The Effect of Abiotic Stress Conditions on Expression of Calmodulin (CaM) and Calmodulin-Like (CML) Genes in Wild-Growing Grapevine Vitis amurensis.

Plant calmodulins (CaMs) and calmodulin-like proteins (CMLs) are important plant Ca2+-binding proteins that sense and decode changes in the intracellular Ca2+ concentration arising in response to environmental stimuli. Protein Ca2+ sensors are presented by complex gene families in plants and perform diverse biological functions. In this study, we cloned, sequenced, and characterized three CaM and 54 CML mRNA transcripts of Vitis amurensis Rupr., a wild-growing grapevine with a remarkable stress tolerance. Using real-time quantitative RT-PCR, we analyzed transcript abundance of the identified VaCaMs and VaCMLs in response to water deficit, high salinity, high mannitol, cold and heat stresses. Expression of VaCaMs and 32 VaCMLs actively responded to the abiotic stresses and exhibited both positive and negative regulation patterns. Other VaCML members showed slight transcriptional regulation, remained essentially unresponsive or responded only after one time interval of the treatments. The substantial alterations in the VaCaM and VaCML transcript levels revealed their involvement in the adaptation of wild-growing grapevine to environmental stresses.

1. Introduction

Plants, as sessile organisms, have to develop multiple biochemical and physiological reactions in order to adapt to the constantly changing environmental conditions. Different abiotic stress stimuli, including unfavorable temperatures, drought, flooding, or soil salinity, affect plant growth and productivity. These and other abiotic stresses evoke spatially and temporally distinct alterations in the intracellular Ca2+ concentrations, i.e., “Ca2+ signatures”, which are perceived and decoded by Ca2+ binding proteins referred to as Ca2+ sensors [1,2]. The majority of Ca2+ sensor proteins contain several EF-hand motifs, conserved helix-loop-helix structures, in which the Ca2+ ions are coordinated within the acidic Ca2+-coordinating loop [3]. Furthermore, recent studies indicated that Ca2+ signals are implicated in the signal transmission at long distances or even at the organismic level [4,5]. The major plant EF-hand-containing Ca2+-binding proteins are divided into calmodulins (CaMs), calmodulin-like proteins (CML), Ca2+-dependent protein kinases (CDPKs), and calcineurin B-like proteins (CBLs) [1,6,7].

CaM is a functionally important and highly conserved Ca2+ sensor present in all eukaryotic organisms, while CMLs are Ca2+ sensors closely related to CaMs but present only in higher plants [8,9]. CaMs are small proteins containing four EF-hands; CMLs greatly vary in their length and EF-hand number containing from one to six EF-hand motifs. Plant CaMs and CMLs do not exhibit catalytic activity and act as sensor relays that transmit the information encoded by the Ca2+ signatures to downstream events, such as protein interaction, protein phosphorylation, metabolic changes, or gene regulation [8,10].

Plant CaMs and CMLs are known to function in plant developmental processes and in response to both biotic and abiotic stresses [2,11,12,13]. It is known that gene expression levels of plant CaMs and CMLs are regulated by a variety of abiotic stress stimuli, e.g., in tea, apple and soybean in response to cold, drought, flooding, or high salinity [14,15,16]. Transgenic plants overexpressing certain stress-responsive CaMs and CMLs showed greater tolerance to the respective or even multiple abiotic stress treatments [17,18,19,20] or, in turn, were rendered more sensitive to the applied abiotic stresses [21]. There is also evidence that some plant CaMs and CMLs interact with the protein targets that are known to regulate abiotic stress adaptation either positively or negatively [22,23,24].

A recent study has identified and characterized CaM and CML gene families in cultured grapevine Vitis vinifera L. based on the genome sequencing data and publicly available expression profiling datasets [25]. The purpose of the present study was to identify the CaM and CML genes actively expressed in wild-growing grape Vitis amurensis Rupr. in response to various abiotic stress stimuli and analyze their expression under these stress conditions. Wild-growing relatives of cultured plant species often exhibit higher tolerance to abiotic stresses, and genes of wild plant species represent an important source for improving abiotic stress tolerance of cultivated counterparts.

2. Results

2.1. Isolation, Molecular Cloning, and Sequencing of VaCaM and VaCML Transcripts

To clone and sequence the full-length coding sequences of CaMs and CMLs of V. amurensis, we designed specific primers to the 5′ and 3′ ends of the highly homologous CaMs and CMLs of V. vinifera (Table 1; Table S1). V. vinifera is a close species to V. amurensis, and its genome was sequenced [26,27]. To design the specific primers, we retrieved the predicted CaMs and CMLs of V. vinifera PN20024 genotype (V2 proteome prediction) present in the Grapevine Genome Centro Di Ricerca Interdipartimentale Per Le Biotecnologie Innovative (CRIBI) Biotech Centre database using the CaM and CML protein sequences of A. thaliana downloaded from the Arabidopsis Information Resource (TAIR) database as queries as described [25]. In addition, we downloaded all CaM and CML protein sequences of V. vinifera predicted by an automated computational analysis and deposited to the National Centre for Biotechnology Information (NCBI) GenBank. After the removal of all duplicate sequences and sequences containing functional domains other than EF-hands, we obtained a total of three VviCaMs and 68 VviCMLs.

