Characterization and the comprehensive expression analysis of tobacco valine-glutamine genes in response to trichomes development and stress tolerance

doi:10.21203/rs.3.rs-2229831/v1

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Research Article

Characterization and the comprehensive expression analysis of tobacco valine-glutamine genes in response to trichomes development and stress tolerance

https://doi.org/10.21203/rs.3.rs-2229831/v1

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published 09 Jul, 2023

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Valine-glutamine genes (VQ) acted as transcription regulators and played the important roles in plant growth and development, and stress tolerance through interacting with transcription factors and other co-regulators. In this study, sixty-one VQ genes containing the FxxxVQxxTG motif were identified and updated in the Nicotiana tobacum genome. Phylogenetic analysis indicated that NtVQ genes were divided into seven groups and genes of each group had highly conserved exon-intron structure. Expression patterns analysis firstly showed that NtVQ genes expressed individually in different tobacco tissues including mixed-trichome (mT), glandular-trichome (gT), and nonglandular-trichome (nT), and the expression levels were also distinguishing in response to methyl jasmonate (MeJA), salicylic acid (SA), gibberellic acid (GA), ethylene (ETH), high salinity and PEG stresses. Besides, only NtVQ17 of its gene family was verified to have acquired autoactivating activity. This work will not only lead a foundation on revealing the functions of NtVQ genes in tobacco trichomes but also provided references to VQ genes related stress tolerance research in more crops.

Valine-glutamine gene (NtVQ)

mixed-trichome (mT)

glandular-trichome (gT)

nonglandular-trichome (nT)

stress tolerance

Nicotiana tobacum

Valine-glutamine genes (VQ) were kinds of transcription regulators and played the important roles in plant growth, development and responses to various environmental stimulus. VQ proteins were labeled with a conserved single short FxxxVQxxTG amino acid sequence motif (pfam 05678), which was essential between protein interaction with transcript factors like WRKY (Cheng et al. 2012), and dispensable in the interaction with other genes like MAPKs (Pecher et al. 2014). Multiple VQ gene families from plant species have been identified and studied. Taking monocotyledons for examples, there were forty VQ genes in Oryza sativa (Kim et al. 2013; Jiang et al. 2018), sixty-one in Zea mays (Song et al. 2016), and eighteen in Vitis vinifera (Wang et al. 2015); taking dicotyledons for another examples, there were thirty-four VQ genes in Arabidopsis thaliana (Cheng et al. 2012), twenty-six in Solanum lycopersicum (Ding et al. 2019), and eight-nine in Gossypium hirsutum (Chen et al. 2020b).

VQ genes were involved in multiple regulatory aspects of plant growth and development. For instance, AtVQ8 mutants showed pale-green and stunted-growth, which was similar with the roles of AtVQ17/18/22 in overexpression Arabidopsis plants (Cheng et al. 2012). AtVQ14 (HAIKU1, IKU1) regulated endosperm growth and seeds size by interacting with AtWRKY10 (Wang et al. 2010). AtVQ20 participated in pollen development via inhibiting the expression of downstream MYBs genes (Lei et al. 2017). AtVQ29 acted as a repressor in the light-meditated inhibition of hypocotyl elongation during early seedling development (Cheng et al. 2012). Moreover, OsVQ13 could positively regulate grain size in rice (Uji et al. 2019). Soybean VQ genes overexpression plants showed altered leaf morphology and flowering time (Zhou et al. 2016).

VQ genes were also verified to manage responses to plant biotic and abiotic stress. In Arabidopsis, both AtVQ4 (MPK3/6-targeted VQ protein1, MVQ1) and AtVQ21 (Mitogen-activated Protein Kinnase4 Substrate1, MKS1) met with the quick response to WRKY mediated immune defense (Andreasson et al. 2005; Pecher et al. 2014). AtVQ5/20, AtVQ16 (Sigma Factor-Interacting Protein2, SIB2) and AtVQ23 (SIB1) were proved to be involved in the resistance to Botrytis cinera (Lai et al. 2011; Cheng et al. 2012). AtVQ9 and AtVQ15 (Arabidopsis CaM-binding protein, AtCaMBP25) were demonstrated to regulate salinity and osmotic stresses, responsively (Hu et al. 2013b; Perruc et al. 2004). AtVQ22 (Jasmonate-associated VQ motif gene1, JAV1) negatively defined the transcriptional activity of WRKY28/51 responsive to injury (Hu et al. 2013a; Yan et al. 2018). Moreover, OsVQ13/14/32 overexpression plants increased resistance to rice bacterial blight (Uji et al. 2019; Li et al. 2021). BnVQ7 from Brassica napus, a MKS1 homologous gene, enhanced disease resistance to Leptosphaeria maculans (Zou et al. 2021). The function of soybean GmVQ58 were similar with AtVQ4 and AtVQ21 to participate in WRKY meditated immune defense responses (Li et al. 2020). These results indicated that most VQ genes were efficiently responsive to environmental conditions (Cheng et al. 2012).

As the epidermal outgrowths, trichomes have been divided into glandular- (gT) and nonglandular- (nT) type according to the secretory ability. Glandular-trichome were the site of biosynthesis and storage of large quantities of specialized metabolites (Chalvin et al. 2020), and played essential roles in the defense against biotic and abiotic factors such as pathogens attack and osmotic stress (Schuurink and Tissier 2020). However, little studies about VQ genes have focused on the trichomes development.

As one worldwide cultivated industrial crop, tobacco leaves were the main source of economic value surrounding with high density of trichomes. Tobacco glandular-trichome were related to various responses to salinity and heavy metal stresses (Yan et al. 2021; Zhang et al. 2021), and the industrial quality of flue-cured leaves (Li et al. 2017). In this study, the information of tobacco NtVQ gene family were collected and updated for the performance of bioinformatics analysis. Then, the wild flue-cured tobacco K326 covered with mixed trichomes (mT) contained gT and nT, Tobacco Introduction 1112 (T.I.1112) characterized only by nT and T.I.1068 only shown gT were used to manifest the NtVQ genes comprehensive expression patterns, thus further indicating the importance of NtVQ in trichomes formation and development process. Meanwhile, several hormone treatments and abiotic stresses were conducted to evaluate NtVQ genes values in order to demonstrate the broad spectrum resistance functions. After the above analysis, the representative NtVQ genes were tested for autoactivation activity to facilitating a complete research system. This work would lay a solid theoretical foundation for exploring the crucial functions of VQ genes in more plants.

2.1 Plant material and growth conditions

K326 was stored in the lab, Tobacco Introduction 1112 (T.I.1112) and T.I.1068 were provided from Oxford Tobacco Research Station (Oxford, North Carolina, USA). K326, T.I.1112 and T.I.1068 seedlings were grown at 25℃, 70–80% proportional humidity under 12 h / 12 h day / night cycles. For all experiments, four-week old seedlings were used. All experiments were repeated in triplicate.

2.2 Identification of tobacco VQ genes

The VQ proteins of Arabidopsis were obtained from TAIR database (http://www.arabidopsis.org/). A Hidden Markov Model (HMM) with VQ motif was extracted from Pfam database to identify all putative NtVQ genes from tobacco genome database (https://solgenomics.net/) and NCBI (https://www.ncbi.nlm.nih.gov/). The identified VQ proteins were determined using the SMART server (http://smart.embl.de/).

