ISSN: 2329-9029
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Research Article - (2018) Volume 6, Issue 3
Keywords: Brachypodium distachyon; Diadenosine polyphosphate; Drought stress; Nudix hydrolase; UV irradiation
Diadenosine polyphosphate (ApnA) is a ubiquitous family of nucleotides in which two nucleoside moieties are linked 5-5’ through a polyphosphate chain containing 3-7 phosphoryl groups [1,2]. Ap4A is implicated in coupling DNA replication to cell division [3,4], initiation of DNA replication [5,6], recovery from stress by modulating protein refolding [2,7,8], and regulation of ATP-sensitive K+ channels [9,10]. Because the Ap4A level is increased in cells exposed to stress conditions such as oxidative, heat, nutritional, and DNA damage [7,8,11-13] and because Ap4A increases the gene expression of phenylalanine ammonia-lyase and 4-coumarate: CoA ligase consisting of phenylpropanoid pathway by heavy metals in Arabidopsis [14], Ap4A has been proposed as an ‘alarmone’. The long-chain (ApnA) (n=5-6) produces cytotoxic effects, although it is also an intracellular and extracellular signaling molecule [2,15-18]. Distributed among humans, bacteria, fungi, and plants, Ap4A hydrolase metabolizes and regulates (ApnA) levels. It is classified into two groups. One group cleaves Ap4A symmetrically to produce two moles of ADP. Its structure is related to serine/threonine protein phosphatase [19-23]. The other group, which shows asymmetrical Ap4A hydration to produce ATP and AMP, belongs to the nudix hydrolase (NUDX) family [24]. Some asymmetrical Ap4A hydrolases catalyze not only Ap4A but also long-chain (ApnA) and other nucleotide polyphosphates, for which specific activities and their products depend on enzyme characteristics [24-27].
In plants, AtNUDX13, AtNUDX25, AtNUDX26, and AtNUDX27 from Arabidopsis thaliana belong to (ApnA) hydrolase of the NUDX family [28-31]. Actually, AtNUDX13 is active toward Ap6A and Ap5A, but it has no activity to Ap4A and other substrates for Ap4A hydrolases. AtNUDX25 hydrolyzes NADH, coenzyme A (CoA), and guanosine-3′, 5′-tetraphosphate (ppGpp), whereas AtNUDX26 hydrolyzes ppGpp, in addition to the activities of AtNUDX25 and 26 toward Ap5A and Ap4A. AtNUDX27 hydrolyzes only Ap5A. These NUDXs have a well-conserved nudix motif, GX5EX7REUXEEXGU, where U is usually Ile, Leu, or Val [24]. AtNUDX25, AtNUDX26, and AtNUDX27 had a tyrosine residue downstream of the nudix motif found in other Ap4A hydrolases and located in chloroplasts, whereas AtNUDX13 had a glycine tripeptide motif downstream of the nudix motif. The subcellular location was in mitochondria [28,29]. These results suggest that enzymatic properties and biological functions differ between (ApnA) hydrolases that have long-chain (ApnA)-specific activity and which have wide substrate specificity, but most studies of enzymatic properties and diversity of Ap4A hydrolases and long-chain (ApnA) hydrolases have scope that is limited to Arabidopsis NUDXs in plants.
Brachypodium distachyon is a model plant of Pooideae subfamily including wheat and barley, which has tractable features such as small genome size with diploid, small plant size, and short life cycle [32]. It is expected to serve as a useful function model for identification of genes and biological functions related to agronomic interest from Triticeae crops. This study identified the putative gene from Brachypodium which encodes the homologue of AtNUDX13 that hydrolyzes Ap6A and Ap5A specifically. Furthermore, this study elucidated the structure, enzymatic properties, subcellular location, and expression profiles under stress conditions.