Table 1

Characteristics of CaM and CML transcripts and deduced amino acid sequences of Vitis amurensis and Vitis vinifera.

Vitis amurensiscDNA Clone				Vitis viniferaGene Prediction				Protein I/S (%)	No. of EF Hands	N-Myrist	N-Palmit	MW (kDa)	Group
Transcript Name	ID GeneBank	CDS (bp)	Amino Acids	Gene Name	Gene ID	CDS (bp)	Amino Acids
VaCaM8	MN515154	450	149	VviCaM8	VIT_208s0040g00470.6	450	149	100/100	4	-	-	16.8	1
VaCaM9	MN478368	450	149	VviCaM9	VIT_217s0000g00580.1	450	149	100/100	4	-	-	16.9	1
VaCaM10	MN515156	450	149	VviCaM10	VIT_206s0009g01910.1	462	153	100/100	4	-	-	16.8	1
VaCML9a	MN515159	462	153	VviCML9a	VIT_214s0030g02150.1	462	153	97/98	4	-	-	17.5	2
VaCML9b	MN515160	450	149	VviCML9b	VIT_200s0179g00280.1	450	149	100/100	4	-	Yes	17.0	2
VaCML79	MN515161	450	149	VviCML79	VIT_205s0020g04420.1	450	149	100/100	4	-	-	17.0	2
VaCML80	MN515162	444	147	VviCML80	VIT_202s0241g00140.1	444	147	100/100	3	Yes	-	16.6	2
VaCML107	MN562253	438	145	VviCML107	VIT_207s0031g00700.3	438	145	99/98	3	-	Yes	16.8	2
VaCML108	MN562252	522	173	VviCML108	VIT_205s0094g01240.3	522	173	99/99	4	-	-	19.9	2
VaCML72	MN515163	483	160	VviCML72	VIT_217s0000g04460.1	483	160	99/99	4	Yes	-	17.6	3
VaCML73	MN515164	489	162	VviCML73	VIT_201s0011g02470.1	489	162	100/100	4	-	-	17.8	3
VaCML74	MN537892	492	163	VviCML74	VIT_216s0039g01880.1	492	163	100/100	4	-	-	18.2	3
VaCML75	MN537893	492	163	VviCML75	VIT_202s0012g02060.1	492	163	100/100	4	-	-	18.1	3
VaCML1	MN537894	552	183	VviCML1	VIT_203s0063g00530.1	549	182	98/98	4	-	-	21.0	4
VaCML41a	MN537895	561	186	VviCML41a	VIT_204s0023g01100.1	558	185	97/98	3	Yes	-	21.0	4
VaCML41b	MN537896	576	191	VviCML41b	VIT_218s0001g11830.1	576	191	98/98	3	-	-	21.2	4
VaCML44	MN537897	489	162	VviCML44	VIT_218s0001g01630.1	489	162	99/99	4	-	-	18.3	4
VaCML60	MN537898	669	222	VviCML60	VIT_208s0007g05790.1	669	222	98/99	4	-	-	24.3	4
VaCML86	MN540577	492	163	VviCML86	VIT_217s0000g02480.1	282	93	98/98 a	4	-	-	18.1	4
VaCML88	MN540578	579	192	VviCML88	VIT_208s0056g00290.1	579	192	100/100	3	Yes	-	21.4	4
VaCML89	MN540579	663	220	VviCML89	VIT_211s0016g05740.1	771	256	99/100	4	-	Yes	24.5	4
VaCML90	MN540580	465	154	VviCML90	VIT_207s0289g00040.1	465	154	100/100	4	-	Yes	17.5	4
VaCML91				VviCML91	VIT_207s0141g00300.1
VaCML92	MN540581	507	168	VviCML92	VIT_218s0122g00180.1	507	168	100/100	4	-	Yes	18.4	4
VaCML93	MN540582	429	142	VviCML93	VIT_214s0171g00150.1	429	142	99/99	4	-	-	15.9	4
VaCML94	MN540583	432	143	VviCML94	VIT_217s0000g01630.1	432	143	99/100	4	-	-	16.1	4
VaCML105	MN562248	453	150	VviCML105	VIT_214s0006g01400.1	453	150	100/100	4	-	Yes	16.5	4
VaCML106	MN562254	255	84	VviCML106	VIT_208s0007g08830.1	255	84	100/100	2	-	-	9.5	4
VaCML109	MN562249	615	204	VviCML109	VIT_215s0048g00790.1	615	204	99/99	4	-	Yes	22.9	4
VaCML110	MN562246	648	215	VviCML110	VIT_205s0102g00450.1	645	214	99/99	4	-	Yes	24.6	4
VaCML95	MN540584	423	140	VviCML95	VIT_201s0010g03000.1	423	140	98/99	4	-	Yes	16.1	5
VaCML96	MN540585	423	140	VviCML96	VIT_201s0010g02990.1	423	140	96/97	4	-	Yes	16.1	5
