3 and HvG6PDH4 in leaves (Figure 7C) and the expression of HvG6PDH1, HvG6PDH2, HvG6PDH3, and HvG6PDH4 in roots (Figure 7D).

3.6. G6PDH Is Involved in ROS Scavenging under Salt and Drought Stresses

To elucidate the role of G6PDH in salt and drought stresses, Glucm, a competitive inhibitor of G6PDH [58], was used in this study. As expected, the activity of plastidic G6PDH was significantly decreased, while the activity of cytosolic G6PDH had a slight decline in both leaves and roots under Glucm treatment alone (Supplementary Figure S4). Supplementary Figure S4 showed that the activities of cytosolic and plastidic G6PDH were obviously stimulated by 150 mM NaCl or 10% PEG treatment; and the 200 mM NaCl or 20% PEG treatment induced the higher increase of their activities in the leaves and roots of highland barley. Application of Glucm (10 mM) could markedly inhibit the G6PDH activities induced by NaCl and PEG treatments (Supplementary Figure S4).
G6PDH is the main source of NADPH production and NADPH regulates the balance of ROS level in animals and yeast [59,60]. To declare G6PDH function in ROS production in highland barley, we examined the effect of Glucm on H2O2 and O2− contents under salt and drought stresses. Compared to the control, the application of Glucm alone had almost no effects on H2O2 and O2− contents (Figure 8). Salt and drought stress significantly increased the H2O2 and O2− contents in highland barley (Figure 8). For example, under 200 mM NaCl treatment, the H2O2 and O2− contents in leaves increased to 2.2-fold and 1.6-fold, respectively; while in roots they increased to 2.5-fold and 2.2-fold, respectively. Similarly, under 20% PEG treatment, the H2O2 and O2− contents in leaves were increased to 1.9-fold and 1.4-fold, respectively; in roots, they were increased to 2.4-fold and 2.1-fold, respectively (Figure 8). The application of Glucm under NaCl or PEG treatment further increased the H2O2 and O2− contents in comparison with the NaCl treatment alone or PEG treatment alone in highland barley, respectively (Figure 8). The results suggested that G6PDH probably has a critical role in ROS removal in highland barley.