Plant cultivation and stress treatment
Seeds of Brachypodium, Brachypodium distachyon Bd21, were incubated on filter paper kept moist with water at 23°C for 5 days in the dark. Seedlings were selected randomly from the germinated seeds. Three seedlings were planted on one Wagner pot (1/5000 a) filled with soil under a metal halide light (350 μmol/m2/s) with a light/dark cycle of 16 h/8 h in a growth chamber. After 7 days of cultivation, plants were irradiated with 186, 431, and 438 μW/cm2 of 340, 312, and 260 nm of UV light for 6 h to induce UV stress. The plants were pulled up and dehydrated on a paper towel for 6 h to stimulate drought stress, were soaked in a pot with 100 mM NaCl solution for 24 h to stimulate salt stress or were cultivated under metal halide light as a control. After exposure to stress conditions, the shoots were harvested, frozen in liquid nitrogen, and stored at -80°C.
Quantitative RT-PCR analysis
Total RNA was isolated from shoot samples using the RNeasy Plant mini kit (Qiagen Inc., Tokyo, Japan) following the manufacturer’s instructions. Poly(A)+ RNA was purified from total RNA with the Poly (A) Purist MAG (Ambion Inc., Austin, Texas). Then the purified poly(A)+ RNA was dissolved in the RNA storage solution. First-strand cDNA was synthesized from poly(A)+ RNA using a PrimeScript RT Master Mix (Takara Bio Inc., Shiga, Japan). Quantitative RT-PCR was performed in a mixture of 20 μl containing first-strand cDNA, SYBR Premix Ex Taq (Takara Bio Inc.), and 0.2 μmol of each forward primer, 5′-TGCACTGCTGGAGCGGTTAT-3′, and reverse primer, 5′- ATCAGATGTCGTTTGGAGCA-3′ using LightCycler 2.0 (Roche Applied Science, Mannheim, Germany). The thermal cycle profile was 1 cycle of 95°C for 10 s, followed by 40 cycles of 95°C for 5 s and 60°C for 20 s. The cDNA quantities of each gene were calculated using software (LightCycler 4.0; Roche Applied Science) and were normalized with that of the S-adenosylmethionine decarboxylase gene [33]. The expression analysis was conducted three times.
Expression and purification of BraNUDX15
The active form of HvNUDX 15 genes was amplified with firststrand cDNA from control shoots and the primers, 5′- CCATATGAAGAAGGACGAGGGGAACCC-3′, which creates a Nde I site (denoted as underlined), and 5′- CCTCGAGGCACAATGCAACTGCGCC-3′, which creates a Xho I site (denoted as underlined). The PCR product of 525 bp length was cloned into the pGEM-T vector. Then the fragments of the plasmids digested by Nde I and Xho I were subcloned into a pET-20b (+) vector, in which a polyhistidine tag gene is fused upstream from the start codon. The resulting plasmid, pBraNUDX15-ACT, was transformed into E. coli BL21 cells. E. coli cells harboring pBraNUDX15-ACT were grown at 37°C in Luria-Bertani (LB) medium containing 50 μg/ml ampicillin. When the OD600 reached 0.5, isopropyl-μ-Dthiogalactopyranoside (IPTG) was added to the culture at a final concentration of 0.5 mM. After cultivation at 25°C for 18 h, the cells were harvested by centrifugation and were frozen at -80°C for at least 2 h. The frozen cell pellets were suspended in a protein extraction reagent (BugBuster™ HT; Merck, Darmstadt, Germany) according to the manufacturer’s instructions. The resulting recombinant protein, which showed an insoluble form, was dissolved in 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl, 5 mM imidazole, and 6 M guanidine HCl (Buffer A) was purified using an Ni-NTA agarose column (Qiagen Inc.) initially equilibrated in Buffer A. The column was washed with Buffer A, followed by 60 mM imidazole in Buffer A, with the absorbed protein eluted with 200 mM imidazole in Buffer A. The protein solution was dialyzed against 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl, 5 mM imidazole, and 3 M guanidine HCl, followed by 20 mM Tris-HCl buffer (pH 8.0) containing 1 mM DTT, 100 mM NaCl, 0.5% n-dodecyl-μ-D-maltoside, and 10% ethylene glycol. The dialyzed solution was then concentrated (Vivaspin 4; Sartorius, Goettingen, Germany).