nd				VviCML97	VIT_201s0010g02940.1	423				-	-	16.1	5
nd				VviCML98	VIT_201s0010g02970.1	423				-	-	16.1	5
nd				VviCML99	VIT_201s0010g03010.1	423				-	-	16.1	5
VaCML100	MN540586	423	140	VviCML100	VIT_201s0010g02980.1	423	140	96/97	4	-	Yes	16.1	5
nd				VviCML101	VIT_201s0010g02930.1	423				-	-	16.1	5
nd				VviCML102	VIT_201s0010g02950.1	423				-	-	16.1	5
VaCML103	MN540587	423	140	VviCML103	VIT_201s0010g03040.1	423	140	95/96	4	-	Yes	16.1	5
VaCML104	MN540588	423	140	VviCML104	VIT_201s0010g03020.1	423	140	96/97	4	-	Yes	16.1	5
VaCML55	MN540589	294	97	VviCML55	VIT_218s0001g10670.1	294	97	100/100	2	-	Yes	11.4	6 a
VaCML83	MN540590	543	180	VviCML83	VIT_201s0026g02590.1	543	180	98/98	2	-	Yes	20.5	6 a
VaCML84	MN540591	471	156	VviCML84	VIT_214s0108g01000.1	471	156	99/99	2	-	Yes	18.3	6 a
VaCML85	MN540592	591	196	VviCML85	VIT_217s0000g06325.1	591	196	99/99	2	-	Yes	22.0	6 a
VaCML87	MN540593	294	97	VviCML87	VIT_207s0031g00760.1	294	97	99/98	2	-	Yes	11.5	6 a
VaCML51	MN540594	288	95	VviCML51	VIT_218s0001g10605.1	288	95	96/98	2	-	-	10.6	6 b
VaCML52	MN540595	279	92	VviCML52	VIT_218s0001g10600.1	279	92	100/100	2	-	-	10.6	6 b
VaCML53	MN540596	270	89	VviCML53	VIT_218s0001g10595.1	270	89	98/100	2	-	-	10.6	6 b
VaCML54	MN540597	270	89	VviCML54	VIT_218s0001g10645.1	270	89	98/98	2	-	Yes	10.3	6 b
nd				VviCML56	VIT_218s0001g10630.1								6 b
VaCML57	MN540598	294	97	VviCML57	VIT_218s0001g10620.1	285	94	97/96	2	-	-	11.2	6 b
nd				VviCML58	VIT_218s0001g10640.1	435							6 b
nd				VviCML59	VIT_218s0001g10610.1	300							6 b
nd				VviCML21v.1	VIT_219s0015g01200.1	708
VaCML21v.2	MN540599	699	232	VviCML21v.2	VIT_219s0015g01200.2	699	232	99/99	4	Yes	Yes	26.4	7 a
VaCML22	MN540602	729	242	VviCML22	VIT_205s0029g00070.1	747	248	97/97	4	Yes	Yes	27.3	7 a
VaCML62	MN540605	414		VviCML62	VIT_212s0059g00360.1	414		97/98	2	Yes	-	15.5	7 a
nd				VviCML63	VIT_212s0059g00320.1	414							7 a
nd				VviCML64	VIT_212s0059g00370.1	414							7 a
VaCML65	MN540606	414	137	VviCML65	VIT_212s0059g00430.1	414	137	99/99	2	Yes	-	15.6	7 a
VaCML66	MN540607	420	139	VviCML66	VIT_213s0156g00120.1	420	139	100/100	2	Yes	-	15.5	7 a
nd				VviCML67	VIT_212s0059g00340.1	414							7 a
nd				VviCML68	VIT_212s0059g00420.1	414							7 a
nd				VviCML69	VIT_212s0059g00400.1	414							7 a
nd				VviCML70	VIT_212s0059g00350.1	414							7 a
VaCML48	MN562247	678	225	VviCML48	VIT_206s0080g00450.1	678	225	100/100	2	-	-	25.3	7 b
VaCML61	p.s.b			VviCML61	VIT_205s0077g00300.1	909	302	100/100	2	Yes	-	33.5	7 b
nd				VviCML77v.1	VIT_200s0252g00130.1	711							7 b
VaCML77v.2	MN540608	831	276	VviCML77v.2	VIT_200s0252g00130.2	831	276	98/98	2	Yes	-	29.7
VaCML78	MN540610	1119	372	VviCML78	VIT_210s0071g00670.1	1119	372	98/99	5	Yes	-	43.4	7 b
VaCML81	MN540611	1425	474	VviCML81	VIT_204s0008g06280.1	1425	474	99/99	4	-	Yes	54.2	7 b
VaCML82	MN540612	1065	354	VviCML82	VIT_211s0118g00540.1	1065	354	99/100	5	-	-	40.6	7 b
VaCML71	MN548771	594	72	VviCML71	VIT_219s0014g04650.1	453	150	100/100 a	0	-	-	8.0	8
VaCML76	MN540595	363	120	VviCML76	VIT_202s0012g00660.1	363	120	99/99	1	Yes	-	13.5	8