4. Discussion

In this study, we first identified five HvG6PDH genes in highland barley, and their sequences were confirmed with the published highland barley genome sketch [38]. The substrate-binding (IDHYLG) and NADP-binding (NEFVIRLQP) motifs are necessary for G6PDH activity [14,56]. Indeed, we found that the five deduced HvG6PDH protein sequences contain both “IDHYLG” and “NEFVIRLQP” motifs (Figure 1), indicating the corresponding G6PDH proteins should be active. Expression of the five HvG6PDH genes in E. coli indicated that these genes encode the active G6PDH proteins (Supplementary Figure S1). The online software and subcellular localization analyses revealed that HvG6PDH2 and HvG6PDH5 are localized in the cytosol, while the other three HvG6PDHs (HvG6PDH1, 2 and 4) belong to the plastidic isoforms (Table 1; Figure 3). The cytosolic and plastidic HvG6PDH isoforms are consistent with reported orthologues in A. thaliana, O. sativa, T. urartu, Z. mays, and H. brasiliensis [14,52,53,54,56]. Moreover, the numbers of cytosolic and plastidic G6PDH isoforms are different in the diverse plant species. For example, plastidic G6PDH isoforms in A. thaliana, O. sativa, T. urartu, Z. mays, and B. distachyon are 4, 3, 3, 3, and 2, respectively, which is probably associated with the protein functional redundancy.
As a reductive coenzyme, NADPH not only provides the reduced power for the regeneration of reduced glutathione (GSH) but is the substrate of the plasma membrane NADPH oxidase to participate in H2O2 production [44,61,62]. NADPH is originated from photosynthesis and the OPPP pathway. In non-photosynthetic tissues, NADPH production is sourced through the OPPP [13]. Numerous literature have reported that G6PDH is widely induced in aluminum, drought, salt, and low-temperature stresses [23,24,25,26,27,28,29,30,33,34]. Under salinity or drought conditions, the activity of cytosolic and plastidic G6PDH significantly increases in highland barley seedlings, and the cytosolic G6PDH is more sensitive than the plastidic G6PDH (Figure 4). Liu et al. reported that G6PDH regulates H2O2 metabolism by maintaining GSH and ASC contents under drought in soybean [23]. Moreover, it was thought that the NO-modulated cytosolic G6PDH is responsible for ROS accumulation through the PM NADPH oxidase, resulting in the inhibition of soybean root elongation under high aluminum stress [31]. The results suggest cytosolic G6PDH should be involved in the regulation of the cellular redox homeostasis by supplying NADPH against abiotic stress.
In plants, the cytosolic and plastidic G6PDH genes have different expression patterns, and their gene expression does not positively correlate with the enzymatic activity [21,56]. Thus, the G6PDH isoforms should be regulated at the transcription and translation level. In the study, gene expression analysis indicated the mRNA abundance of cytosolic HvG6PDH2 is significantly increased, but HvG6PDH5 does not change in highland barley under salt and drought stresses (Figure 5). The significant enhancement in cytosolic G6PDH genes during salinity or drought conditions has also been reported in Para rubber tree and sugarcane [34,56]. Moreover, overexpression of cytosolic PsG6PDH from poplar confers cold tolerance in transgenic tobacco [33]. The Saccharomyces cerevisiae overexpressing CvG6PDH from Chlorella vulgaris increased the tolerance to cold [63]. Although the plastidic HvG6PDH genes (HvG6PDH1 and 4) were largely induced, the activity of the plastidic enzyme had no change under salt and drought stresses (Figure 4 and Figure 5). These results imply that HvG6PDH2, as a cytosolic G6PDH, mainly participated in the tolerance to abiotic stresses.
ABA and JA are widely involved in plant response to stresses, such as salinity, drought, cold, wounding, and so on [57,64,65]. ABA response elements (ABREs) and CGTCA motif have been identified in the promoters of ABA and JA-induced genes, respectively [66,67]. In this study, we found that promoters of HvG6PDH1, HvG6PDH2, HvG6PDH3, and HvG6PDH4 have the CGTCA motif and the promoters of HvG6PDH2-HvG6PDH4 have ABRE motif. HvG6PDH5 has neither CGTCA nor ABRE motif (Supplementary Figure S3). As expected, ABA treatment induced the expression of HvG6PDH2, HvG6PDH3, and HvG6PDH4, but HvG6PDH1 and HvG6PDH5 had no responses to ABA treatments (Figure 7). Moreover, the activities of cytosolic and plastidic G6PDH were significantly increased in highland barley under ABA treatment (Figure 6A,B). ABRE motif also has been found in the promoters of P2-G6PDH in rice and barley [68]. Cardi et al. reported that ABA can activate the activity of the P2-isoform G6PDH, but not the cytosolic G6PDH in barley [28]. However, the down-regulation of HbG6PDHs transcription was also found in latex from H. brasiliensis after ABA treatment [56]. In addition to ABA, the cytosolic and plastidic G6PDH activities were significantly increased in highland barley exposed to JA treatment (Figure 6C,D). Moreover, except for HvG6PDH5, the expression level of all HvG6PDHs was obviously induced by JA treatment in both leaves and roots (Figure 7C, D). Long et al. found that JA treatment activated the gene expression of cytosolic and plastidic G6PDH [56]. These results imply that the cytosolic and plastidic G6PDH play an important role in ABA and JA responses in highland barley seedlings.
With the environmental changes, plant cells need to alter the cellular metabolism to adapt to stresses. One of these metabolic changes is in the ROS level, which is a universal phenomenon in plant response to environmental stresses [2,3,9,10]. At a low level, ROS can act as the signaling molecules, but at an excessive level, it can lead to oxidative damages [2,10,19,69]. G6PDH can supply massive NADPH to regulate the ROS level [20,47,61,70]. Moreover, G6PDH is also required for plant development by regulating carbon and nitrogen flow [21,56].
In animals and yeast, NADPH is mainly produced through the OPPP to maintain the ROS homeostasis [59,60]. Here, we found that Glucm (a competitive inhibitor of G6PDH) treatment significantly decreased the activity of cytosolic and plastidic G6PDH (Supplementary Figure S4), accompanied by the high ROS contents (Figure 8) in highland barley under salt and drought stresses. Dal Santo et al. assumed that photosynthesis could provide reducing equivalents in plants [2]. When plants are exposed to unfavorable conditions, photosynthesis may be impaired. In this case, the reducing power for the regulation of intracellular redox balance may be mainly provided by OPPP. The GSH production is impaired in the Atg6pd6 mutant, leading to enhanced sensitivity to salt stress in Arabidopsis [2]. Scharte et al. reported that overexpression of a plastidic G6PD gene in tobacco enhanced the tolerance to drought [58]. When the G6PDH activity is disrupted, mouse embryonic stem cells are highly sensitive to oxidative stress [59,71]. Similarly, G6PDH-defective yeast mutants are very susceptible to oxidative stress [60,72]. These results, together with our observation in highland barley, suggest that G6PDH, a key enzyme of OPPP, plays a pivotal role in maintaining the cellular redox balance in stress acclimation.
In summary, G6PDH is a primary regulator of ROS removal and plays an important role in plant acclimatization to stresses. We cloned five HvG6PDH genes from highland barley and confirmed that HvG6PDH1-HvG6PDH4 are involved in response to salt and drought stresses. Among them, the cytosolic HvG6PDH2 is the major isoform against oxidative stress. Moreover, JA and ABA are probably critical regulators of HvG6PDHs (except HvG6PDH5) expression under salt and drought stresses.

Supplementary Materials

The following are available online at https://www.mdpi.com/2223-7747/9/12/1800/s1.

Author Contributions

R.F. and Y.B. designed the research. R.F., S.W., J.L., J.J., and L.H. conducted experiments. R.F. and J.L. analyzed data. R.F., X.W. and Y.B. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (31972494; 31671595; 31670244), Foundation of science and technology of Gansu Province (20JR5RA288), and Fundamental Research Funds for the Central Universities (lzujbky-2020-sp04).

Conflicts of Interest

The authors declare that they have no conflicts of interests.