Enzyme and protein assays
The hydrolytic activity of BraNUDX15 was assayed according to a method described previously [34]. The reaction mixture (100 μl), DTT, 100 μM substrate, and recombinant protein, was incubated at which consists of 10 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 10 mM 37°C for 30 min. After the reaction was terminated by 17 μl of 100 mM EDTA, the reaction mixture was subjected to HPLC using a column (Cosmosil C18, 4.6 × 250 mm, Nacalai Tesque Inc., Kyoto, Japan) equilibrated with 100 mM phosphate buffer (pH 6.0) and 5% methanol at a flow rate of 0.6 ml/min. The reaction products were detected by absorption at 293 nm for 8-oxo-dGTP, 8-oxo-dGDP, at 260 nm for (ApnA) (n=4–6), ADP-ribose, NADH, UDP-Gal, dATP, ppGpp, and CoA, at 252 nm for dGTP, at 264 nm for dTTP, and at 271 nm for dCTP. Protein concentrations were quantified according to Bradford [35] with bovine serum albumin as the standard.
GFP transient assay
A DNA fragment encoding a putative transit peptide of BraNUDX15 predicted to the residue within the first 45 N-terminal amino acid residues was amplified by PCR with the first-strand cDNA from control shoot and primers, 5′- GGTCGACATGTCCAGCCTCGTTCTCGC-3′, which creates a Sal I site (denoted as underlined), and 5′- TCCATGGTGTACGGGACACACCCTGCA-3′, which creates a Nco I site (denoted as underlined). The PCR product of 144 bp length was cloned into the pGEM-T vector. Then the fragments of the plasmids digested by Sal I and Nco I were subcloned into a plasmid pTH-2, in which a GFP is fused downstream from the transit peptide [36]. The plasmid, pBraNUDX15-SIG+GFP, was transformed into Arabidopsis protoplasts according to the method explained by Miura et al. [37].
Identification of Brachypodium (ApnA) hydrolase genes
The genes, which encode amino acid sequences showing homology with those of 28 Arabidopsis NUDX families (AtNUDX1–27 and AtDCP2) and nudix motif (e<0.0001), were searched using a BLAST program [38,39] and the RIKEN Brachypodium distachyon Full- Length cDNA Clone Database. The full-length cDNAs of 19 putative Brachypodium NUDX genes, BraNUDX1-19, were identified. The deduced amino acid sequences of BraNUDX1-19 showed 71-43% identities with those of AtNUDXs and nudix motif (Table 1).
Gene | Gene ID | Identity (%) | Subfamily | |
---|---|---|---|---|
BraNUDX1 | Bradi1g35490.1 | AtNUDX2: | 55 | ADP-ribose/NADH |
AtNUDX10: | 50 | |||
AtNUDX7: | 45 | |||
AtNUDX6: | 43 | |||
BraNUDX2 | Bradi3g53887.1 | AtNUDX3: | 66 | n.d. |
BraNUDX3 | Bradi1g44170.1 | n.d. | ||
BraNUDX4 | Bradi2g37517.1 | AtNUDX9: | 60 | GDP-mannose |
BraNUDX5 | Bradi2g32550.1 | n.d. | ||
BraNUDX6 | Bradi5g08460.1 | n.d. | ||
BraNUDX7 | Bradi1g49810.1 | AtNUDX14: | 58 | ADP-ribose/ADP-glucose |
BraNUDX8 | Bradi3g56830.1 | AtNUDX17: | 51 | n.d. |
AtNUDX4: | 49 | |||
BraNUDX9 | Bradi1g51060.1 | AtNUDX19: | 59 | NADPH |
BraNUDX10 | Bradi4g28030.2 | AtNUDX20: | 61 | Thiamin diphosphate |
AtNUDX24: | 58 | |||
BraNUDX11 | Bradi4g37360.1 | AtNUDX23: | 53 | FAD |
BraNUDX12 | Bradi5g26560.2 | AtNUDX26: | 58 | ApnA/ppGpp |
AtNUDX25: | 49 | |||
AtNUDX27: | 47 | |||
BraNUDX13 | Bradi3g35160.1 | AtNUDX22: | 55 | CoA |
AtNUDX11: | 51 | |||
BraNUDX14 | Bradi5g17500.1 | AtNUDX8: | 50 | n.d. |
BraNUDX15 | Bradi3g44460.1 | AtNUDX13: | 47 | ApnA/ppGpp |
AtNUDX12: | 46 | |||
BraNUDX16 | Bradi3g35150.1 | AtNUDX15: | 57 | CoA |
BraNUDX17 | Bradi1g54020.1 | AtNUDX16: | 71 | n.d. |
BraNUDX18 | Bradi3g56830.1 | AtNUDX18: | 52 | n.d. |
AtNUDX21: | 47 | |||
BraNUDX19 | Bradi3g54700.1 | AtDCP2: | 58 | mRNA cap |
n.d., not detected. |
Table 1: Identity of deduced amino acid sequences of BraNUDX genes with those of AtNUDX genes.