Note: No. of EF hands—the number of EF hands predicted by PROSITE scan [28,29]; N-Myrist and N-Palmit—the number of myristoylation and palmitoylation motifs identified with GPS-Lipid [30,31]; CDS—coding DNA sequences; MW (kDa)—molecular mass calculated using the Compute pI/Mw tool [32]; I and S—identities and similarities of the deduced V. amurensis and V. vinifera amino acid sequences. a the protein I and S were obtained without taking into account insertions; b partially sequenced (Figure S7).

We compared our results with the computational analysis of CaMs and CMLs of V. vinifera published by Vandelle et al. [25]. In addition to the three VviCaMs and 62 VviCMLs described previously for V. vinifera, our analysis of the V. vinifera CaMs and CMLs sequences available at the CRIBI and NCBI databases identified seven additional VviCMLs, including VviCML48, VviCML105, VviCML106, VviCML107, VviCML108, VviCML109, and VviCML110 (Table 1; Figure 1). The seven newly identified VviCMLs were named according to the procedure described by Vandelle et al. [25] following the instructions of the international Super-Nomenclature Committee for Grape Gene Annotation [28]. The deduced amino acid sequences of the newly identified VviCMLs did not contain functional domains other than EF-hands (Table 1). We also noted that nucleotide sequences of the previously identified VviCML90 and VviCML91 shared 100% identity with each other at the nucleotide level, and they should be treated as one VviCML.

Figure 1

Phylogenetic tree of plant calmodulins (CaMs) and calmodulin-like proteins (CMLs) of Vitis amurensis, Vitis vinifera, and Arabidopsis thaliana created using the full-length protein sequences. The phylogenetic tree was constructed using the MEGA-X program by the neighbor joining method with 1000 bootstrap replicates. The CaMs and CMLs were categorized into nine subgroups highlighted with different colors. Red asterisks denote CaMs and CMLs of V. amurensis identified in this study. Dark blue asterisks denote CaMs and CMLs of V. vinifera firstly described in this study.

Using RT-PCRs with the primers designed to the three VviCaMs and 68 VviCMLs (Table S1), we cloned and sequenced cDNAs containing full-length coding DNA sequences (CDS) of CaM and CML transcript variants from wild grape V. amurensis using RNA isolated from unstressed leaves and stems of V. amurensis. The analysis identified three VaCaMs and 54 VaCMLs expressed in the cuttings of V. amurensis. Table 1 shows a comparison of the cloned and sequenced VaCaMs and VaCMLs with those identified for V. vinifera. The deduced amino acid sequences of the cloned and sequenced VaCaMs and VaCMLs shared high identities (95–100%) with the homologous CaMs and CMLs of V. vinifera predicted by the automated computational analysis (Table 1).

A phylogenetic analysis was performed on the full-length amino acid sequences of the CaMs and CMLs from V. vinifera, V. amurensis, and A. thaliana (Figure 1). The proteins were categorized into nine subgroups based on sequence similarities and relationships of CaMs and CMLs of the analyzed plant species and taking into attention the described classification for V. vinifera proteins [25]. The deduced amino acid sequences of the newly identified VviCMLs clustered into the subgroup 2 (VviCML107, VviCML108), subgroup 4 (VviCML105, VviCML109), subgroup 6 (VviCML106, VviCML110), and subgroup 7 (VviCML48). The newly identified VviCMLs possessed from two to four putative EF-hand motifs (Table 1).

The identified three VaCaMs and 54 VaCMLs shared a common structure with other plant and V. vinifera CaMs and CMLs and possessed from one to five putative EF-hand motifs, except for VaCML71. The deduced amino acid sequence of VaCML71 did not contain any putative EF-hand motifs due to the presence of a premature stop codon after a 141 bp insertion, which resembled intron retention due to the presence of canonical 5′ and 3′ splice sites. Notably, the VaCML86 transcript contained a 210-bp insertion (no frameshift) and contained a higher number of putative EF-hands in comparison with the homologous VviCML86. Most of the VaCMLs were predicted to be myristoylated or palmitoylated proteins (Table 1), which indicates possible protein-membrane interactions of these CMLs.

2.2. Expression of VaCaMs and VaCMLs in Response to Abiotic Stress Conditions

To test the involvement of VaCaM and VaCML genes in the responses of V. amurensis to the water deficit, high salt, high mannitol, cold, and heat abiotic stress conditions, we used the V. amurensis cuttings (excised young stems with one healthy leaf) for the control non-stress and abiotic stress treatments. Total RNA was isolated from the leaves of the treated cuttings of V. amurensis 6 h, 12 h, and 24 h post-treatments. The V. amurensis cuttings were placed in filtered water at +25 °C (non-stress treatment), on a paper towel at +25 °C (desiccation or water deficit stress), in 0.4 M NaCl at +25 °C (high salt stress) and 0.4 M mannitol at +25 °C (high mannitol stress). To apply cold and heat temperature stress, the cuttings were incubated in filtered water in a growth chamber at +4 °C, +10 °C, and +37 °C. We used a similar experimental design and chemical concentrations as in Chung et al. [30] and Dubrovina et al. [33] for studying CDPK gene expression in Capsicum annuum and V. amurensis, respectively. Then, we applied qRT-PCR for the analysis of VaCaM and VaCML gene expression.

The qRT-PCR data revealed that VaCaM8 transcript levels considerably increased under the high salt stress conditions in 1.4–1.8 times at all time intervals post-treatment (Figure 2 and Figure S1). Also, the VaCaM8 expression was affected under +37 °C, high mannitol, and +4 °C stress conditions but only at one time interval. The VaCaM9 gene was responsive to low temperature stress (+10 °C), with considerable increases in transcript levels detected after 6 h and 24 h of treatment (Figure 2 and Figure S1). However, incubation at +4 °C did not cause such elevation to the VaCaM9 expression. Transcript levels of VaCaM9 also responded to mannitol and heat but at one time interval after being exposed to the stresses (Figure 2 and Figure S1). The VaCaM10 gene responded to the abiotic stresses more actively than VaCaM8 and VaCaM9, but the detected alterations were considerable only after 6 h of treatments (Figure 2 and Figure S1).

