The RabGAP Gene Family in Tomato (Solanum lycopersicum) and Wild Relatives: Identification, Interaction Networks, and Transcriptional Analysis during Plant Development and in Response to Salt Stress.

RabGTPase activating proteins (RabGAP) are responsible for directing the deactivation of vesicular trafficking master regulators associated to plant development, the RabGTPase proteins. Recently, RabGAPs were identified in Arabidopsis and rice, but studies were not yet reported in tomato. Herein, we identified 24 RabGAP-encoding genes in cultivated tomato (Solanum lycopersicum) and its wild relative genomes (Solanum pimpinellifolium and Solanum pennellii). We analyzed them based on their exon-intron structures, conserved protein motifs, putative subcellular localizations, phylogenetic and gene duplications analyses, interaction networks, and gene expression patterns in tomato. Phylogenetic relationship analysis also indicated that RabGAP family is classified into seven subclasses, of which subclasses I and II are plant-exclusive. Furthermore, segmental duplication events and positive evolutionary forces are associated with the maintenance of the number and function of their members. On the other hand, the protein-protein interaction networks on tomato suggested that members of subclasses I, II, and III could be associated to endocytic traffic routes. In addition, the qRT-PCR experiments in S. lycopersicum and Solanum chilense exposed to a salt stress treatment validated the differential expression patterns of 20 RabGAP genes in different tissues, development stages, and stress conditions obtained through extensive microarray-based analyses. This work suggests the critical role of RabGAP family in the context of intracellular vesicular trafficking in tomato, particularly under conditions of abiotic stress. It also contributes to the breeding programs associated with the development of crops tolerant to salt stress.

1. Introduction

Abiotic stress represents the main environmental challenge in the production of agronomically important crops. Salt, drought, or heat, among other stresses, alter the internal homeostasis of plants, damaging their tissues and organs, as well as reducing their yield [1]. To counteract these negative effects, mechanisms for synthesis and accumulation of compatible osmolytes [2], detoxification of reactive oxygen species [3], mobilization of ion and water transporters [4,5], or synthesis and mobilization of lipids are activated [6]. The performance of these mechanisms depends on the efficient vesicular traffic between the different subcellular compartments [7,8,9]. The small GTPases of the Rab family (RabGTPases) regulate the vesicular traffic, alternating between an “active” state GTP-bound and an “inactive” state GDP-bound, as a molecular switch. The activated state is dependent on guanine nucleotide exchange factors (GEF) proteins, which facilitate the GDP dissociation by GTP [10]. Then, RabGTPase proteins are recognized by effector proteins such as homotypic fusion and protein sorting (HOPS), transport protein particle (TRAPP), and class C core vacuole/endosome tethering (CORVET) complexes, among others, helping membrane fusions [11]. The “inactive” state of RabGTPase is promoted by association with RabGAP proteins. The RabGAP proteins hydrolyze GTP, allowing RabGTPases to be bio-available for a new cycle of vesicular trafficking. In plants, each RabGTPase directs a particular traffic route, but the knowledge of the routes in which many RabGAP proteins participate is still very limited [12].

Unlike yeast and animals, little is known about the RabGAP proteins in plants, the mechanisms that they modulate, their transcriptional regulation, or their role in the stress tolerance. From a genomic analysis in yeast, all RabGAPs should have a conserved TBC-like domain (Tre-2/Bub2/Cdc16) with a hydrolytic activity directed by an arginine and glutamine that act as a dual-finger mechanism on GTP-bound RabGTPases [13]. Other domains associated with RabGAP proteins are the Rab3-GTPase_cat and Rab3GAP2_N domains, which are specific for RabGTPase proteins of subfamily D (RabD) [14]. In plants, 25 and 24 RabGAP genes have been identified in rice and Arabidopsis thaliana, respectively [15], whereas in Vitis vinifera, only five RabGAPs have been found by EST analysis [16]. In general, the RabGAP family has been subdivided into nine subclasses (I to IX), of which the subclasses III, IV, V, VI, and VIII are present in plants, yeast, insects, and animals, whereas the subclasses I and II are plant-specific [15].