2.3 Bioinformatics analysis of tobacco VQ genes

A phylogenetic tree of NtVQ proteins was constructed with MEGA 7.0 software using the neighbor-joining method (Kumar et al. 2016). The exon-intron structures were analyzed with their coding regions and full-length sequences and generated using the Gene Structure Display SERVER 2.0 (http://gsds.cbi.pku.edu.cn/). The NtVQ protein structures were determined using SMART server. NtVQ genes duplications were identified as previously described (Yan et al. 2017). The syntenic blocks were used to construct a synteny analysis map of the NtVQ genes from the Plant Genome Duplication Database (Tang et al. 2008). Diagrams were generated using Circos version 0.63 (http://circos.ca/).

2.4 Acquisition of tobacco tissues and trichomes phenotyping

Root, stem, leaf (with its epidermis) and flower from K326 were collected for tissue-specific expression analysis. Trichomes from K326, T.I.1112 and T.I.1068 leaves were removed using freeze-thawing method with liquid nitrogen (Yan et al. 2021). After staining by 2% Rhodamine B for 30 min, 1 cm wide leaf filaments without veins were cut and observed using the depth-of-field digital microscope (VHX-2000; KEYENCE, Osaka, Japan).

2.5 Phytohormone treatments and abiotic stress

For exogenous phytohormone treatment, nine four-week old seedlings were sprayed with 150 µM methyl jasmonate (MeJA), 2.0 mM salicylic acid (SA), 150 µM gibberellic acid (GA), and 100 µM ethylene (ETH), respectively. Controls were cultured without any treatment. Samples were collected at 0, 1, 3, 6, and 12 h after phytohormone treatments and stored at -80℃. For abiotic stress, nine four-week old seedlings were separately treated with 300 mM NaCl and PEG-6000 (-0.5 MPa) solutions. Samples were collected at 0, 6, 24, 48, and 72 h after stress treatments and stored at -80℃.

2.6 Transcriptional activity assay

The full-length coding sequences (CDS) of NtVQ genes were cloned using the tobacco genome as the template and the relative primers in Table S1. CDS of NtVQ genes were fused into the pGBKT7 vector, and the empty pGBKT7 vector was used as the control. All constructions were transformed into the Y2H Gold yeast strain and selected on SD/-Trp/X (X: X-α-gal) and SD/-Trp/X/A medium (A: Aba), respectively (Yan et al. 2021).

2.7 RNA extraction and sqRT-PCR analysis

Tobacco RNA was extracted using the Total RNA Extraction Kit (R6827-01, Omega Bio-tek, USA). First-strand cDNAs were synthesized using a PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa Biotechnology, Dalian, China). NtVQ gene-specific primers were designed using Primer Premier 5.0 and listed in Table S1. NtL25 was used as the reference gene. The semi-quantitative RT-PCR reactions profiles and methods were described in previous studies (Yan et al. 2017; Yan et al. 2021). The data were analyzed with 2^−△△Ct method, quantified using the Gene Tools software, and visualized into heat maps with TBtools software (Chen et al. 2020a).

2.8 Data statistics

Data were presented using Microsoft Excel and Sigma Plot 10.0. One-way ANOVA analysis was performed using the SPSS Statistics 20.0 software (IBM China Company Ltd. Beijing, China) to assess significant differences.

3.1 Identification and synteny analysis of VQ genes families

Sixty-one candidate NtVQ genes were identified in the tobacco genome sequence. The coding length of sixty-one NtVQ gene sequences ranged from 216 to 1404 bp (Table 1). The exon numbers of NtVQ genes ranged from one to nine, and 77.05% of these genes had one exon. The isoelectric points of thirty NtVQ proteins were alkaline and these of the remaining proteins were acidic, which indicated that NtVQ gene family had nearly harmonious relationship between alkaline and acidic amino acids.

As shown in Fig. 1a and Table 2, a total of nineteen NtVQ genes were clustered into twelve tandem duplication event regions on tobacco chromosome 2, 4, 5, 8, 10, 14, 15, 17, 20, 21, 22 and 24, indicating that less than half of the NtVQ genes were generated by tandem duplication. A synteny analysis from tobacco and Arabidopsis further showed five syntenic relations that contained four NtVQ genes and five AtVQ genes (Fig. 1b, Table 3).