References

  1. Boscaiu, M.; Vicente, O. Mechanisms of drought and salt stress tolerance in plants. J. Biotechnol. 2017, 256, S7. [Google Scholar] [CrossRef]
  2. Dal Santo, S.; Stampfl, H.; Krasensky, J.; Kempa, S.; Gibon, Y.; Petutschnig, E.; Rozhon, W.; Heuck, A.; Clausen, T.; Jonak, C. Stress-induced GSK3 regulates the redox stress response by phosphorylating glucose-6-phosphate dehydrogenase in Arabidopsis. Plant Cell 2012, 24, 3380–3392. [Google Scholar] [CrossRef] [Green Version]
  3. Hasegawa, P.M.; Bressan, R.A.; Zhu, J.K.; Bohnert, H.J. Plant cellular and molecular responses to high salinity. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000, 51, 463–499. [Google Scholar] [CrossRef] [Green Version]
  4. Munns, R. Comparative physiology of salt and water stress. Plant Cell Environ. 2002, 25, 239–250. [Google Scholar] [CrossRef]
  5. Tahi, H.; Wahbi, S.; Wakrim, R.; Aganchich, B.; Serraj, R.; Centritto, M. Water relations, photosynthesis, growth and water-use efficiency in tomato plants subjected to partial rootzone drying and regulated deficit irrigation. Plant Biosyst. 2007, 141, 265–274. [Google Scholar] [CrossRef]
  6. Foyer, C.H.; Noctor, G. Redox regulation in photosynthetic organisms: Signaling, acclimation, and practical implications. Antioxid. Redox Signal. 2009, 11, 861–905. [Google Scholar] [CrossRef] [PubMed]
  7. Mittler, R.; Vanderauwera, S.; Suzuki, N.; Miller, G.; Tognetti, V.B.; Vandepoele, K.; Gollery, M.; Shulaev, V.; Van Breusegem, F. ROS signaling: The new wave? Trends Plant Sci. 2011, 16, 300–309. [Google Scholar] [CrossRef] [PubMed]
  8. Wang, H.; Yang, L.; Li, Y.; Hou, J.; Huang, J.; Liang, W. Involvement of ABA- and H2O2-dependent cytosolic glucose-6-phosphate dehydrogenase in maintaining redox homeostasis in soybean roots under drought stress. Plant Physiol. Biochem. 2016, 107, 126–136. [Google Scholar] [CrossRef] [PubMed]
  9. Moller, I.M.; Jensen, P.E.; Hansson, A. Oxidative modifications to cellular components in plants. Annu. Rev. Plant Biol. 2007, 58, 459–481. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  10. Choudhury, F.K.; Rivero, R.M.; Blumwald, E.; Mittler, R. Reactive oxygen species, abiotic stress and stress combination. Plant J. 2017, 90, 856–867. [Google Scholar] [CrossRef] [PubMed]
  11. Foyer, C.H. Reactive oxygen species, oxidative signaling and the regulation of photosynthesis. Environ. Exp. Bot. 2018, 154, 134–142. [Google Scholar] [CrossRef]
  12. Von Schaewen, A.; Langenkamper, G.; Graeve, K.; Wenderoth, I.; Scheibe, R. Molecular characterization of the plastidic glucose-6-phosphate-dehydrogenase from potato in comparison to its cytosolic counterpart. Plant Physiol. 1995, 109, 1327–1335. [Google Scholar] [CrossRef] [Green Version]
  13. Thom, E.; Mohlmann, T.; Quick, W.P.; Camara, B.; Neuhaus, H.E. Sweet pepper plastids: Enzymic equipment, characterisation of the plastidic oxidative pentose-phosphate pathway, and transport of phosphorylated intermediates across the envelope membrane. Planta 1998, 204, 226–233. [Google Scholar] [CrossRef]
  14. Meyer, T.; Holscher, C.; Schwoppe, C.; von Schaewen, A. Alternative targeting of Arabidopsis plastidic glucose-6-phosphate dehydrogenase G6PD1 involves cysteine-dependent interaction with G6PD4 in the cytosol. Plant J. 2011, 66, 745–758. [Google Scholar] [CrossRef]
  15. Rao, X.J.; Duan, X.T.; Mao, W.M.; Li, X.X.; Li, Z.H.; Li, Q.; Zheng, Z.G.; Xu, H.M.; Chen, M.; Wang, P.G.; et al. O-GlcNAcylation of G6PD promotes the pentose phosphate pathway and tumor growth. Nat. Commun. 2015, 6, 8468. [Google Scholar] [CrossRef] [Green Version]
  16. Zhang, Z.Y.; Zheng, X.X.; Yang, J.; Messing, J.; Wu, Y.R. Maize endosperm-specific transcription factors O2 and PBF network the regulation of protein and starch synthesis. Proc. Natl. Acad. Sci. USA 2016, 113, 10842–10847. [Google Scholar] [CrossRef] [Green Version]
  17. Yuzbasheva, E.Y.; Mostova, E.B.; Andreeva, N.I.; Yuzbashev, T.V.; Laptev, I.A.; Sobolevskaya, T.I.; Sineoky, S.P. Co-expression of glucose-6-phosphate dehydrogenase and acyl-CoA binding protein enhances lipid accumulation in the yeast Yarrowia lipolytica. New Biotechnol. 2017, 39, 18–21. [Google Scholar] [CrossRef]
  18. Acero-Navarro, K.E.; Jimenez-Ramirez, M.; Villalobos, M.A.; Vargas-Martinez, R.; Perales-Vela, H.V.; Velasco-Garcia, R. Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa. Protein Expr. Purif. 2018, 142, 53–61. [Google Scholar] [CrossRef]
  19. Foyer, C.H.; Noctor, G. Redox homeostasis and antioxidant signaling: A metabolic interface between stress perception and physiological responses. Plant Cell 2005, 17, 1866–1875. [Google Scholar] [CrossRef] [Green Version]