During environmental stress the vesicular trafficking mechanisms are up-regulated. For example, RabGTPase genes are regulated in a positive way, increasing the endocytosis, the vesicular traffic, and the vacuolar compartmentation of ions [9]. Among them, Rab5 of Mesembryanthemum crystallinum, a salt tolerant species, or OsRab7 of rice, increase their expression during in cold, dehydration or salt stress [17,18]. Other studies have demonstrated that the over-expression of RabGTPase genes associated to endocytosis mechanisms, like RabG3e and RabF1 in A. thaliana or PgRAB7 in tobacco, also increase the vacuolar compartmentalization of Na+ ions and the transport of proteins between organelles [9,19,20]. Likewise, the participation of proteins associated to molecular switching during abiotic stress has been evaluated. Among them, the overexpression of SchRabGDI1 of Solanum chilense is capable of increasing the endocytosis and salt tolerance of A. thaliana [21]. While the absence of AtVPS9, a GEF protein, compromises its salt stress tolerance [22]. However so far, the OsGAP1 protein from rice is the only RabGAP protein characterized in the context of abiotic stress [23]. Specifically, OsGAP1 interacts with RAB11, being both essential for vesicle trafficking of OsVHA-a1 (a vacuolar H+-ATPase) from trans-Golgi network (TGN) to the central vacuole and salt stress tolerance [24].

The cultivated tomato (Solanum lycopersicum) is one of the most important crops in the world. However, abiotic stress is the main limitation of their yield, development and agronomic properties [25]. On the other hand, wild tomato species such as Solanum pimpinellifolium, Solanum habrochaites, Solanum pennellii, and S. chilense, among others, are tolerant species to abiotic stress and thrive in extreme environments due to differential expression of a diverse set of genes that activate molecular and physiological mechanisms that allow them to effectively adapt [26,27]. The recent sequencing of the S. pennellii, S. lycopersicum, and S. pimpinellifolium genomes provides an excellent opportunity to identify and analyze the RabGAP gene family in the context of abiotic stress [28,29]. In this sense, we have identified each RabGAP gene from these tomato species, reporting amino-acid characteristics, phylogenetics, and the interaction analysis between RabGTPase–RabGAP proteins. Furthermore, through a comprehensive study of the transcriptional profiles in different tissues and stages of development, as well as during salt stress conditions, we provide a global overview of its putative biological and physiological functions. This research supplies an important framework for future research in tomatoes as well as for the development of genetic improvement strategies.

2. Materials and Methods

2.1. Identification and Phylogenetic Analysis of RabGAPs from Tomatoes

To determine the sequences corresponding to RabGAP proteins, a local database was established using the proteomes of S. lycopersicum (SlRabGAP), S. pennellii (SpeRabGAP), and S. pimpinellifolium (SpiRabGAP) obtained from the SolGenomics Network database (https://solgenomics.net) [30]. Here, the RabGAP proteins were determined by the specific TBC domain (Pfam PF00566) as a profile for Hidden Markov chains (HMM), with a cut-off value of 1e−10 in the HMMERv3.1 software [31]. Each protein was manually validated by comparative analyses with the Solgenomics, BLASTp (https://blast.ncbi.nlm.nih.gov), CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), and Pfam databases (http://pfam.xfam.org/) [32].

To assign a name to each RabGAP protein and determine the subclass to which it belongs, the complete length of the amino-acid sequence was aligned with its closest homolog of A. thaliana using the software ClustalO [15,33]. The phylogenetic tree was constructed with MEGA7 software using the neighbor-joining method with 5000 iterations bootstrap [34].

2.2. Sequence Amino-Acid Properties, Synteny and Gene Duplication Analysis

The isoelectric point (pI) and molecular weight (MW) were predicted with Expasy Compute pI/Mw tool (http://web.expasy.org/compute_pi/). The exon-intron structure of the genes was performed with the GSDS software (http://gsds.cbi.pku.edu.cn) [35]. In addition, the conserved motifs in the amino-acid sequence were identified using the web tool MEME (http://meme-suite.org) and analyzed with InterPro Scan 5 (https://www.ebi.ac.uk/interpro/) [36,37]. Values of hydropathy (GRAVY value) were calculated with the help of Expasy ProtParam tool (http://expasy.org/tools/protparam.html). The subcellular locations were determined using CELLO V2.5 (http://cello.life.nctu.edu.tw) [38], Wolf PSORT (https://wolfpsort.hgc.jp) [39], and MultiLoc2 tools (https://abi.inf.uni-tuebingen.de/Services/MultiLoc2) [40]. The predictions of protein-protein interactions were made with the STRING database (https://string-db.org) [41].

Synteny analysis was carried out using the chromosomal locations of homologous and paralogous RabGAP genes in S. lycopersicum and A. thaliana obtained from the PGDD database (http://chibba.agtec.uga.edu/duplication/) [42]. The graphic representation was made using the web-based service ClicO FS [43]. Along with this, the selection pressure of RabGAP genes was determined by the ratio of nonsynonymous to synonymous nucleotide substitutions (Ka/Ks) between paralogs in S. lycopersicum. The approximate date of the duplication events was estimated using T = (Ks/2λ) × 10−6 million years ago (mya), based on the clock-like rates (λ) in Solanaceae of 1.5 × 10–8 [44].