Table 1

Tobacco VQ genes and accession numbers. CDS: coding sequence.
Gene Locus	Gene Length	Start	End	Exon Number	CDS (bp)	Protein (aa)	Amino acid proportion (%)			pI
Gene Locus	Gene Length	Start	End	Exon Number	CDS (bp)	Protein (aa)	Alpha helix (Hh)	Beta-sheet (E)	Loop/Random coil (L/C)	pI
Nitab4.5_0002305g0070.1	588	399194	399781	1	588	195	28/14.36%	33/16.92%	134/68.72%	5.83
Nitab4.5_0000138g0140.1	564	1093775	1094338	1	564	187	29/15.51%	28/14.97%	130/69.52%	5.92
Nitab4.5_0000443g0160.1	627	826332	826958	1	627	208	27/12.98%	36/17.31%	145/69.71%	4.98
Nitab4.5_0008965g0010.1	648	78700	79347	1	648	215	26/12.09%	45/20.93	144/66.98%	6.29
Nitab4.5_0008708g0020.1	675	84655	85329	1	675	224	54/24.11%	43/19.20%	127/56.70%	4.72
Nitab4.5_0000443g0140.1	675	852301	852975	1	675	224	60/26.79%	43/19.20%	121/54.02%	4.97
Nitab4.5_0002570g0040.1	603	93406	94008	1	603	200	34/17.00%	28/14.50%	137/68.50%	10.6
Nitab4.5_0000073g0510.1	600	1448350	1448949	1	600	199	27/13.57%	30/15.08%	142/71.36%	8.34
Nitab4.5_0000170g0400.1	744	926895	927638	2	529	202	31/15.35%	32/15.84%	139/68.81%	10.46
Nitab4.5_0002232g0070.1	612	396497	397108	1	612	203	25/12.32%	31/15.27%	147/72.41%	10.32
Nitab4.5_0005431g0010.1	1203	72449	73651	1	1203	400	68/17.00%	37/9.25%	295/73.75%	6.45
Nitab4.5_0000421g0040.1	894	164669	165562	1	894	297	31/10.44%	25/8.42%	241/81.14%	6.22
Nitab4.5_0002098g0010.1	879	132258	133136	1	879	292	24/8.22%	31/10.62%	237/81.16%	5.95
Nitab4.5_0008140g0010.1	1674	39111	40784	3	1338	445	43/9.66%	25/5.62%	377/84.72%	7.33
Nitab4.5_0001729g0010.1	1404	94292	95695	1	1404	467	32/6.85%	44/9.42%	391/83.73%	6.49
Nitab4.5_0001270g0170.1	660	438569	439228	1	660	219	49/22.37%	31/14.16%	139/63.47%	5.91
Nitab4.5_0000123g0220.1	660	409929	410588	1	660	220	45/20.55%	21/9.59%	15./69.86%	6.3
Nitab4.5_0000062g0280.1	657	1034829	1035485	1	657	218	35/16.06%	19/8.72%	164/75.23%	9.16
Nitab4.5_0010768g0030.1	10278	14633	24910	9	1347	449	144/32.07%	90/20.04%	215/47.88%	5.57
Nitab4.5_0016059g0010.1	593	9637	10229	2	321	106	21/19.81%	11/10.38%	74/69.81%	9.74
Nitab4.5_0008385g0010.1	863	402586	41148	2	645	214	33/15.42%	24/11.21%	157/73.36%	8.34
Nitab4.5_0000680g0150.1	516	696743	697258	1	516	171	30/17.54%	18/10.53%	123/71.93%	6.56
Nitab4.5_0000680g0130.1	7468	604651	612118	2	1167	388	84/21.65%	36/9.28%	268/69.07%	9.08
Nitab4.5_0008385g0020.1	9020	59372	68391	2	1143	380	98/25.79%	26/6.84%	256/67.37%	9.28
Nitab4.5_0008558g0010.1	2332	7920	10251	3	969	322	93/28.88%	40/12.42%	189/58.70%	10.24
Nitab4.5_0000786g0140.1	875	3998189	399045	2	804	267	89/33.33%	35/13.11%	143/53.56%	10.46
Nitab4.5_0004604g0010.1	948	94021	94968	1	948	315	21/6.67%	14/4.44%	280/88.89%	10.67
Nitab4.5_0000256g0320.1	948	966518	967465	1	948	315	22/6.98%	16/5.08%	277/87.94%	10.67
Nitab4.5_0003157g0050.1	558	13932	139880	1	558	185	26/14.05%	22/11.89%	137/74.05%	9.51
Nitab4.5_0001271g0090.1	537	577237	577773	1	537	178	30/16.85%	23/12.92%	125/70.22%	9.79
Nitab4.5_0003072g0110.1	567	198841	199407	1	567	188	22/11.70%	29/15.43%	137/72.87%	9.99
Nitab4.5_0004013g0040.1	567	191866	192432	1	567	188	24/12.77%	23/12.23%	141/75.00%	9.9
Nitab4.5_0001652g0050.1	783	103509	104291	1	783	260	28/10.77%	26/10.00%	206/79.23%	9.42
Nitab4.5_0009099g0040.1	783	73650	74432	1	783	360	30/11.54%	21/8.08%	203/78.00%	9.45
Nitab4.5_0001241g0040.1	687	263071	263757	1	687	228	32/14.04%	23/10.09%	173/75.88%	9.77
Nitab4.5_0001336g0060.1	684	557974	558657	1	684	227	32/14.10%	26/11.45%	169/74.45%	9.77
Nitab4.5_0003481g0110.1	453	209282	209734	1	453	150	58/38.67%	16/10.67%	76/50.67%	6.31
Nitab4.5_0005808g0010.1	474	8127	8600	1	474	157	54/34.39%	21/13.38%	82/52.23%	5.41
Nitab4.5_0000812g0090.1	426	586770	587195	1	426	141	31/21.99%	17/12.06%	93/65.96%	5
Nitab4.5_0004588g0030.1	474	228189	228662	1	474	157	44/28.03%	31/19.75%	82/52.23%	5.96
Nitab4.5_0000349g0050.1	10775	152011	162785	2	216	71	39/54.93%	7/9.86%	25/35.21%	8.85
Nitab4.5_0002640g0020.1	1582	61688	63269	2	294	97	49/50.52%	15/15.46%	33/34.02%	9.94
Nitab4.5_0000687g0100.1	345	345785	346129	1	345	114	33/28.95%	15/13.61%	66/57.89%	6.59
Nitab4.5_0008548g0020.1	351	19094	19444	1	351	116	33/28.45%	15/12.93%	68/58.62%	5.87
Nitab4.5_0000930g0040.1	309	607049	607357	1	309	102	29/28.43%	14/13.73%	59/57.84%	5.47
Nitab4.5_0022515g0010.1	309	2522	2830	1	309	102	23/22.55%	10/9.80%	69/67.65%	6.58
Nitab4.5_0000930g0050.1	309	662450	662758	1	309	102	31/30.39%	9/8.82%	62/60.78%	6.25
Nitab4.5_0009599g0020.1	318	77300	77617	1	318	105	29/27.62%	12/11.43%	64/60.95%	5.4
Nitab4.5_0000639g0190.1	306	579499	579804	1	306	101	36/35.64%	15/14.85%	50/49.50%	9.63
Nitab4.5_0000505g0120.1	297	773483	773779	1	297	98	37/37.76%	15/15.31%	46/46.94%	9.17
Nitab4.5_0005080g0020.1	360	140722	141081	1	360	119	45/37.82%	15/12.61%	59/49.58%	5.34
Nitab4.5_0001924g0010.1	375	39051	39425	1	375	124	51/41.13%	12/9.68%	61/49.19%	5.79
Nitab4.5_0002906g0010.1	471	82252	82722	1	471	156	14/8.97%	26/16.67%	116/74.36%	5.45
Nitab4.5_0000489g0090.1	552	590481	590132	1	552	183	24/13.11%	23/12.57%	136/74.32%	5.7
Nitab4.5_0001674g0050.1	417	331009	331425	1	417	138	14/10.14%	23/16.67%	101/73.19%	9.17
Nitab4.5_0006679g0010.1	1554	144320	145873	2	455	184	28/20.65%	24/13.64%	122/66.30%	9.3
Nitab4.5_0000633g0120.1	702	299447	300148	2	510	169	38/22.49%	14/8.28%	117/69.23%	9.41
Nitab4.5_0000299g0270.1	765	884677	885441	1	765	254	58/22.83%	26/9.45%	172/67.72%	7.83
Nitab4.5_0000427g0090.1	753	785763	786515	2	506	167	38/22.75%	15/8.98%	114/68.26%	9.41
Nitab4.5_0004813g0010.1	678	31487	32164	1	678	225	43/19.11%	25/11.11%	157/69.78%	5.89
Nitab4.5_0002121g0030.1	678	283134	283811	1	678	225	38/16.89%	25/11.11%	162/72.00%	5.89

Table 2

The details of syntenic regions of tobacco VQ genes.
Number	Gene of Nicotiana tobacum	Block 1			Gene of Nicotiana tobacum	Block 2
Number	Gene of Nicotiana tobacum	Chr	Start	End	Gene of Nicotiana tobacum	Chr	Start	End
1	Nitab4.5_0002098g0010.1	20	68708367	73291850	Nitab4.5_0000421g0040.1	15	20669917	21423296
2	Nitab4.5_0000639g0190.1	8	99547870	101524099	Nitab4.5_0000505g0120.1	21	52580931	54121620
3	Nitab4.5_0000073g0510.1	21	70839788	71923681	Nitab4.5_0000170g0400.1	2	104854013	107232470
4	Nitab4.5_0000073g0510.1	21	71824456	72399223	Nitab4.5_0002570g0040.1	8	103491312	103894013
5	Nitab4.5_0000073g0510.1	21	70130728	72003347	Nitab4.5_0002232g0070.1	10	107561581	113593166
6	Nitab4.5_0000138g0140.1	17	57874512	58956217	Nitab4.5_0000443g0140.1	17	199091884	200293163
7	Nitab4.5_0001270g0170.1	5	138747	1534446	Nitab4.5_0000123g0220.1	24	50020237	50809798
8	Nitab4.5_0000812g0090.1	17	66442094	66154875	Nitab4.5_0004588g0030.1	5	43332771	43890275
9	Nitab4.5_0001674g0050.1	22	70052300	78283777	Nitab4.5_0000489g0090.1	14	73104160	79702893
10	Nitab4.5_0000170g0400.1	2	107224367	108805080	Nitab4.5_0002232g0070.1	10	106939084	107784401
11	Nitab4.5_0000062g0280.1	24	8997624	9896916	Nitab4.5_0000123g0220.1	24	48998617	50487060
12	Nitab4.5_0003072g0110.1	4	15579277	17796517	Nitab4.5_0001271g0090.1	20	92064219	93658586