  20. Kruger, N.J.; von Schaewen, A. The oxidative pentose phosphate pathway: Structure and organisation. Curr. Opin. Plant Biol. 2003, 6, 236–246. [Google Scholar] [CrossRef]
  21. Wakao, S.; Benning, C. Genome-wide analysis of glucose-6-phosphate dehydrogenases in Arabidopsis. Plant J. 2005, 41, 243–256. [Google Scholar] [CrossRef]
  22. Sindelar, L.; Sindelarova, M. Correlation of viral RNA biosynthesis with glucose-6-phosphate dehydrogenase activity and host resistance. Planta 2002, 215, 862–869. [Google Scholar]
  23. Liu, J.; Wang, X.M.; Hu, Y.F.; Hu, W.; Bi, Y.R. Glucose-6-phosphate dehydrogenase plays a pivotal role in tolerance to drought stress in soybean roots. Plant Cell Rep. 2013, 32, 415–429. [Google Scholar] [CrossRef]
  24. Wang, X.M.; Ruan, M.J.; Wan, Q.; He, W.L.; Yang, L.; Liu, X.Y.; He, L.; Yan, L.L.; Bi, Y.R. Nitric oxide and hydrogen peroxide increase glucose-6-phosphate dehydrogenase activities and expression upon drought stress in soybean roots. Plant Cell Rep. 2019, 39, 69–73. [Google Scholar] [CrossRef]
  25. Valderrama, R.; Corpas, F.J.; Carreras, A.; Gomez-Rodriguez, M.V.; Chaki, M.; Pedrajas, J.R.; Fernandez-Ocana, A.; Del Rio, L.A.; Barroso, J.B. The dehydrogenase-mediated recycling of NADPH is a key antioxidant system against salt-induced oxidative stress in olive plants. Plant Cell Environ. 2006, 29, 1449–1459. [Google Scholar] [CrossRef]
  26. Wang, X.; Ma, Y.; Huang, C.; Wan, Q.; Li, N.; Bi, Y. Glucose-6-phosphate dehydrogenase plays a central role in modulating reduced glutathione levels in reed callus under salt stress. Planta 2008, 227, 611–623. [Google Scholar] [CrossRef]
  27. Zhang, L.; Liu, J.; Wang, X.M.; Bi, Y.R. Glucose-6-phosphate dehydrogenase acts as a regulator of cell redox balance in rice suspension cells under salt stress. Plant Growth Regul. 2013, 69, 139–148. [Google Scholar] [CrossRef]
  28. Cardi, M.; Castiglia, D.; Ferrara, M.; Guerriero, G.; Chiurazzi, M.; Esposito, S. The effects of salt stress cause a diversion of basal metabolism in barley roots: Possible different roles for glucose-6-phosphate dehydrogenase isoforms. Plant Physiol. Biochem. 2015, 86, 44–54. [Google Scholar] [CrossRef]
  29. Yang, L.; Wang, X.M.; Chang, N.; Nan, W.B.; Wang, S.W.; Ruan, M.J.; Sun, L.L.; Li, S.F.; Bi, Y.R. Cytosolic glucose-6-phosphate dehydro-genase is involved in seed germination and root growth under salinity in Arabidopsis. Front. Plant Sci. 2019, 10, 182. [Google Scholar] [CrossRef] [Green Version]
  30. Slaski, J.J.; Zhang, G.C.; Basu, U.; Stephens, J.L.; Taylor, G.J. Aluminum resistance in wheat (Triticum aestivum) is associated with rapid, Al-induced changes in activities of glucose-6-phosphate dehydrogenase and 6-pbosphogluconate dehydrogenase in root apices. Physiologia. Plantarum. 1996, 98, 477–484. [Google Scholar] [CrossRef]
  31. Wang, H.H.; Hou, J.J.; Li, Y.; Zhang, Y.Y.; Huang, J.J.; Liang, W.H. Nitric oxide-mediated cytosolic glucose-6-phosphate dehydrogenase is involved in aluminum toxicity of soybean under high aluminum concentration. Plant Soil. 2017, 416, 39–52. [Google Scholar] [CrossRef]
  32. Gong, H.L.; Chen, G.S.; Li, F.J.; Wang, X.M.; Hu, Y.F.; Bi, Y.R. Involvement of G6PDH in regulation of heat stress tolerance in the calli from Przewalskia tangutica and Nicotiana tabacum. Biol. Plantarum 2012, 56, 422–430. [Google Scholar] [CrossRef]
  33. Lin, Y.Z.; Lin, S.Z.; Guo, H.; Zhang, Z.Y.; Chen, X.Y. Functional analysis of PsG6PDH, a cytosolic glucose-6-phosphate dehydrogenase gene from Populus suaveolens, and its contribution to cold tolerance improvement in tobacco plants. Biotechnol. Lett. 2013, 35, 1509–1518. [Google Scholar] [CrossRef] [PubMed]
  34. Yang, Y.T.; Fu, Z.W.; Su, Y.C.; Zhang, X.; Li, G.Y.; Guo, J.L.; Que, Y.X.; Xu, L.P. A cytosolic glucose-6-phosphate dehydrogenase gene, ScG6PDH, plays a positive role in response to various abiotic stresses in sugarcane. Sci. Rep. 2014, 4, 7090. [Google Scholar] [CrossRef]
  35. He, L.; Wang, X.; Feng, R.; He, Q.; Wang, S.; Liang, C.; Yan, L.; Bi, Y. Alternative Pathway is Involved in Nitric Oxide-Enhanced Tolerance to Cadmium Stress in Barley Roots. Plants 2019, 8, 557. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Esposito, S.; Guerriero, G.; Vona, V.; Di Martino Rigano, V.; Carfagna, S.; Rigano, C. Glutamate synthase activities and protein changes in relation to nitrogen nutrition in barley: The dependence on diferent plastidic glucose-6P dehydrogenase isoforms. J. Exp. Bot. 2005, 56, 55–64. [Google Scholar] [CrossRef] [Green Version]
  37. Nyima, T.; Tang, Y.; Zeng, X. Food preparation from hulless barley in Tibet. Adv. Barley Sci. 2013, 151–158. [Google Scholar]