2.3. RabGAP Gene Expression Profiles in Databases

Tissue-specific expression profiles of the RabGAP genes of S. lycopersicum and S. pennellii were obtained from the Bio-Analytic Resource database from the University of Toronto (http://bar.utoronto.ca). The data were obtained from flowers, shoots, leaves, vegetative meristems, seedling shoots, seedling roots, mature fruit, and developing fruit [45]. To establish a relation between the expression profiles of the RabGAP genes and the capacity of tolerance to abiotic stress among S. lycopersicum and its wild relatives (S. pimpinellifolium, S. habrochaites, and S. pennellii), values normalized in respect to S. lycopersicum were obtained from the “Tomato Expression” database (http://malooflab.phytonetworks.org/apps/tomato-expression/) [45]. In addition, to study the expression profiles of RabGAP genes in response to abiotic stress, we used the PLEX database (http://www.plexdb.org). The data were extracted from L6 (GEO accession GSE16401), L12 and L13 experiments (GEO accession GSE22304). The L6 experiment corresponded to a salt stress assay considering samples of leaves from 6-week-old S. lycopersicum and S. pimpinellifolium treated with NaCl 200 mM for 5 h [46]. The L12 experiment correspond to drought stress considering samples of leaves from four-week-old S. lycopersicum and S. habrochaites, previously non-irrigated for 14 days. Finally, the L13 experiment corresponded to a heat stress assay considering samples of leaves from four-week-old S. lycopersicum and S. habrochaites treated at 40 °C [47]. The profiles are relative to the control without stress.

2.4. Plant Material and Gene Expression Analysis

Seeds of S. chilense (Dunal) and S. lycopersicum were germinated in a mixture of perlite, vermiculite and peat moss (1:1:1), grown under greenhouse conditions at 23–25 ºC, with photoperiod of 16/8 h light/dark, irrigated with 400 mL every five days and fertilized with a commercial Hoagland’s solution (1/4 strength) every 10 days. At the sixth week, a saline stress of NaCl 300 mM was applied to a group of plants. Leaves and roots were collected at 0, 3, 6, 12, 24, 48, and 72 h for the gene expression analyses. Then, total RNA from both tissues of S. chilense was extracted, treated, and quantified following the protocol of San Martín-Davison et al. (2017) [21]. To obtain the first strand of cDNA, 2 μg of RNA was retro-transcribed in 20 μL of reaction using the First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) and following the manufacturer’s instructions.

Relative levels of gene expression were measured by qRT-PCR, using the Stratagene Mx3000p thermocycler (Agilent Technologies, Santa Clara, CA, USA). Maxima SYBR Green/ROX qRT-PCR Master MIX (Thermo Scientific) was used for all reactions according to the protocol described by the manufacturer. For each sample (three biological replicates), qRT-PCR was performed in triplicate (technical replicates) using 10 μL of Master MIX, 0.5 μL of 250 nM of primers, 1 μL of cDNA and nuclease-free water in a final volume of 20 μL [48]. The amplification was followed by a melting curve with continuous fluorescence measurement at 55 to 95 °C. The data were manually analyzed, and the expression normalized with the Ubiquitin3 gene [49]. All primers are listed in Table S1. The transcript levels of each gene were evaluated using the 2−ΔΔCT method [50]. The qRT-PCR data were analyzed using ANOVA (one-way analysis of variance) tests (with a significance of p-value < 0.05). Data management and standardization were performed with R version 3.2.5 [51].

3. Results

3.1. 24 Proteins Organized in 7 Subclasses Constitute the RabGAP Family in Tomato

An analysis of HMM on the proteomes of S. lycopersicum, S. pimpinellifolium, and S. pennellii allowed us to identify 24 putative RabGAP proteins in each species. They were named according to the closest ortholog of A. thaliana [15]. Among them, 21 RabGAPs possessed the TBC domain, two the Rab3-GTPase_cat domain and one the Rab3GAP2_N domain [14,52]. At the amino-acid level, all RabGAP proteins had the conserved residues of arginine and glutamine, critical for catalytic activity. The exceptions were RabGAP23b and those with the Rab3-GTPase_cat and Rab3GAP2_N domains, see in Figure S1. In S. lycopersicum, the RabGAP proteins had a length between 352 (SlRabGAP18) and 942 amino-acids (SlRab3GAP2) and a theoretical isoelectric point ranging from 4.79 (SlRabGAP6) to 8.97 (SlRabGAP18). Similar characteristics were also observed in the RabGAP protein families of S. pennellii and S. pimpinellifolium. On the other hand, the GRAVY index, associated to the hydrophobicity level of a protein, was less than 0, revealing that all RabGAPs are hydrophilic. In this same sense, the prediction of subcellular localization using three different web servers indicated that the main locations for all RabGAP proteins were in the nucleus and cytoplasm, as shown as in Table 1.