Table 3

The details of syntenic regions between tobacco and *Arabidopsis thaliana VQ* genes.
Number	Gene of Nicotiana tobacum	Block 1			Gene of Arabidopsis thaliana	Block 2
Number	Gene of Nicotiana tobacum	Chr	Start	End	Gene of Arabidopsis thaliana	Chr	Start	End
1	Nitab4.5_0001336g0060.1	10	67145787	75458550	AT3G15300	3	5025184	5196591
2	Nitab4.5_0001336g0060.1	10	67187464	71464670	AT5G53830	5	21822323	21914866
3	Nitab4.5_0002906g0010.1	4	8900255	13115923	AT3G18360	3	6154363	6399337
4	Nitab4.5_0003072g0110.1	4	15178772	15579843	AT1G80450	1	30243430	30280359
5	Nitab4.5_0001924g0010.1	10	74607412	80668737	AT1G78410	1	29540984	29468394

3.2 Phylogenetic analysis of VQ gene families

According to the phylogenetic tree (Fig. 2), ninety-five VQ proteins derived from N. tobacum and A. thaliana were clustered into seven groups named from I to VII. Group I - VI contained VQ proteins from both tobacco and Arabidopsis, and group VII contained only tobacco VQ proteins. Group III (29.50%) contained the largest number of genes, followed by group I (16.39%), group IV (14.75%), group II (13.11%), group VII (13.11%), group V (6.56%) and group VI (6.56%). Moreover, a subset of the Arabidopsis VQ proteins phosphorylated by the MPK3a and MPK6 (named as MVQs) were uniformly clustered into the group I and II (Pecher et al. 2014), which predicated that eighteen tobacco VQ proteins from group I and II may be involved in the cellular process of protein phosphorylation. This comparison between VQ proteins clarified that genes in the same group may have similar functions.

3.3 Structure and phylogenetic analysis of NtVQ gene family

A phylogenetic tree was constructed using sixty-one NtVQ protein sequences (Fig. 3a). The NtVQ genes could be divided into thirty-four categories (NtVQ1 to NtVQ34) marked with different colours. The analysis of protein domain organization showed that all NtVQ proteins delineated the VQ-motif using SMART database and NCBI, and protein structures were highly similar (Fig. 3b). Protein structure of NtVQ3 (Nitab4.5_0000443g0140.1) contained a coiled-coil region. Protein structures of NtVQ16 (Nitab4.5_0008558g0010.1 and Nitab4.5_0000786g0140.1) had two transmembrane helix regions, respectively. The analysis of gene structure showed that NtVQ genes in the same branch shared a similar exon-intron distribution, except NtVQ5, NtVQ8, NtVQ16 and NtVQ33 (Fig. 3c). Fourteen NtVQ genes contained introns in the genomic sequences, the number of introns varied significantly from one to eight, indicating that NtVQ genes usually varied in the exon-intron distribution profile and gene length of the tobacco genomic sequences.

3.4 Tissue expression pattern of NtVQ genes

To determine the potential functions of NtVQ genes in tobacco development, the expression profiles of thirty-four NtVQ genes were conducted in four tissues and three types of trichomes. Lots of trichomes were shown on the surface of stem and leaf, and tobacco root and flower were also covered with epidermis (Fig. 4a). As shown in Fig. 4b, the result of tissue expression profiles showed that NtVQ11 and NtVQ30 were barely expressed in different tissues including trichomes, NtVQ16 were barely expressed in tissues, and NtVQ19 were barely expressed in different type of trichomes. Seven genes (NtVQ3, NtVQ8, NtVQ17, NtVQ25, NtVQ27, NtVQ29 and NtVQ32) were highly expressed in root, and NtVQ28 was expressed only in root. Five genes (NtVQ9, NtVQ13, NtVQ18, NtVQ21 and NtVQ26) were highly expressed in stem. Eight genes (NtVQ2, NtVQ5, NtVQ10, NtVQ12, NtVQ20, NtVQ22, NtVQ24 and NtVQ33) were highly expressed in leaf. NtVQ15 was highly expressed in flower, NtVQ1 and NtVQ8 were barely expressed in flower. These results forecasted that NtVQ genes played essential roles in tobacco tissues growth and development. Besides, eleven genes (NtVQ8, NtVQ9, NtVQ12, NtVQ13, NtVQ16, NtVQ22, NtVQ24, NtVQ25, NtVQ26, NtVQ31 and NtVQ34) were highly expressed in mT. Four genes (NtVQ1, NtVQ2, NtVQ17 and NtVQ20) were highly expressed in gT. Five genes (NtVQ3, NtVQ4, NtVQ5, NtVQ28 and NtVQ33) were highly expressed in nT. The expression profiles in different type of trichomes firstly clarified the important functions of NtVQ genes in glandular- and nonglandular-trichome formation and development.

3.5 Expression patterns of NtVQ genes following phytohormone treatments and abiotic stresses

To elucidate the roles of NtVQ genes under different phytohormone, sqRT-PCT was conducted to achieve the relative expression values of each NtVQ gene (Fig. 5). The expression levels of thirteen genes (NtVQ3, NtVQ4, NtVQ6, NtVQ13, NtVQ15, NtVQ16, NtVQ20, NtVQ22, NtVQ28, NtVQ29, NtVQ31, NtVQ33 and NtVQ34) increased following MeJA treatment. The expression levels of twenty-two genes (NtVQ3, NtVQ4, NtVQ5, NtVQ7, NtVQ9, NtVQ10, NtVQ12, NtVQ14, NtVQ15, NtVQ16, NtVQ17, NtVQ18, NtVQ19, NtVQ21, NtVQ23, NtVQ24, NtVQ25, NtVQ29, NtVQ31, NtVQ32, NtVQ33 and NtVQ34) increased following SA treatment. The expression levels of eleven genes (NtVQ2, NtVQ7, NtVQ10, NtVQ16, NtVQ20, NtVQ23, NtVQ26, NtVQ27, NtVQ29, NtVQ33 and NtVQ34) increased following GA treatment. The expression levels of eight genes (NtVQ1, NtVQ4, NtVQ6, NtVQ7, NtVQ9, NtVQ14, NtVQ15 and NtVQ29) increased following ETH treatment. Among these genes, the expression levels of NtVQ29 was simultaneously up-regulated following the four phytohormones treatments. These results uncovered that all NtVQ genes were involved in the intricate signaling pathways and each gene had different regulatory characteristics.