  38. Zeng, X.Q.; Long, H.; Wang, Z.; Zhao, S.C.; Tang, Y.W.; Huang, Z.Y.; Wang, Y.L.; Xu, Q.J.; Mao, L.K.; Deng, G.B.; et al. The draft genome of Tibetan hulless barley reveals adaptive patterns to the high stressful Tibetan Plateau. Proc. Natl. Acad. Sci. USA 2015, 112, 1095–1100. [Google Scholar] [CrossRef] [Green Version]
  39. Dai, F.; Nevo, E.; Wu, D.Z.; Comadran, J.; Zhou, M.X.; Qiu, L.; Chen, Z.H.; Beiles, A.; Chen, G.X.; Zhang, G.P. Tibet is one of the centers of domestication of cultivated barley. Proc. Natl. Acad. Sci. USA 2012, 109, 16969–16973. [Google Scholar] [CrossRef] [Green Version]
  40. Zhao, C.Z.; Wang, X.M.; Wang, X.Y.; Wu, K.L.; Li, P.; Chang, N.; Wang, J.F.; Wang, F.; Li, J.L.; Bi, Y.R. Glucose-6-phosphate dehydrogenase and alternative oxidase are involved in the cross tolerance of highland barley to salt stress and UV-B radiation. J. Plant. Physiol. 2015, 181, 83–95. [Google Scholar] [CrossRef]
  41. Zeng, X.Q.; Bai, L.J.; Wei, Z.X.; Yuan, H.J.; Wang, Y.L.; Xu, Q.J.; Tang, Y.W.; Nyima, T. Transcriptome analysis revealed the drought-responsive genes in Tibetan hulless barley. BMC Genom. 2016, 17, 386. [Google Scholar] [CrossRef] [Green Version]
  42. Yuan, H.J.; Zeng, X.Q.; Ling, Z.H.; Wei, Z.X.; Wang, Y.L.; Zhuang, Z.H.; Xu, Q.J.; Tang, Y.W.; Tashi, N. Transcriptome profiles reveal cold acclimation and freezing tolerance of susceptible and tolerant hulless barley genotypes. Acta. Physiol. Plant. 2017, 39, 275. [Google Scholar] [CrossRef]
  43. Liu, Y.; Wu, R.; Wan, Q.; Xie, G.; Bi, Y. Glucose-6-phosphate dehydrogenase plays a pivotal role in nitric oxide-involved defense against oxidative stress under salt stress in red kidney bean roots. Plant Cell Physiol. 2007, 48, 511–522. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Li, J.S.; Chen, G.C.; Wang, X.M.; Zhang, Y.L.; Jia, H.L.; Bi, Y.R. Glucose-6-phosphate dehydrogenase-dependent hydrogen peroxide production is involved in the regulation of plasma membrane H+-ATPase and Na+/H+ antiporter protein in salt-stressed callus from Carex moorcroftii. Physiol. Plant. 2011, 141, 239–250. [Google Scholar] [CrossRef] [PubMed]
  45. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods. 2001, 25, 402–408. [Google Scholar] [CrossRef]
  46. Wendt, U.K.; Wenderoth, I.; Tegeler, A.; von Schaewen, A. Molecular characterization of a novel glucose-6-phosphate dehydrogenase from potato (Solanum tuberosum L.). Plant J. 2000, 23, 723–733. [Google Scholar] [CrossRef]
  47. Hauschild, R.; von Schaewen, A. Differential regulation of glucose-6-phosphate dehydrogenase isoenzyme activities in potato. Plant Physiol. 2003, 133, 47–62. [Google Scholar] [CrossRef] [Green Version]
  48. Koide, H.; Oda, T. Pathological occurrence of glucose-6-phosphatase in serum in liver diseases. Clin. Chim. Acta 1959, 4, 554–561. [Google Scholar]
  49. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef]
  50. Veljovic-Jovanovic, S.; Noctor, G.; Foyer, C.H. Are leaf hydrogen peroxide concentrations commonly overestimated? The potential influence of artefactual interference by tissue phenolics and ascorbate. Plant Physiol. Biochem. 2002, 40, 501–507. [Google Scholar] [CrossRef]
  51. Elstner, E.F.; Heupel, A. Inhibition of nitrite formation from hydroxylammoniumchloride: A simple assay for superoxide dismutase. Anal. Biochem. 1976, 70, 616–620. [Google Scholar] [CrossRef]
  52. Ling, H.Q.; Zhao, S.C.; Liu, D.C.; Wang, J.Y.; Sun, H.; Zhang, C.; Fan, H.; Li, D.; Dong, L.L.; Tao, Y.; et al. Draft genome of the wheat A-genome progenitor Triticum urartu. Nature 2013, 496, 87–90. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Valenti, V.; Stanghellini, M.A.; Pupillo, P. Glucose-6-phosphate-dehydrogenase isozymes of maize leaves—some comparative properties. Plant Physiol. 1984, 5, 521–526. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Luo, M.C.; Gu, Y.Q.; Puiu, D.; Wang, H.; Twardziok, S.O.; Deal, K.R.; Huo, N.X.; Zhu, T.T.; Wang, L.; Wang, Y.; et al. Genome sequence of the progenitor of the wheat D genome Aegilops tauschii. Nature 2017, 551, 498–502. [Google Scholar] [CrossRef]
  55. Cardi, M.; Chibani, K.; Castiglia, D.; Cafasso, D.; Pizzo, E.; Rouhier, N.; Jacquot, J.P.; Esposito, S. Overexpression, purification and enzymatic characterization of a recombinant plastidial glucose-6-phosphate dehydrogenase from barley (Hordeum vulgare cv. Nure) roots. Plant Physiol. Biochem. 2013, 73, 266–273. [Google Scholar] [CrossRef] [Green Version]
  56. Long, X.Y.; He, B.; Fang, Y.J.; Tang, C.R. Identification and characterization of the glucose-6-phosphate dehydrogenase gene family in the para rubber tree, Hevea brasiliensis. Front Plant Sci. 2016, 7, 215. [Google Scholar] [CrossRef] [Green Version]
  57. Zhu, J. Plant salt tolerance. Trends Plant Sci. 2001, 6, 66–71. [Google Scholar] [CrossRef]