Figure 2

Heatmap of VaCaM expression levels after 6 h, 12 h, and 24 h of treatments in Vitis amurensis cuttings exposed to abiotic stress conditions. The VaCaM expression levels were determined by quantitative RT-PCR. The color scale represents increased (green) and decreased (red) log2 fold changes of the expression values under abiotic stress treatments relative to the control. Control—non-stress conditions (filtered water, +25 °C); WD—water-deficit stress (cuttings laid on a paper towel, +25 °C); NaCl—salt stress (0.4 M NaCl, +25 °C); Mannitol—osmoticum (0.4 M mannitol, +25 °C); +37 °C—heat stress (filtered water, +37 °C); +10 °C, and +4 °C—cold stress (filtered water, +10 °C, and +4 °C). *, **—significantly different from the values of CaM expression in V. amurensis under the control conditions after 6 h, 12 h, or 24 h of treatments at p ≤ 0.05 and 0.01 according to the Student’s t-test.

Then, we analyzed transcript levels of the 54 identified VaCMLs in response to desiccation, high salt, high mannitol, cold, and heat stresses (Figure 3, Figure 4, Figure 5 and Figures S1–S6). The VaCMLs were divided into four groups based on their responsiveness to the abiotic stresses:

Figure 3

Heatmap of expression levels of 13 VaCMLs significantly up-regulated at least at two time intervals after one or several abiotic stress treatments in Vitis amurensis cuttings. The VaCaM expression levels were determined by quantitative RT-PCR and analyzed after 6 h, 12 h, and 24 h of treatments. The color scale represents increased (green) and decreased (red) log2 fold changes of the expression values under abiotic stress treatments relative to the control. The VaCML genes were ordered by their subgroup numbers. Control—non-stress conditions (filtered water, +25 °C); WD—water-deficit stress (cuttings laid on a paper towel, +25 °C); NaCl—salt stress (0.4 M NaCl, +25 °C); Mannitol—osmoticum (0.4 M mannitol, +25 °C); +37 °C—heat stress (filtered water, +37 °C); +10 °C, and +4 °C—cold stress (filtered water, +10 °C, and +4 °C). *, **—significantly different from the values of CML expression in V. amurensis under the control conditions after 6 h, 12 h, or 24 h of treatments at p ≤ 0.05 and 0.01 according to the Student’s t-test.

Figure 4

Heatmap of expression levels of five VaCMLs significantly down-regulated at least at two time intervals after one or several abiotic stress treatments in Vitis amurensis cuttings. The VaCaM expression levels were determined by quantitative RT-PCR and analyzed after 6 h, 12 h, and 24 h of treatments. The color scale represents increased (green) and decreased (red) log2 fold changes of the expression values under abiotic stress treatments relative to the control. The VaCML genes were ordered by their subgroup numbers. Control—non-stress conditions (filtered water, +25 °C); WD—water-deficit stress (cuttings laid on a paper towel, +25 °C); NaCl—salt stress (0.4 M NaCl, +25 °C); Mannitol—osmoticum (0.4 M mannitol, +25 °C); +37 °C—heat stress (filtered water, +37 °C); +10 °C and +4 °C—cold stress (filtered water, +10 °C, and +4 °C). *, **—significantly different from the values of CML expression in V. amurensis under the control conditions after 6 h, 12 h, or 24 h of treatments at p ≤ 0.05 and 0.01 according to the Student’s t-test.

Figure 5

Heatmap of expression levels of 14 VaCMLs displaying both up- and down-regulation at least at two time intervals after one or several abiotic stress treatments in Vitis amurensis cuttings. The VaCaM expression levels were determined by quantitative RT-PCR and analyzed after 6 h, 12 h, and 24 h of treatments. The color scale represents increased (green) and decreased (red) log2 fold changes of the expression values under abiotic stress treatments relative to the control. The VaCML genes were ordered by their subgroup numbers. Control—non-stress conditions (filtered water, +25 °C); WD—water-deficit stress (cuttings laid on a paper towel, +25 °C); NaCl—salt stress (0.4 M NaCl, +25 °C); Mannitol—osmoticum (0.4 M mannitol, +25 °C); +37 °C—heat stress (filtered water, +37 °C); +10 °C and +4 °C—cold stress (filtered water, +10 °C and +4 °C). *, **—significantly different from the values of CML expression in V. amurensis under the control conditions after 6 h, 12 h, or 24 h of treatments at p ≤ 0.05 and 0.01 according to the Student’s t-test.

Thirteen VaCML genes were significantly up-regulated at a minimum of two time intervals after one or several abiotic stress treatments (Figure 3 and Figure S2);

Five VaCML genes were significantly down-regulated at a minimum of two time intervals after one or several abiotic stress treatments (Figure 4 and Figure S3);

Fourteen VaCML genes were differentially regulated displaying both up- and down-regulation at a minimum of two time intervals post-treatment (Figure 5 and Figure S4);

Expression levels of 22 VaCML genes showed slight effects or occasional regulation mainly at one time interval (Figure S5) or were not essentially changed (Figure S6).