A systematic expression analysis of NtVQ genes was conducted following the abiotic stresses. The expression levels of eight genes (NtVQ5, NtVQ18, NtVQ20, NtVQ21, NtVQ24, NtVQ26, NtVQ28 and NtVQ34) increased following high salinity stress. The expression levels of six genes (NtVQ2, NtVQ7, NtVQ12, NtVQ16, NtVQ20 and NtVQ24) increased following PEG stress. NtVQ20 and NtVQ24 were simultaneously up-regulated following these two stresses. While the expression levels of fifteen genes (NtVQ1, NtVQ3, NtVQ4, NtVQ6, NtVQ8, NtVQ13, NtVQ15, NtVQ17, NtVQ19, NtVQ22, NtVQ23, NtVQ25, NtVQ29, NtVQ32 and NtVQ33) were simultaneously down-regulated. These results predicated that most NtVQ genes showed the negative regulatory responses to abiotic stress.

3.6 Transcriptional activity analysis of NtVQ genes

Several NtVQ genes have been chosen to investigate the potential transcriptional activity (Fig. 6). Compared with the control, pGBKT7-NtVQ4, pGBKT7-NtVQ28 and pGBKT7-NtVQ29 could only grow on SD/-Trp medium, while pGBKT7-NtVQ17 grew normally on both selective media and showed alpha-galactosidase activity, which speculated that most NtVQ genes might have no transcription activity.

VQ gene has been extensively identified in various plants including angiosperm, gymnosperm and moss. Some VQ gene were known to be related to the plant development and stress tolerance (Jiang et al. 2018; León et al. 2021). According to the tobacco genome data, sixty-one NtVQ genes containing VQ-motif were identified and their protein lengths ranged from 71 to 467 amino acids in the current study (Table 1), which was similar with these in Arabidopsis but smaller than these in moss (Li et al. 2014; Jing and Lin, 2015). Then NtVQ gene structures, gene expression patterns, protein characteristics and their primary functions have already been confirmed.

4.1 Gene duplication and phylogenetic analysis of NtVQ and AtVQ genes

Gene duplication event played a major role in plant genome rearrangement and expansion (Vision et al. 2000), and segregation duplication events were shown to provide references for the evolutionary relationship between VQ genes (Fig. 1), thereby enabling functional predictions (Panchy et al. 2016). For example, AtVQ11 (MVQ5, AT1G80450), AtVQ19 (MVQ4, AT3G15300) and AtVQ33 (MVQ3, AT5G53830) interacted with specific subgroups of WRKY transcription factors and their proteins stability were mediated by phosphorylation (Pecher et al. 2014). The phylogenetic analysis revealed that the VQ genes from tobacco and Arabidopsis were classified into seven groups and the orthologous genes were clad in the same group (Fig. 2). Pecher (2014) has also proved that AtVQ4, AtVQ6, AtVQ9, AtVQ13, AtVQ14, AtVQ31 and AtVQ32 were all targeted by MPK3 and MPK6, which speculated that tobacco NtVQ proteins from the group I and II might be involved in the phosphorylation process. These results indicated that the related VQ proteins in each group could be equipped with the similar functions.

4.2 Structural and conserved domain analysis of NtVQ genes

Cultivated tobacco N. tobacum was an allotetraploid plant, thereby one gene might have two homologous sequences from two ancestors of N. sylvestris and N. tomentosiformis (Renny-Byfield et al. 2011). In this study, thirty-four NtVQ genes were grouped referring to the phylogenetic tree (Fig. 3a). Structure analysis showed that NtVQ proteins in the same group contained the similar type of motifs (Fig. 3b), indicating that close proteins shared similar functions. NtVQ3 contained a coiled-coil region which was found in more than two hundred proteins and used to predicate regions of protein discontinuity and folding stability (Lupas et al. 1991). NtVQ16 had two transmembrane helix regions and marked as the integral membrane protein (Krogh et al. 2001). Interestingly, most VQ genes in higher plants did not have any intron (Jing and Lin, 2015), for instance, 90% AtVQ genes (Cheng et al. 2012) and 76% PoVQ genes (Chu et al. 2016) had no introns. Consistently, introns of twenty NtVQ genes were lost (Fig. 3c), which meant that NtVQ genes might have experienced different selective pressures during evolution.

4.3 Analysis of NtVQ gene expression patterns in different tissues and trichomes

Tissue transcription patterns could exhibit genes involvement in functional or differential events. Thirty-four AtVQ genes were induced and differentially expressed in different tissues (Cheng et al. 2012). Herein, NtVQ11 and NtVQ30 were barely expressed in different tissues including trichomes. Consistently, the homologous gene AtVQ20 expressed strongly in the male gametophytic tissues, but barely in seedling, leaf, stem and root (Lei et al. 2017). AtVQ12 was mainly expressed in the root and leaf (Wang et al. 2015). AtVQ29 was mainly expressed in the root, leaf, hypocotyl and silique base (Jing and Lin 2015). Moreover, GmVQ58 was highest expressed in the cotton leaf and root (Li et al. 2020). Almost all CsVQ genes were more highly expressed in root, stem and leaf of tea plant, while only a few genes were more highly expressed in the flower (Guo et al. 2018). Some uncharacterized VQ proteins were also found in response to meditate plant growth. In our results, the tissues expression profiles showed that almost all NtVQ genes were responsive to organogenesis, thus pointing to the important regulatory roles of NtVQ genes in tobacco development.

Trichomes were one of the most important accommodative traits in plants (Ishida et al. 2008). Trichomes were associated with several important features that were involved in phytohormone responses, resistance to biotic and abiotic stress (Schuurink and Tissier, 2020; Yan et al. 2021). gT and nT not only formed the physical barriers against UV radiation, waster loss and excess light (Schuurink and Tissier 2020), but also were crucial in the process of chemical secretion such as phenylpropanoids, alkaloids, sugars and some other metabolic compounds against insects and arthropods (Yang and Ye, 2013; Maurya et al. 2019). However, the expression patterns and molecular regulatory mechanisms of NtVQ genes in trichomes had never been published. Here, eleven NtVQ genes highly expressed in mT from K326, four NtVQ genes highly expressed in gT from T.I.1068, and five NtVQ genes highly expressed in nT from T.I.1112, which made it clear that NtVQ genes had unique potentials and momentous values to participate in trichomes development responsive to the multiple external stimulus.

4.4 Gene expression patterns under environmental stresses

Plants had generated kinds of effective defense systems against biotic and abiotic stresses. JA and SA were two of the best-known signaling molecules that regulated plant development and triggered defense responses (Dubrovsky 2005). Jing and Lin (2015) pointed that VQ genes were involved in JA- and SA-meditated defense responses.

In this study, NtVQ4 was significantly and consistently up-regulated upon JA treatment (Fig. 5), and its highly homologous gene AtVQ22 (JAV1) transcript significantly increased and negatively regulated the transcriptional activity of WRKY28/51 involved in the JA-meditated defense response (Andreasson et al. 2005; Hu et al. 2013a; Yan et al. 2018). Moreover, OsVQ13 positively regulated JA-meditated grain size by activating the OsMPK6-OsWRKY45 signaling pathway in rice (Uji et al. 2019). The expression levels of MaVQ5 was up-regulated after MeJA treatment (Ye et al. 2016). Moreover, the roles of JA in promoting leaf senescence and epidermogenesis were affirmed in many plant species (Zhang et al. 2019; Wang et al. 2022). AtVQ18, AtVQ26 and ZmVQ52 could effectively regulate JA-mediated leaf senescence (Pan et al. 2018; Yu et al. 2019). Herein, a detailed expression analysis revealed that nineteen NtVQ genes were obviously involved in the formation and development of gT and nT (Fig. 4), and thirteen NtVQ genes were up-regulated after MeJA treatment (Fig. 5). These results proved that NtVQ genes emitted important response functions to JA signaling, which was similar with the result of around 32% AtVQ genes up-regulated in senescing leaves (Schmid et al. 2005).