  58. Scharte, J.; Schon, H.; Tjaden, Z.; Weis, E.; von Schaewen, A. Isoenzyme replacement of glucose-6-phosphate dehydrogenase in the cytosol improves stress tolerance in plants. Proc. Natl. Acad. Sci. USA 2009, 106, 8061–8066. [Google Scholar] [CrossRef] [Green Version]
  59. Pandolfi, P.P.; Sonati, F.; Rivi, R.; Mason, P.; Grosveld, F.; Luzzatto, L. Targeted disruption of the housekeeping gene encoding glucose-6-phosphate-dehydrogenase (G6PD): G6PD is dispensable for pentose synthesis but essential for defense against oxidative stress. EMBO. J. 1995, 14, 5209–5215. [Google Scholar] [CrossRef]
  60. Juhnke, H.; Krems, B.; Kotter, P.; Entian, K.D. Mutants that show increased sensitivity to hydrogen peroxide reveal an important role for the pentose phosphate pathway in protection of yeast against oxidative stress. Mol. Gen. Genet. 1996, 252, 456–464. [Google Scholar] [CrossRef]
  61. Leopold, J.A.; Zhang, Y.Y.; Scribner, A.W.; Stanton, R.C.; Loscalzo, J. Glucose-6-phosphate dehydrogenase overexpression decreases endothelial cell oxidant stress and increases bioavailable nitric oxide. Arterioscl. Throm. Vas. 2003, 23, 411–417. [Google Scholar] [CrossRef] [Green Version]
  62. Mendez, D.; Linares, M.; Diez, A.; Puyet, A.; Bautista, J.M. Stress response and cytoskeletal proteins involved in erythrocyte membrane remodeling upon Plasmodium falciparum invasion are differentially carbonylated in G6PD A(-) deficiency. Free Radical. Biol. Med. 2011, 50, 1305–1313. [Google Scholar] [CrossRef] [PubMed]
  63. Honjoh, K.; Machida, T.; Hagisako, T.; Suga, K.; Yonekura, M.; Shimizu, H.; Ohashi, N.; Miyamoto, T.; Hatano, S.; Iio, M. Molecular cloning and characterization of a cDNA for low-temperature inducible cytosolic glucose 6-phosphate dehydrogenase gene from Chlorella vulgaris and expression of the gene in Saccharomyces cerevisiae. Plant Sci. 2007, 172, 649–658. [Google Scholar] [CrossRef]
  64. Savchenko, T.; Kolla, V.A.; Wang, C.Q.; Nasafi, Z.; Hicks, D.R.; Phadungchob, B.; Chehab, W.E.; Brandizzi, F.; Froehlich, J.; Dehesh, K. Functional convergence of oxylipin and abscisic acid pathways controls stomatal closure in response to drought. Plant Physiol. 2014, 164, 1151–1160. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Verma, V.; Ravindran, P.; Kumar, P.P. Plant hormone-mediated regulation of stress responses. BMC. Plant Biol. 2016, 16, 86. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  66. Fu, J.; Wu, H.; Ma, S.Q.; Xiang, D.H.; Liu, R.Y.; Xiong, L.Z. OsJAZ1 attenuates drought resistance by regulating JA and ABA signaling in rice. Front. Plant Sci. 2017, 8, 2108. [Google Scholar] [CrossRef] [Green Version]
  67. Wu, H.; Ye, H.Y.; Yao, R.F.; Zhang, T.; Xiong, L.Z. OsJAZ9 acts as a transcriptional regulator in jasmonate signaling and modulates salt stress tolerance in rice. Plant Sci. 2015, 232, 1–12. [Google Scholar] [CrossRef]
  68. Cardi, M.; Chibani, K.; Cafasso, D.; Rouhier, N.; Jacquot, J.P.; Esposito, S. Abscisic acid effects on activity and expression of barley (Hordeum vulgare) plastidial glucose-6-phosphate dehydrogenase. J. Exp. Bot. 2011, 62, 4013–4023. [Google Scholar] [CrossRef] [Green Version]
  69. Dietz, K.J. Redox control, redox signaling, and redox homeostasis in plant cells. Int. Rev. Cytol. 2003, 228, 141–193. [Google Scholar]
  70. Bowsher, C.G.; Boulton, E.L.; Rose, J.K.C.; Nayagam, S.; Emes, M.J. Reductant for glutamate synthase is generated by the oxidative pentose-phosphate pathway in non-photosynthetic root plastids. Plant J. 1992, 2, 893–898. [Google Scholar] [CrossRef]
  71. Filosa, S.; Fico, A.; Paglialunga, F.; Balestrieri, M.; Crooke, A.; Verde, P.; Abrescia, P.; Bautista, J.M.; Martini, G. Failure to increase glucose consumption through the pentose-phosphate pathway results in the death of glucose-6-phosphate dehydrogenase gene-deleted mouse embryonic stem cells subjected to oxidative stress. Biochem. J. 2003, 370, 935–943. [Google Scholar] [CrossRef] [PubMed]
  72. Izawa, S.; Maeda, K.; Miki, T.; Mano, J.; Inoue, Y.; Kimura, A. Importance of glucose-6-phosphate dehydrogenase in the adaptive response to hydrogen peroxide in Saccharomyces cerevisiae. Biochem. J. 1998, 330, 811–817. [Google Scholar] [CrossRef] [PubMed] [Green Version]
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Figure 1. Amino acid sequence alignment of the five HvG6PDH isoforms from highland barley. The strictly conserved and different amino acid residues were indicated with black and white backgrounds, respectively. Green and red shades show the conserved amino acid residues with ≥75% and ≥50%, respectively. The three putative conserved sequences with (GA × G × LA), (RIGH × LG), and (NE × VR × P) of HvG6PDH isoforms are underlined and labeled as Rossman fold, substrate binding site, and NADP+ binding site, respectively. The sequences were aligned using the DNAMAN 6.0 software (Lynnon Biosoft, USA). The numbers on the right indicate amino acid positions.