Notably, expression levels of VaCML95, VaCML96, VaCML100, VaCML103, and VaCML104 (Figure S5; Table S2) were analyzed together using one primer pair due to a high identity among the corresponding nucleotide sequences.

Water deficit was one of the strongest stimuli for induction of VaCML expression with a marked elevation in 1.8–13.1 times in transcript levels of nine genes (VaCML62, VaCML72, VaCML79, VaCML83, VaCML92, VaCML93, VaCML105, VaCML106, and VaCML110) detected after 12 h and 24 h of treatment (Figure 3, Figure 5, Figures S2 and S4). In addition, incubation under water deficit resulted in a progressive and considerable down-regulation in 1.7–3.1 times of three CMLs, including VaCML57, VaCML66, and VaCML88, which was detected after two periods of treatment (Figure 4, Figure 5, Figures S3 and S4).

Similar to the effect of water deficit, high salt stress considerably affected VaCML expression with both up- and down-regulation. Transcript levels of VaCML44, VaCML61, VaCML78, VaCML82, VaCML86, and VaCML92 were significantly induced in 2.1–15 times at a minimum of two time intervals (Figure 3, Figure 5, Figures S2 and S4), while the transcript abundance of VaCML57, VaCML60, VaCML77, and VaCML89 was progressively suppressed in 1.8–2.8 times at a minimum of two time intervals (Figure 4, Figure 5, Figures S3 and S4). Notably, the VaCML44, VaCML61, and VaCML57 genes progressively and remarkably responded after 6 h, 12 h and 24 h of high salt exposure (Figure 3, Figure 4, Figures S2 and S3).

High mannitol stress had a weaker effect on VaCML gene expression in comparison with other abiotic stress treatments. We detected a considerable activation of only three CMLs (VaCML79, VaCML92, and VaCML105) in 1.9–5.9 times after two time periods of the high mannitol treatment (Figure 5 and Figure S4). As for negative regulation, only VaCML57 gene was progressively down-regulated under high mannitol conditions (Figure 4 and Figure S3). Other VaCML genes responded with considerable changes only at one time interval post-treatment or remained unresponsive to the high mannitol treatment (Figure 3, Figure 4, Figure 5 and Figures S2–S4).

Heat and cold stress conditions markedly affected VaCML expression. In response to heat stress, expression of VaCML1, VaCML21, VaCML22, VaCML52, VaCML107, and VaCML108 was distinctly modulated in 1.7–5.9 times at two time intervals post-treatment (Figure 3, Figure 5 and Figures S2 and S4), while expression of VaCML60, VaCML79, VaCML88, VaCML89, and VaCML105 was progressively repressed under the high temperature conditions 6 h and 12 h after the treatment in most cases (Figure 4, Figure 5 and Figures S3 and S4). Incubation at lowered temperatures (+4 °C or +10 °C) strongly induced transcript levels of six CML genes (VaCML21, VaCML44, VaCML61, VaCML78, VaCML86, and VaCML89) and suppressed eight CML genes (VaCML9a, VaCML48, VaCML57, VaCML75, VaCML82, VaCML85, VaCML92, and VaCML107) at a minimum of two incubation intervals post-treatment (Figure 3, Figure 4 and Figure 5 and Figures S2–S4). Notably, although these low temperature stresses are similar, expression of only VaCML61, VaCML75, and VaCML89 genes was significantly and progressively affected by both these incubation temperatures at least at two time periods of the cold treatments. Transcript levels of VaCML44 and VaCML92 markedly responded exclusively to the incubation at +10 °C, while transcript levels of VaCML9a, VaCML57, VaCML85, VaCML86, and VaCML107 were affected only at +4 °C (Figure 3, Figure 4, Figure 5 and Figures S2–S4).

The data obtained revealed that the detected changes in transcription levels of 16 CML genes were not consistent and exhibited rather slight effects (VaCML9b, VaCML41b, VaCML54, VaCML65, VaCML71, VaCML73, VaCML74, VaCML76, and VaCML90/91), or considerable effects were observed only 24 h after stress application (VaCML41a, VaCML81, VaCML95, VaCML96, VaCML100, VaCML103, and VaCML104) (Figure S5). Expression of six CML genes (VaCML51, VaCML53, VaCML80, VaCML87, VaCML94, VaCML109) remained unresponsive to the abiotic stress treatments (Figure S6). We also noted that some genes originating from the same subgroup of the phylogenetic tree (Figure 1) showed similar expression pattern under the abiotic stress treatments. For example, VaCML9a, VaCML79, VaCML107 (subgroup 2); VaCML92, VaCML93, VaCML105, VaCML110 (subgroup 4); VaCML83, VaCML84, VaCML89, VaCML106 (subgroup 6), VaCML48, and VaCML82 (subgroup 7) (Figure 3, Figure 5, Figures S2 and S4).