NtVQ34 were highly expressed after SA treatment (Fig. 5), and its homologous gene AtVQ21 played a positive role in SA signaling (Andreasson et al. 2005). While NtVQ22 and NtVQ23 were not sensitive to SA, which was opposite to the functions of their highly homologous genes AtVQ16 (SIB2) and AtVQ23 (SIB1) strongly induced by SA treatment (Xie et al. 2010; Lai et al. 2011). These results verified that NtVQ genes interlaced to form a variety of complex regulatory mechanisms in tobacco. In addition, most NtVQ genes were up-regulated after SA treatment and conjectured to play the important roles in SA-meditated plant growth, development and resistance responses, which was consistent with the results that thirty-four AtVQ genes were responsive to SA treatment (Cheng et al. 2012), and sixteen VvVQ genes were induced by SA treatment (Wang et al. 2015).

Recent studies in Arabidopsis and other crop species highlighted the emerging key roles for GA and ETH in the regulation of nearly all aspects of plant organs growth and yield under abiotic stress (Yamaguchi 2008; Dubois et al. 2018). In this study, part of NtVQ genes were positively induced after these two phytohormone treatments. The VQ genes from other plant also showed the similar function, for example, PbrVQ9 was the top highly expressed gene after GA treatment (Cao et al. 2018), VvVQ2 was highly up-regulated in grapevine after ETH treatment (Wang et al. 2015).

Salt and drought were the main factors of reducing crop production. The expression levels of NtVQ16 and NtVQ17 decreased after NaCl treatment (Fig. 5), while their homologous gene AtVQ9 transcript increased (Perruc et al. 2004; Hu et al. 2013b). Some studies have indicated that some up-regulated VQ genes might have a negative effect on abiotic stress resistance. AtVQ9 expression was induced by salinity stress, but AtVQ9 overexpression increased plants hypersensitive to salinity stress (Hu et al. 2013b), which conjectured that the functional roles of NtVQ genes to abiotic stresses were characterized by inconsistency. Moreover, AtVQ15 (AtCaMBP25) was induced by various abiotic stresses, including dehydration and high salinity, and AtVQ15 overexpression plants displayed increasing sensitivity to both NaCl and mannitol (Perruc et al. 2004; Hu et al. 2013b). AtVQ24, a closed protein of AtVQ15, was clarified to have the negative role in osmotic stress (Hu et al. 2013b). And their homologous tobacco genes NtVQ9 and NtVQ10 showed the similar negative regulation. Besides, GhVQ18 and GhVQ84 were highly expressed under NaCl and PEG treatments (Chen et al. 2020b). Bamboo PeVQ28 overexpression in Arabidopsis showed increased resistance to salinity stress (Cheng et al. 2020). Here, eight and six NtVQ genes were up-regulated after NaCl and PEG treatments, respectively. While the expression profiles of most NtVQ genes decreased. Previous studies showed the consistent results that ten BrVQ genes (Zhang et al. 2015), eight PtVQ genes (Chu et al. 2016), eighteen VvVQ genes (Wang et al. 2015), and twenty-two OsVQ genes (Kim et al. 2013) were up-regulated after drought stress. And most VQ genes from Poplour (Chu et al. 2016), maize (Song et al. 2016), and tea (Guo et al. 2018) were also responsive to drought or PEG and NaCl stress.

4.5 Most NtVQ genes lacked the transcriptional activity

Studies have proved that AtVQ1 and AtVQ10 did not have transcriptional activity (Jing and Lin 2015). Consistently, the homologous gene NtVQ28 and NtVQ29 were shown no self-activating activity (Fig. 6), and NtVQ4 also showed no self-activating activity in the Y2H assay. Acted as the homologous gene of AtVQ14 (IKU1/MVQ9), NtVQ17 displayed the transcriptional activity, which was same with the result of the most prominent interactions between MPK3, MPK6 and MVQ proteins from group I and II (Pecher et al. 2014). All these data predicted that most NtVQ genes acted as transcription regulators might have no transcriptional activity, and functions of NtVQ genes, especially NtVQ17, needed further verification and evaluation.

The transcriptions of VQ genes were modulated by multiple endogenous and environmental signals, consistent with their diverse roles in stress responses, plant growth and development. Study about NtVQ gene family provided a glimpse into the potential biological functions in tissues development, trichomes formation, and resistance to abiotic stress, indicating that members of NtVQ family played important roles in plant growth and responses to environmental conditions.

Acknowledgments

This work was supported by the National Natural Science Foundation of China [32102361], the Science and Technology Department of Henan Province, China [212102110046], the State Tobacco Monopoly Administration of China [110202101005 (JY-05)], the Science and Technology Project of China National Tobacco Corporation Hunan Tobacco Company [202143000834089], and the Innovation and Entrepreneurship Training Program for College Students of Henan Province [202110466042].

Author Contribution

Xiaoxiao Yan (XY) and Hong Cui (HC) designed the study and wrote the paper. Xiaoxiao Yan (XY), Rui Luo (RL) and Xiangyang Liu (XL) performed the experiments. Rui Luo (RL), Xiangyang Liu (XL), Wenyi Pei (WP) and Wenqi Zhu (WZ) contributed to data analysis.

Data Availability Statement

All data analyzed during this study are included in this published article and its supplementary information files.