Figure 1. Amino acid sequence alignment of the five HvG6PDH isoforms from highland barley. The strictly conserved and different amino acid residues were indicated with black and white backgrounds, respectively. Green and red shades show the conserved amino acid residues with ≥75% and ≥50%, respectively. The three putative conserved sequences with (GA × G × LA), (RIGH × LG), and (NE × VR × P) of HvG6PDH isoforms are underlined and labeled as Rossman fold, substrate binding site, and NADP+ binding site, respectively. The sequences were aligned using the DNAMAN 6.0 software (Lynnon Biosoft, USA). The numbers on the right indicate amino acid positions.
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Figure 2. Phylogenetic profiling of plant G6PDH isoforms. A distance cladogram using the Neighbour-Joining method shows the majority consensus of the G6PDH proteins from highland barley (Hordeum vulgare), Oryza sativa (Os), Zea mays (Zm), T. aestivum (Ta), Triticum urartu (Tu), Aegilops tauschii (Ata), Brachypodium distachyon (Bd), Sorghum bicolor (Sb), Populus trichocarpa (Pt), Ricinus communis (Rc), and Arabidopsis thaliana (At). The four G6PDH clades are indicated and clades P (0, 1, 2) and Cyt correspond to plastidic and cytosolic G6PDH isoforms, respectively. Bootstrap values are indicated at the branch points. Barley sequences are marked in bold dots.
Figure 2. Phylogenetic profiling of plant G6PDH isoforms. A distance cladogram using the Neighbour-Joining method shows the majority consensus of the G6PDH proteins from highland barley (Hordeum vulgare), Oryza sativa (Os), Zea mays (Zm), T. aestivum (Ta), Triticum urartu (Tu), Aegilops tauschii (Ata), Brachypodium distachyon (Bd), Sorghum bicolor (Sb), Populus trichocarpa (Pt), Ricinus communis (Rc), and Arabidopsis thaliana (At). The four G6PDH clades are indicated and clades P (0, 1, 2) and Cyt correspond to plastidic and cytosolic G6PDH isoforms, respectively. Bootstrap values are indicated at the branch points. Barley sequences are marked in bold dots.
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Figure 3. Subcellular localization of HvG6PDHs-GFP in N. benthamiana leaves. The photographs were taken after transient expression of HvG6PDHs-GFP genes for 48 h. Scale bar = 10 µm. Green indicates the fluorescence of HvG6PDHs-GFP proteins. Red and yellow show the chloroplast autofluorescence and merged GFP and chloroplast colors, respectively.
Figure 3. Subcellular localization of HvG6PDHs-GFP in N. benthamiana leaves. The photographs were taken after transient expression of HvG6PDHs-GFP genes for 48 h. Scale bar = 10 µm. Green indicates the fluorescence of HvG6PDHs-GFP proteins. Red and yellow show the chloroplast autofluorescence and merged GFP and chloroplast colors, respectively.
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Figure 4. The activity of cytosolic and plastidic G6PDH in leaves and roots of highland barley under NaCl (A,B) and PEG (C,D) treatments for 0 h to 144 h. Data are mean ± SE (n = 3). Statistical differences between Control (0 h, the untreated seedlings) and NaCl/PEG treatment at every time point were analyzed on the basis of Student’s t-test (p < 0.05 * and p < 0.01 **).
Figure 4. The activity of cytosolic and plastidic G6PDH in leaves and roots of highland barley under NaCl (A,B) and PEG (C,D) treatments for 0 h to 144 h. Data are mean ± SE (n = 3). Statistical differences between Control (0 h, the untreated seedlings) and NaCl/PEG treatment at every time point were analyzed on the basis of Student’s t-test (p < 0.05 * and p < 0.01 **).
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Figure 5. Time courses of HvG6PDHs gene expression in leaves and roots of highland barley under NaCl (A,B) and PEG (C,D) treatments for 0–120 h. The relative expression level of HvG6PDHs was normalized to HvACTIN, and then they were expressed as a ratio of the value of the untreated seedlings (0 h) for each treatment (n = 3).
Figure 5. Time courses of HvG6PDHs gene expression in leaves and roots of highland barley under NaCl (A,B) and PEG (C,D) treatments for 0–120 h. The relative expression level of HvG6PDHs was normalized to HvACTIN, and then they were expressed as a ratio of the value of the untreated seedlings (0 h) for each treatment (n = 3).