Alignment analysis of amino acid sequences of BraNUDX1-19 obtained using the Clustal W program showed that the nudix motif comprising 23 amino acid residues was conserved in the amino acid sequences of Brachypodium NUDXs, except for the insertion of 22 amino acid residues in BraNUDX4 (Figure 1).
Figure 1: Alignment of the deduced amino acid sequences around the nudix motif in putative Brachypodium NUDXs. Gaps, denoted by a dash were introduced into the sequences to maximize the homology. The nudix motif is shown below the sequence. Identical amino acid residues to those of nudix motif are shown as reversed letters.
According to the substrate specificities of Arabidopsis NUDXs, 15 Brachypodium NUDXs are classified into the following subfamilies: BraNUDX1 belongs to ADP-ribose/NADH hydrolase; BraNUDX4 belongs to GDP-mannose hydrolase; BraNUDX7 belongs to ADPribose/ ADP-glucose hydrolase; BraNUDX9 belongs to NADPH hydrolase; BraNUD×10 belongs to thiamin diphosphate hydrolase; BraNUDX11 belongs to FAD hydrolase; BraNUDX12 and 15 belong to (ApnA)/ppGpp hydrolase; BraNUDX13 and 16 belong to CoA hydrolase; BraNUDX19 belongs to mRNA cap; although BraNUDX2, 3, 5, 6, 8, 14, 17, and 18 were not assigned to any established subfamily (Table 1). These results suggest that two (ApnA) hydrolase genes, BraNUDX12 and 15, are present in Brachypodium . BraNUDX12 conserved Tyr downstream of nudix motif as did Arabidopsis (ApnA) hydrolases, AtNUDX25, 26, and 27, whereas BraNUDX15 conserved glycine tripeptide motif, GX2GX6G, as did AtNUDX13, which hydrolyzes Ap6A and Ap5A specifically (Figure 2).
Figure 2: Alignment of the deduced amino acid sequences around the nudix motif in Brachypodium and Arabidopsis ApnA hydrolases. Glycine residues of glycine tripeptide motif and a tyrosine residue downstream from nudix motif are shown respectively by white and black triangles.
Purification and enzymatic characterization of recombinant BraNUDX15
BraNUDX15 protein, which was encoded by the open reading frame of cDNA, was produced in E. coli cells. However, the protein made an inclusion body and could not be made soluble by refolding (data not shown). Results of alignment analysis of the amino acid sequence of BraNUDX15 with that of AtNUDX13, which had the transit peptide [29], suggest that the N-terminal peptide of 46 amino acid residues of BraNUDX15 is the transit peptide (Figure 3).
The mature form of BraNUDX15 protein, of which the transit peptide at the N-terminus was eliminated, tagged with a His-Tag at its C-terminus, was produced in E. coli cells. An extra protein with a molecular mass of 22 kDa, which is similar to that calculated from the amino acid sequence, was produced as an inclusion body in E. coli cells and was refolded to soluble form (Figure 4).
Figure 4: Analysis of the expression of BraNUDX15 in E. coli cells using SDS-polyacrylamide gel. E. coli cells harboring pBraNUDXACT were harvested after IPTG induction at 25°C for 18 h. The soluble protein (1), insoluble protein (2), and refolded BraNUDX15 purifying with Ni-NTA column (3) were subjected to 12% SDSPAGE with molecular mass marker (M) followed by Coomassie Brilliant Blue R-250 staining.