Declaration of Competing Interest

All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Andreasson E, Jenkins T, Brodersen P, Thorgrimsen S, Petersen NH, Zhu S, Qiu JL, Micheelsen P, Rocher A, Petersen M, Newman MA, Bjorn Nielsen H, Hirt H, Somssich I, Mattsson O, Mundy J (2005) The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J 24:2579–2589. doi: 10.1038/sj.emboj.7600737
Cao Y, Meng D, Abdullah M, Jin Q, Lin Y, Cai Y (2018) Genome wide identification, evolutionary, and expression analysis of VQ genes from two Pyrus Species. Genes (Basel) 9:224. doi: 10.3390/genes9040224
Chalvin C, Drevensek S, Dron M, Bendahmane A, Boualem A (2020) Genetic control of glandular trichome development. Trends Plant Sci 25(5):477–487. doi: 10.1016/j.tplants.2019.12.025
Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R (2020a) TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant 13(8):1194–1202. doi: 10.1016/j.molp.2020.06.009
Chen P, Wei F, Cheng S, Ma L, Wang H, Zhang M, Mao G, Lu J, Hao P, Ahmad A, Gu L, Ma Q, Wu A, Wei H, Yu S (2020b) A comprehensive analysis of cotton VQ gene superfamily reveals their potential and extensive roles in regulating cotton abiotic stress. BMC Genomics 21:795. doi: 10.1186/s12864-020-07171-z
Cheng X, Wang Y, Xiong R, Gao Y, Yan H, Xiang Y (2020) A Moso bamboo gene VQ28 confers salt tolerance to transgenic Arabidopsis plants. Planta 251:99. doi: 10.1007/s00425-020-03391-5
Cheng Y, Zhou Y, Yang Y, Chi YJ, Zhou J, Chen JY, Wang F, Fan B, Shi K, Zhou YH, Yu JQ, Chen Z (2012) Structural and functional analysis of VQ motif-containing proteins in Arabidopsis as interacting proteins of WRKY transcription factors. Plant Physiol 159:810–825. doi: 10.1104/pp.112.196816
Chu W, Liu B, Wang Y, Pan Y, Chen Z, Yan H, Xiang Y (2016) Genome-wide analysis of poplar VQ gene family and expression profiling under PEG, NaCl, and SA treatments. Tree Genet Genomes 12:124–140. doi: 10.1007/s11295-016-1082-z
Ding H, Yuan G, Mo S, Qian Y, Wu Y, Chen Q, Xu X, Wu X, Ge C (2019) Genome-wide analysis of the plant-specific VQ motif-containing proteins in tomato (Solanum lycopersicum) and characterization of SlVQ6 in thermotolerance. Plant Physiol Biochem 143:29–39. doi: 10.1016/j.plaphy.2019.08.019
Dubois M, Van den Broeck L, Inzé D (2018) The pivotal role of ethylene in plant growth. Trends Plant Sci 23:311–323. doi: 10.1016/j.tplants.2018.01.003
Dubrovsky EB (2005) Hormonal cross talk in insect development. Trends Endocrinol Metab 16:6–11. doi: 10.1016/j.tem.2004.11.003
Guo J, Chen J, Yang J, Yu Y, Yang Y, Wang W (2018) Identification, characterization and expression analysis of the VQ motif-containing gene family in tea plant (Camellia sinensis). BMC Genomics 19:710. doi: 10.1186/s12864-018-5107-x
Hu P, Zhou W, Cheng Z, Fan M, Wang L, Xie D (2013a) JAV1 controls jasmonate-regulated plant defense. Mol Cell 50:504–515. doi: 10.1016/j.molcel.2013.04.027
Hu Y, Chen L, Wang H, Zhang L, Wang F, Yu D (2013b) Arabidopsis transcription factor WRKY8 functions antagonistically with its interacting partner VQ9 to modulate salinity stress tolerance. Plant J 74:730–745. doi: 10.1111/tpj.12159
Ishida T, Kurata T, Okada K, Wada T (2008) A genetic regulatory network in the development of trichomes and root hairs. Annu Rev Plant Biol 59:365–386. doi: 10.1146/annurev.arplant.59.032607.092949
Jiang SY, Sevugan M, Ramachandran S (2018) Valine-glutamine (VQ) motif coding genes are ancient and non-plant-specific with comprehensive expression regulation by various biotic and abiotic stresses. BMC Genomics 19:342. doi: 10.1186/s12864-018-4733-7
Jing Y, Lin R (2015) The VQ motif-containing protein family of plant-specific transcriptional regulators. Plant Physiol 169:371–378. doi: 10.1104/pp.15.00788
Kim DY, Kwon SI, Choi C, Lee H, Ahn I, Park SR, Bae SC, Lee SC, Hwang DJ (2013) Expression analysis of rice VQ genes in response to biotic and abiotic stresses. Gene 529:208–214. doi:10.1016/j.gene.2013.08.023
Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580. doi: 10.1006/jmbi.2000.4315
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. doi: 10.1093/molbev/msw054
Lai Z, Li Y, Wang F, Cheng Y, Fan B, Yu JQ, Chen Z (2011) Arabidopsis sigma factor binding proteins are activators of the WRKY33 transcription factor in plant defense. Plant Cell 23:3824–3841. doi: 10.1105/tpc.111.090571
Lei R, Li X, Ma Z, Lv Y, Hu Y, Yu D (2017) Arabidopsis WRKY2 and WRKY34 transcription factors interact with VQ20 protein to modulate pollen development and function. Plant J 91:962–976. doi: 10.1111/tpj.13619
León J, Gayubas B, Castillo MC (2021) Valine-glutamine proteins in plant responses to oxygen and nitric oxide. Front Plant Sci 11:632678. doi: 10.3389/fpls.2020.632678
Li N, Yang Z, Li J, Xie W, Qin X, Kang Y, Zhang Q, Li X, Xiao J, Ma H, Wang S (2021) Two VQ proteins are substrates of the OsMPKK6-OsMPK4 cascade in rice defense against bacterial blight. Rice 14:39. doi: 10.1186/s12284-021-00483-y
Li X, Qin R, Du Q, Cai L, Hu D, Du H, Yang H, Wang J, Huang F, Wang H, Yu D (2020) Knockdown of GmVQ58 encoding a VQ motif-containing protein enhances soybean resistance to the common cutworm (Spodoptera litura Fabricius). J Exp Bot 71:3198–3210. doi: 10.1093/jxb/eraa095
Li Y, Jing Y, Li J, Xu G, Lin R (2014) Arabidopsis VQ MOTIF-CONTAINING PROTEIN29 represses seedling deetiolation by interacting with PHYTOCHROME-INTERACTING FACTOR1. Plant Physiol 164:2068–2080. doi: 10.1104/pp.113.234492
Li YH, Zhang HY, WEI YW, Cui H (2017) Comparison analysis of trichome morphology and secretion of typical flue- cured tobacco varieties. Acta Tabacaria Sinica 23(2):84–91. doi: 10.16472/j.chinatobacco.2016.315
Lupas A, Van Dyke M, Stock J (1991) Predicting coiled coils from protein sequences. Science 252:1162–1164. doi: 10.1126/science.252.5009.1162
Maurya S, Chandra M, Yadav RK, Narnoliya LK, Sangwan RS, Bansal S, Sandhu P, Singh U, Kumar D, Sangwan NS (2019) Interspecies comparative features of trichomes in Ocimum reveal insights for biosynthesis of specialized essential oil metabolites. Protoplasma 256:893–907. doi: 10.1007/s00709-018-01338-y
Pan J, Wang H, Hu Y, Yu D (2018) Arabidopsis VQ18 and VQ26 proteins interact with ABI5 transcription factor to negatively modulate ABA response during seed germination. Plant J 95:529–544. doi: 10.1111/tpj.13969
Panchy N, Lehti-Shiu M, Shiu SH (2016) Evolution of gene duplication in plants. Plant Physiol 171:2294–2316. doi: 10.1104/pp.16.00523
Pecher P, Eschen-Lippold L, Herklotz S, Kuhle K, Naumann K, Bethke G, Uhrig J, Weyhe M, Scheel D, Lee J (2014) The Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6 target a subclass of 'VQ-motif'-containing proteins to regulate immune responses. New Phytol 203:592–606. doi: 10.1111/nph.12817
Perruc E, Charpenteau M, Ramirez BC, Jauneau A, Galaud JP, Ranjeva R, Ranty B (2004) A novel calmodulin-binding protein functions as a negative regulator of osmotic stress tolerance in Arabidopsis thaliana seedlings. Plant J 38:410–420. doi: 10.1111/j.1365-313X.2004.02062.x
Renny-Byfield S, Chester M, Kovařík A, Le Comber SC, Grandbastien MA, Deloger M, Nichols RA, Macas J, Novák P, Chase MW, Leitch AR (2011) Next generation sequencing reveals genome downsizing in allotetraploid Nicotiana tabacum, predominantly through the elimination of paternally derived repetitive DNAs. Mol Biol Evol 28:2843–2854. doi: 10.1093/molbev/msr112
Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, Scholkopf B, Weigel D, Lohmann JU (2005) A gene expression map of Arabidopsis thaliana development. Nat Genet 37:501–506. doi: 10.1038/ng1543
Schuurink R, Tissier A (2020) Glandular trichomes: micro-organs with model status? New Phytol 225:2251–2266. doi: 10.1111/nph.16283
Song W, Zhao H, Zhang X, Lei L, Lai J (2016) Genome-wide identification of VQ motif-containing proteins and their expression profiles under abiotic stresses in maize. Front Plant Sci 6:1177. doi: 10.3389/fpls.2015.01177
Tang H, Wang X, Bowers JE, Ming R, Alam M, Paterson AH (2008) Unraveling ancient hexaploidy through multiply-aligned angiosperm gene maps. Genome Res 18:1944–1954. doi: 10.1101/gr.080978.108
Uji Y, Kashihara K, Kiyama H, Mochizuki S, Akimitsu K, Gomi K (2019) Jasmonic acid-induced VQ-motif-containing protein OsVQ13 influences the OsWRKY45 signaling pathway and grain size by associating with OsMPK6 in rice. Int J Mol Sci 20:2917. doi: 10.3390/ijms20122917
Vision TJ, Brown DG, Tanksley SD (2000) The origins of genomic duplications in Arabidopsis. Science 290:2114–2117. doi:10.1126/science.290.5499.2114
Wang A, Garcia D, Zhang H, Feng K, Chaudhury A, Berger F, Peacock WJ, Dennis ES, Luo M (2010) The VQ motif protein IKU1 regulates endosperm growth and seed size in Arabidopsis. Plant J 63:670–679. doi: 10.1111/j.1365-313X.2010.04271.x
Wang C, Ding Y, Wang W, Zhao X, Liu Y, Timko MP, Zhang Z, Zhang H (2022) Insights into gene regulation of jasmonate-induced whole-plant senescence of tobacco under non-starvation conditions. Plant Cell Physiol 63:45–56. doi: 10.1093/pcp/pcab140
Wang M, Vannozzi A, Wang G, Zhong Y, Corso M, Cavallini E, Cheng ZM (2015) A comprehensive survey of the grapevine VQ gene family and its transcriptional correlation with WRKY proteins. Front Plant Sci 6:417. doi: 10.3389/fpls.2015.00417
Xie YD, Li W, Guo D, Dong J, Zhang Q, Fu Y, Ren D, Peng M, Xia Y (2010) The Arabidopsis gene SIGMA FACTOR-BINDING PROTEIN 1 plays a role in the salicylate- and jasmonate-mediated defence responses. Plant Cell Environ 33:828–839. doi: 10.1111/j.1365-3040.2009.02109.x
Yamaguchi S (2008) Gibberellin metabolism and its regulation. Annu Rev Plant Biol 59:225–251. doi: 10.1146/annurev.arplant.59.032607.092804
Yan C, Fan M, Yang M, Zhao J, Zhang W, Su Y, Xiao L, Deng H, Xie D (2018) Injury activates Ca²⁺/calmodulin-dependent phosphorylation of JAV1-JAZ8-WRKY51 complex for jasmonate biosynthesis. Mol Cell 70:136–149.
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.. doi: 10.1016/j.molcel.2018.03.013
Yan X, Guan Y, Liu X, Yu J, Lei B, Wang Z, Zhang H, Cui H (2021) NtCycB2 gene knockout enhances resistance to high salinity stress in Nicotiana tabacum. Ind Crop Prod 171:113886. doi: 10.1016/j.indcrop.2021.113886
Yan X, Qiao H, Zhang X, Guo C, Wang M, Wang Y, Wang X (2017) Analysis of the grape (Vitis vinifera L.) thaumatin-like protein (TLP) gene family and demonstration that TLP29 contributes to disease resistance. Sci Rep 7:4269. doi: 10.1038/s41598-017-04105-w
Yang C, Ye Z (2013) Trichomes as models for studying plant cell differentiation. Cell Mol Life Sci 70:1937–1948. doi: 10.1007/s00018-012-1147-6
Ye YJ, Xiao YY, Han YC, Shan W, Fan ZQ, Xu QG, Kuang JF, Lu WJ, Lakshmanan P, Chen JY (2016) Banana fruit VQ motif-containing protein5 represses cold-responsive transcription factor MaWRKY26 involved in the regulation of JA biosynthetic genes. Sci Rep 6:23632. doi: 10.1038/srep23632
Yu T, Lu X, Bai Y, Mei X, Guo Z, Liu C, Cai Y (2019) Overexpression of the maize transcription factor ZmVQ52 accelerates leaf senescence in Arabidopsis. PLoS One 14:e0221949. doi: 10.1371/journal.pone.0221949
Zhang G, Wei B (2019) Characterization of VQ motif-containing protein family and their expression patterns under phytohormones and abiotic stresses in melon (Cucumis melo L.). Plant Growth Regul 89:273–285. doi: 10.1007/s10725-019-00534-x
Zhang H, Lu X, Wang Z, Yan X, Cui H (2021) Excretion from long glandular trichomes contributes to alleviation of cadmium toxicity in Nicotiana tabacum. Environ Pollut 285:117184. doi: 10.1016/j.envpol.2021.117184
Zhou Y, Yang Y, Zhou X, Chi Y, Fan B, Chen Z (2016) Structural and functional characterization of the VQ protein family and VQ protein variants from soybean. Sci Rep 6:34663. doi: 10.1038/srep34663
Zou Z, Liu F, Huang S, Fernando WGD (2021) Genome-wide identification and analysis of the valine-glutamine motif-containing gene family in Brassica napus and functional characterization of BnMKS1 in response to Leptosphaeria maculans. Phytopathology 111:281–292. doi: 10.1094/PHYTO-04-20-0134-R