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Figure 6. The activity of cytosolic and plastidic G6PDH in leaves and roots of highland barley under abscisic acid (ABA) (A,B) and jasmonic acid (JA) (C,D) treatment for 48 h. Data are mean ± SE (n = 3). Statistical differences between Control (0 h, the untreated seedlings) and ABA/ JA treatment at every time point were analyzed on the basis of Student’s t-test (p < 0.05 * and p < 0.01 **).
Figure 6. The activity of cytosolic and plastidic G6PDH in leaves and roots of highland barley under abscisic acid (ABA) (A,B) and jasmonic acid (JA) (C,D) treatment for 48 h. Data are mean ± SE (n = 3). Statistical differences between Control (0 h, the untreated seedlings) and ABA/ JA treatment at every time point were analyzed on the basis of Student’s t-test (p < 0.05 * and p < 0.01 **).
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Figure 7. Time courses of HvG6PDHs gene expression in leaves and roots of highland barley under ABA (A,B) and JA (C,D) treatments. The relative expression levels of HvG6PDHs were normalized to HvACTIN, and then they were expressed as a ratio of the value of the untreated seedlings (0 h) for each treatment (n = 3).
Figure 7. Time courses of HvG6PDHs gene expression in leaves and roots of highland barley under ABA (A,B) and JA (C,D) treatments. The relative expression levels of HvG6PDHs were normalized to HvACTIN, and then they were expressed as a ratio of the value of the untreated seedlings (0 h) for each treatment (n = 3).
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Figure 8. Effect of G6PDH inhibitor (Glucm) on H2O2 and O2− contents in leaves and roots of highland barley under NaCl (A,B) and PEG (C,D) treatments for 48 h. Data are mean ± SE (n = 3). Statistical differences were analyzed on the basis of the Student’s t-test, and bars with different letters were different at the 0.05 level.
Figure 8. Effect of G6PDH inhibitor (Glucm) on H2O2 and O2− contents in leaves and roots of highland barley under NaCl (A,B) and PEG (C,D) treatments for 48 h. Data are mean ± SE (n = 3). Statistical differences were analyzed on the basis of the Student’s t-test, and bars with different letters were different at the 0.05 level.
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Table
Table 1. Protein properties of HvG6PDHs. The subcellular location of HvG6PDHs was predicted using WoLF PSORT (https://www.genscript.com/psort.html). The protein sequences encoded by HvG6PDH genes were predicted for amino acids number (aa), transit peptide length (TP), isoelectric point (pI), and molecular weight (MW) using the protein tools (properties) in GENE INFINITY website (http://www.geneinfinity.org/index.html?dp=5). The location of HvG6PDH genes is based on barley chromosome sequence.
Table 1. Protein properties of HvG6PDHs. The subcellular location of HvG6PDHs was predicted using WoLF PSORT (https://www.genscript.com/psort.html). The protein sequences encoded by HvG6PDH genes were predicted for amino acids number (aa), transit peptide length (TP), isoelectric point (pI), and molecular weight (MW) using the protein tools (properties) in GENE INFINITY website (http://www.geneinfinity.org/index.html?dp=5). The location of HvG6PDH genes is based on barley chromosome sequence.
Gene NameChromosome LocationPredicted LocalizationaaTPpIMW (kD)GenBank
HvG6PDH14H:433572159-433576826Chloroplast596275.467.0MK034128
HvG6PDH22H:608625462-608631046Cytosol509−6.357.9MK034129
HvG6PDH32H:20076557-20081131Chloroplast589398.365.6MK034130
HvG6PDH46H:114688120-114690749Chloroplast574378.264.8MK034131
HvG6PDH56H:342276577-342281388Cytosol516−5.958.3MK034132
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Feng, R.; Wang, X.; He, L.; Wang, S.; Li, J.; Jin, J.; Bi, Y. Identification, Characterization, and Stress Responsiveness of Glucose-6-phosphate Dehydrogenase Genes in Highland Barley. Plants 2020, 9, 1800. https://doi.org/10.3390/plants9121800

AMA Style

Feng R, Wang X, He L, Wang S, Li J, Jin J, Bi Y. Identification, Characterization, and Stress Responsiveness of Glucose-6-phosphate Dehydrogenase Genes in Highland Barley. Plants. 2020; 9(12):1800. https://doi.org/10.3390/plants9121800

Chicago/Turabian Style

Feng, Ruijun, Xiaomin Wang, Li He, Shengwang Wang, Junjie Li, Jie Jin, and Yurong Bi. 2020. "Identification, Characterization, and Stress Responsiveness of Glucose-6-phosphate Dehydrogenase Genes in Highland Barley" Plants 9, no. 12: 1800. https://doi.org/10.3390/plants9121800

APA Style

Feng, R., Wang, X., He, L., Wang, S., Li, J., Jin, J., & Bi, Y. (2020). Identification, Characterization, and Stress Responsiveness of Glucose-6-phosphate Dehydrogenase Genes in Highland Barley. Plants, 9(12), 1800. https://doi.org/10.3390/plants9121800

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