The purified BraNUDX15 incubated with Ap6A showed production of ATP, of which the specific activity was 1.23 μmol/min/mg. The enzyme activity was inhibited completely by EDTA and was recovered by MgCl2. BraNUDX15 had maximum activity toward Ap6A at pH 8.0 and relative activities of 95% for Ap5A and 76% for Ap4A to Ap6A hydrolyzing activity, with barely any activity toward dCTP (Table 2).
Specific activity (mmol/min/mg) | |||||
---|---|---|---|---|---|
Substrate | BraNUDX15 | AtNUX13 [28] | AtNUDX25 [30,31] | AtNUDX26 [30,31] | AtNUDX27 [30] |
Ap3A | n.d. | n.d. | n.d. | n.d. | n.d. |
Ap4A | 0.94 ± 0.02 | n.d. | 0.026 ± 0.02 | 13.3 ± 0.36 | n.d. |
Ap5A | 1.17 ± 0.11 | 4.2 | 0.017 ± 0.001 | 21.6 ± 0.58 | 0.22 ± 0.01 |
Ap6A | 1.23 ± 0.03 | 10.5 | - | - | - |
Ap4G | 0.72 ± 0.09 | - | - | - | - |
Gp4G | 0.74 ± 0.04 | - | - | - | - |
ADP-ribose | n.d. | - | n.d. | n.d. | n.d. |
NADH | n.d. | - | 0.016 ± 0.001 | n.d. | n.d. |
CoA | n.d. | - | 0.012 ± 0.001 | 0.11 ± 0.01 | n.d. |
UDP-Gal | n.d. | - | n.d. | n.d. | n.d. |
ppGpp | n.d. | - | 0.06 ± 0.01 | 0.19 ± 0.05 | - |
8-oxo-dGTP | n.d. | - | n.d. | 0.02 ± 0.01 | n.d. |
dGTP | n.d. | - | n.d. | 0.05 ± 0.01 | n.d. |
dATP | n.d. | - | n.d. | 0.07 ± 0.01 | n.d. |
dTTP | n.d. | - | n.d. | 0.05 ± 0.01 | n.d. |
dCTP | 0.04 ± 0.001 | - | n.d. | 0.07 ± 0.01 | n.d. |
n.d., not detected; -, not reported.
Table 2: Substrate specificities of Brachypodium and Arabidopsis ApnA hydrolases.
Expression of BraNUDX15 gene under abiotic stress
Brachypodium was cultivated under UV irradiation, drought, and salt conditions to evaluate the response of BraNUDX15 gene to environmental stresses (Figure 5).
Figure 5: Expression profiles of BraNUDX15 gene in Brachypodium under abiotic stress. Total RNAs isolated from shoots of Brachypodium under UV-A, UV-B, UV-C, drought, and 150 mM NaCl conditions were subjected to quantitative RT-PCR. Expression levels were normalized with that of S-adenosylmethionine decarboxylase gene as an internal control. The error bar represents the standard error of the mean for three experiments.
The expression level of BraNUDX15 gene was up-regulated considerably: 2.5, 4.8, and 3.7-fold, respectively, by UV-A, UV-B, and UV-C irradiation. Drought stress reduced the expression level to about half. The expression level was unchanged by salt stresses, which increased it about 10%.
Subcellular localization of BraNUDX15 protein
A DNA fragment corresponding to predicted transit peptide from BraNUDX15 cDNA sequence was fused in frame with GFP at the Cterminus and was expressed in protoplasts under the control of the CaMV 35S promoter. The GFP fusion protein fluorescence in the transgenic cells was colocalized with the surface of chloroplasts (Figure 6).
Figure 6: Subcellular localization of BraNUDX15. Arabidopsis cells were transformed with either pBraNUDX15-SIG+GFP or pTH-2 (GFP control). GFP fluorescence (GFP) and chlorophyll autofluorescence (Chlorophyll) signals were merged (Merged).
The subcellular localization of BraNUDX15 was also predicted in chloroplasts using WoLF PSORT server.