TableS1.xlsx
Table S1 All primers used in this study.

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published 09 Jul, 2023

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Editorial decision: Minor revision
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Reviewers invited by journal
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03 Nov, 2022
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Characterization and the comprehensive expression analysis of tobacco valine-glutamine genes in response to trichomes development and stress tolerance

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Abstract

1. Introduction

2. Materials And Methods

2.1 Plant material and growth conditions

2.2 Identification of tobacco VQ genes

2.3 Bioinformatics analysis of tobacco VQ genes

2.4 Acquisition of tobacco tissues and trichomes phenotyping

2.5 Phytohormone treatments and abiotic stress

2.6 Transcriptional activity assay

2.7 RNA extraction and sqRT-PCR analysis

2.8 Data statistics

3. Results

3.1 Identification and synteny analysis of VQ genes families

3.2 Phylogenetic analysis of VQ gene families

3.3 Structure and phylogenetic analysis of NtVQ gene family

3.4 Tissue expression pattern of NtVQ genes

3.5 Expression patterns of NtVQ genes following phytohormone treatments and abiotic stresses

3.6 Transcriptional activity analysis of NtVQ genes

4. Discussion

4.1 Gene duplication and phylogenetic analysis of NtVQ and AtVQ genes

4.2 Structural and conserved domain analysis of NtVQ genes

4.3 Analysis of NtVQ gene expression patterns in different tissues and trichomes

4.4 Gene expression patterns under environmental stresses

4.5 Most NtVQ genes lacked the transcriptional activity

5. Conclusions

Declarations

References

Supplementary Files

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Journal Publication

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