3. Discussion

Wild grape V. amurensis is known to exhibit a high resistance to abiotic stresses, especially to cold stress [34]. Therefore, it would be interesting to investigate transcriptional responses of V. amurensis genes to various abiotic stimuli. After completion of sequencing and annotation of various plant genomes, the presence of CaMs and large CML-encoding gene families has emerged as a typical feature of plant genomes. The grapevine genome was not an exception. Recently, based on the analysis of the available grapevine genome sequencing data, Vandelle et al. [25] determined the presence of three CaM and 62 CML genes in the genome of the commonly cultivated grapevine V. vinifera. Using the publicly available gene expression datasets of different organs and tissues of V. vinifera cv. Corvina (clone 48), Vandelle et al. [25] analyzed expression of VviCaMs and VviCMLs in detail in the different grape organs and after application of drought, shade, heat, glucose, ultraviolet-C, and abscisic acid. In our study, we treated the stem cuttings of wild grape V. amurensis with a number of abiotic stress factors, including desiccation, high salinity, high mannitol, heat and cold stresses. The full-length coding sequences of VaCaMs and VaCMLs were cloned and their transcript levels were analyzed by real-time qRT-PCR after 6 h, 12 h, and 24 h of treatments.

We revised the identified CaM and CML genes in the V. vinifera genome [25] and described seven additional VviCMLs increasing the family size to 68 VviCMLs in V. vinifera. Then, we identified the CaM and CML genes expressed in wild grape V. amurensis in response to desiccation, high salinity, high mannitol, and temperature stresses. The analysis allowed identification and characterization of three CaMs and 54 CMLs of V. amurensis expressed in non-stressed tissues of V. amurensis and under the analyzed abiotic stress conditions. The qRT-PCR analysis of the VaCaM expression profiles revealed that the genes were actively expressed under the high salinity, high mannitol, desiccation stress, heat, and low temperature stress conditions. The applied abiotic stress treatments positively regulated expression of CaM genes in V. amurensis, with the most persistent changes being observed for VaCaM8 in response to salt stress, but the alternations in VaCaM expression were not progressive and remarkable. The data suggest that CaMs are slightly implicated in the abiotic stress resistance of V. amurensis. In contrast to VaCaMs, most of the evaluated VaCMLs (32 CMLs out of 54 analyzed genes) were highly responsive to the analyzed abiotic stress conditions exhibiting both positive and negative regulation patterns. The results obtained indicated that the 32 VaCMLs play distinct positive and negative roles in responses of wild grape to abiotic stresses. The expression patterns of individual VaCMLs have been shown to be induced and/or repressed in response to particular stress stimuli and frequently varied temporally and in magnitude in response to the abiotic stresses, suggesting specificity in their roles in abiotic stress adaptation of V. amurensis. The expression patterns of some VaCMLs were similar under the analyzed stress conditions, which suggests that the genes could perform similar functions and/or are regulated by related molecular mechanisms. The other 22 VaCMLs displayed insensitive expression patterns, showed occasional regulation or were slightly affected, indicating that they are not implicated in V. amurensis abiotic stress responses.

The effects of heat stress on CaM and CML transcript levels were analyzed both V. amurensis in our study and for V. vinifera by Vandelle et al. [25]. The CaM and CML genes of V. amurensis and V. vinifera displayed similar regulation under heat application. For example, heat stress greatly increased expression of the VviCML55 in V. vinifera and the homologous VaCML55 gene in V. amurensis, while it negatively regulated CML60, CML79, and CML92 expression in both these grapevine species. In addition, we noted that both VaCML92 and VvCML92 were strongly induced by water deficit stress [25].

Recently, a number of studies demonstrated that overexpression of plant CaM and CML genes can improve plant resistance to abiotic stresses [17,18,19,20]. For example, overexpression of the ShCML44 gene isolated from cold tolerant wild tomato enhanced the tolerance of a stress-sensitive tomato to cold, drought, and salinity [18]. According to our data, a homologous VaCML44 gene shared 66% protein similarity to ShCML44 and was up-regulated under cold and salinity stress treatments (Figure 2). Overexpression of the CsCaM3 gene, which shared 100% protein similarity with VaCaM8 and VaCaM10, improved heat stress tolerance in cucumber [19]. Heat stress significantly increased transcript levels of the VaCaM8 and VaCaM10 genes after 6 h of treatment in our experiments (Figure 1).

Results obtained in the present study suggested that the VaCML44, VaCML61, VaCML86, and VaCML89 genes were strongly and progressively induced under salt and cold stresses and are promising candidates for use in overexpression experiments to obtain plants with increased tolerance to these abiotic stress stimuli. The VaCML21, VaCML22, and VaCML52 genes are in turn good candidates for increasing heat stress tolerance and VaCML79, VaCML83, VaCML93, 106, and VaCML110—for increasing water deficit tolerance. The assumptions need to be verified by establishing transgenic plants in future experiments.

In conclusion, our study revealed potential importance of a number of CaM/CML genes of V. amurensis in its adaptation to abiotic stresses and indicated that some VaCaM and VaCML genes (mainly presented in Figure 2 and Figure 4) are positive regulators of plant abiotic stress tolerance and can be used in plant biotechnology and molecular biology in overexpression experiments to obtain plants with increased resistance to water-deficit, salinity, osmoticum, and temperature stresses. The VaCML genes with negative responses to the abiotic stimuli may be blockers in the development of the wild grape stress signaling and need additional studies.