Genes encoding homology with (ApnA) hydrolase were searched from Brachypodium. Of 19 putative NUDX genes, BraNUDX12 and 15 genes showed homology with Arabidopsis (ApnA) hydrolases NUDXs. BraNUDX12 showed identity with AtNUDX25, 26, and 27, which conserved the tyrosine residue found in (ApnA) hydrolases and hydrolyzed Ap4A and/or Ap5A, whereas BraNUDX15 showed identity with AtNUDX13, which had the glycine tripeptide motif and hydrolyzed Ap6A and Ap5A but not Ap4A [28,29]. These results suggest BraNUDX15 as the long-chain (ApnA) specific hydrolase.
The purified BraNUDX15, of which the predicted transit peptide was eliminated, required Mg2+ for hydrolyzing (ApnA), as did other (ApnA) hydrolases. The enzyme had the highest activity toward Ap6A, with relative activities of 95% for Ap5A and 76% for Ap4A to Ap6A hydrolyzing activity. It produced ATP from these substrates, whereas Arabidopsis long-chain (ApnA) hydrolase, AtNUDX13, showed activity toward Ap6A, preferentially toward Ap6A, and relative activity of 40% for Ap5A to Ap6A hydrolyzing activity. However, it showed no activity toward Ap4A. It produced ADP+p4A from Ap6A and AMP +p4A from Ap5A [28]. AtNUDX25 and 26 showed activity not only toward Ap4A and Ap5A but also toward NADH, CoA, 8-oxo-dGTP, ppGpp, or dNTPs [30,31], which were not hydrolyzed by BraNUDX15 except for slight activity toward CoA. These results indicate that BraNUDX15 is a unique (ApnA) hydrolase that has different substrate specificity from Arabidopsis (ApnA) hydrolases and indicate that glycine tripeptide motif is necessary for hydrolyzing long-chain (ApnA).
In plant cells, AtNUDX13 was localized in mitochondria; AtNUDX26 and 27 were localized in chloroplasts [28,30]. Reportedly, AtNUDX26 hydrolyzed ppGpp, of which the level in chloroplasts was increased under environmental stress. Moreover, the expression level of the gene increased under drought stress, suggesting that AtNUDX26 regulates the ppGpp level in chloroplasts [31]. Tomato Ap4A hydrolase gene decreased by CdCl2 [40]; Ap4A increased the gene expression of phenylalanine ammonia-lyase and 4-coumarate: CoA ligase consisting of phenylpropanoid pathway by heavy metals in Arabidopsis [14], indicating that the Ap4A level is regulated by Ap4A hydrolase to induce stress tolerance genes as alarmone. An earlier study showed that Ap6A inhibits ATP-sensitive K+ channels [17] and that extracellular Ap6A and Ap5A influence cytosolic free Ca2+ concentrations [18]. The accumulation of long-chain (ApnA) can produce cytotoxic effects through the inhibition of various kinases [15,16]. Our result demonstrated that BraNUDX15 was localized around the chloroplast surface. The gene expression level was induced under UV-A, -B, and - C exposure, but it was reduced by drought stress. Taken together, the evidence shows that BraNUDX15 can be expected to play a role in accumulating Ap4A to induce drought-stress-relieving genes under drought stress and decreasing long-chain (ApnA) before attaining a potentially toxic concentration under UV irradiation in chloroplasts.
Results of this study demonstrated that Brachypodium (ApnA) hydrolase BraNUDX15, which showed homology with Arabidopsis long-chain (ApnA) hydrolase and conserved glycine tripeptide motif, was a unique (ApnA) hydrolase that has different substrate specificity from those of Arabidopsis (ApnA) hydrolases. The expression level of BraNUDX15 gene was increased by UV irradiation and decreased by drought stress. Moreover, the protein was localized in chloroplasts.
These results suggest that BraNUDX15 is a unique (ApnA) hydrolase with different substrate specificity from those of Arabidopsis (ApnA) hydrolases. It might play a role in regulating (ApnA) levels in chloroplasts under drought stress and UV irradiation.
This research was partially supported by the Ohara Foundation of Kurashiki, Japan.