4. Materials and Methods

4.1. Plant Material and Treatments

For the abiotic stress treatments, we used young vines of wild-growing grapevine V. amurensis Rupr. (Vitaceae) sampled from a non-protected natural population near Vladivostok, Russia (Akademgorodok, the southern Primorsky region of the Russian Far East, longitude 43.2242327 and latitude 131.99112300). The vines were collected in August 2018 and identified at the Botany Department of the Federal Scientific Center of the East Asia Terrestrial Biodiversity. The V. amurensis vines were divided into cuttings (excised young stems 7–8 cm long with one healthy leaf) that were placed in individual beakers and used for the stress treatments. For the control non-stress treatment, the V. amurensis cuttings were placed in filtered water at 25 °C. To induce water deficit stress, the cuttings were laid on a paper towel at 25 °C. To induce osmotic stress, the cuttings were placed in 400 mМ NaCl and 400 mМ mannitol solutions at 25 °C. To apply cold and heat stress, the V. amurensis cuttings were placed in filtered water in the growth chamber (Sanyo MLR-352, Panasonic, Osaka, Japan) at +4 °C, +10 °C, and +37 °C. The V. amurensis cuttings were grown under a 16/8 h light/dark phoperiod. The freshly harvested cuttings were acclimated to the “non-stress” condition for 30 min before they were treated with the stress treatments. The experiments were repeated three times for each stress treatment time and for the control treatment.

4.2. RNA Isolation and cDNA Preparation

Leaf samples were harvested after 6 h, 12 h, and 24 h of the abiotic stress treatments and immediately used for RNA extraction. Total RNA extraction was performed using the cetyltrimethylammonium bromide (CTAB)-based extraction as described [35]. Complementary DNAs were synthesized using 1.5 µg of RNA by the Moloney Murine Leukemia Virus (MMLV) Reverse transcription PCR Kit (RT-PCR, Sileks M, Moscow, Russia) as described [36]. The reverse transcription products were amplified by PCR and verified on the absence for DNA contamination using primers for AtActin2 gene (NM_112764) listed in Table S1 (356 bp PCR product from cDNA and 442 bp from DNA).

4.3. Cloning and Sequencing of VaCaM and VaCML Transcripts

To clone and sequence cDNAs containing full-length CDS of the VaCaM and VaCML transcripts, we retrieved the predicted protein and mRNA sequences of V. vinifera CaMs and CMLs from the Grape Genome Database hosted at CRIBI [37]. For this purpose, the CaM and CML protein sequences of Arabidopsis thaliana were downloaded from the TAIR database [38] and used as queries for blastp (V2 gene prediction) search against the deduced proteome of the PN40024 V. vinifera genome as described [25]. For non-redundant protein sequences, we performed a domain analysis by PROSITE scan [29,39] and prediction of myristoylation and palmitoylation motif numbers with GPS-Lipid [31,40]. The coding nucleotide sequences of the selected V. vinifera CaMs and CMLs were downloaded from the CRIBI database using tblastn search with the VviCaMs and VviCMLs protein sequences as queries (V2 mRNA prediction). Specific primers for amplification of the full-length coding cDNA sequences of the VaCaM and VaCML transcripts (Table S1) were designed to the retrieved VviCaMs and VviCMLs.

The coding cDNA sequences of VaCaMs and VaCMLs were amplified using RNA extracted from unstressed leaves of V. amurensis. To clone the full-length cDNAs of the VaCaMs and VaCMLs, RT-PCRs were performed in a T100TM Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in 20 µL aliquots of the reaction mixtures using Ta 50–56 °C, elongation time 40 s—1 min 30 s. For the PCR reactions, we used Pfu polymerase (Sileks M, Moscow, Russia) as described in Kiselev et al. [41].

The obtained PCR products of VaCaM and VaCML cDNAs were subcloned into a pJET1.2 using CloneJET PCR Cloninig Kit (ThermoFisher Scientific, Waltham, MA, USA) and sequenced using an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manuphacturer’s instructions. The sequences of the V. amurensis VaCaM and VaCML transcripts were deposited to GenBank (Table 1).

Multiple sequence alignments were done with the ClustalX program [42]. For classification of the CaMs and CMLs into subfamilies, a phylogenetic tree was created with the Clustal Omega program [43]. The VaCaM and VaCML amino acid sequences were predicted using the Gene Runner 3.05 program. The following online websites were used to predict molecular weights, N-terminal myristoylation and palmitoylation motifs, and domain structure of the VaCaM and VaCML proteins: Compute pI/Mw tool [32], GPS-Lipid [31,40], and PROSITE [29].

4.4. Expression Analysis of VaCaMs and VaCMLs

Quantitative RT-PCR (qRT-PCR) was performed using Real-time PCR kit (Syntol, Moscow, Russia) and EvaGreen Real-time PCR dye (Biotium, Hayward, CA, USA) using cDNAs of VaCaMs and VaCMLs and two internal controls (VaGAPDH and VaActin1) as described [44,45]. The expression was calculated by the 2−ΔΔCT method [46]. The visualized heatmaps were generated using heatmapper.ca [47,48]. Primers used for qRT-PCRsare listed in Table S2. qRT-PCR data shown were obtained from three independent experiments.

4.5. Statistical Analysis

The statistical analysis was carried out using the Microsoft Office Excel 2007 program (Microsoft corporation, Redmond, WA, USA). The data are presented as mean ± standard error (SE) and were tested by paired Student’s t-test. The 0.05 level was selected as the point of minimal statistical significance in all analyses.