Table 1

List of RabGTPase activating proteins (RabGAP) genes found in domesticated and wild tomatoes and their physical and chemical characteristics. Data obtained from Sol Genomics Network database. Gene prefixes represent Solanum lycopersicum (Sl), Solanum pennellii (Spe), and Solanum pimpinellifolium (Spi). Subcellular location: ML2, MultiLoc2; WPS, Wolf PSORT. Nuc, Nuclear; Cyt, Cytoplasmic; Chl, Chloroplast; MP, Plasma Membrane; ER, Endoplasmic Reticulum; and Mit, Mitochondria.

Gene Name	Gene ID	Chr	Chromosome Location		Length (aa)	Mol. Wt. (kDa)	Isolelec. Point	Conserved Domain	Domain Location		GRAVY	Subcellular Location
			Start	End					Start	End		CELLO	ML2	WPS
SlRab3GAP1	Solyc07g064740	7	66763414	66771800	673	74.853	5.14	Rab3-GTPase_cat	377	530	−0.478	Nuc	Cyt	Nuc
SlRab3GAP2	Solyc06g082200	6	48103293	48118442	942	106.62	4.93	Rab3-GTPase_cat	555	720	−0.386	Cyt	Cyt	Nuc
SlRabGAP1a	Solyc05g053710	5	63747738	63754496	393	43.78	5.38	TBC	144	393	−0.486	Nuc	Cyt	Nuc
SlRabGAP1b	Solyc09g092070	9	71223957	71231836	418	47.161	5.98	TBC	88	387	−0.283	PM	Cyt	Nuc
SlRabGAP2a	Solyc07g008840	7	3803764	3808890	822	90.686	4.98	TBC	16	403	−0.491	Nuc	Cyt	Nuc
SlRabGAP2b	Solyc02g014450	2	16619223	16626117	726	80.876	5.31	TBC	3	277	−0.544	Nuc	Cyt	Chl
SlRabGAP3	Solyc06g008250	6	2120441	2130757	432	49.448	8.53	TBC	71	348	−0.352	Cyt	Cyt	Cyt
SlRabGAP4	Solyc11g005010	11	15822	26251	447	51.433	5.53	TBC	147	403	−0.364	Nuc	Cyt	Nuc
SlRabGAP5	Solyc09g007160	9	779711	793133	797	90.48	5.08	TBC	220	457	−0.625	Cyt	Cyt	Cyt
SlRabGAP6	Solyc04g009560	4	2961142	2970580	828	93.555	4.79	TBC	244	482	−0.581	Cyt	Cyt	Chl
SlRabGAP7	Solyc03g058470	3	24712188	24716135	571	64.49	5.58	TBC	182	427	−0.46	Nuc	Cyt	Chl
SlRabGAP9a	Solyc07g049580	7	59919122	59923736	394	44.936	8.38	TBC	108	325	−0.154	Cyt	Cyt	Nuc
SlRabGAP9b	Solyc12g005930	12	554207	559357	394	44.931	7.21	TBC	108	325	−0.163	Cyt	Cyt	Cyt
SlRabGAP10	Solyc07g062450	7	65184937	65189399	455	50.645	8.59	Rab3GAP2_N	28	416	−0.07	PM	Cyt	Chl
SlRabGAP14	Solyc02g071420	2	40880409	40882735	459	53.267	5.93	TBC	122	421	−0.376	Nuc	Cyt	Cyt
SlRabGAP15	Solyc12g005220	12	141022	146306	438	50.259	7.99	TBC	61	344	−0.387	Cyt	Nuc	Nuc
SlRabGAP16	Solyc09g066420	9	64831553	64843975	485	54.757	5.61	TBC	78	392	−0.514	Nuc	Cyt	Nuc
SlRabGAP18	Solyc01g101090	1	90939383	90945694	352	40.937	8.97	TBC	80	297	−0.169	Cyt	Cyt	Cyt
SlRabGAP20	Solyc06g053190	6	35918577	35924309	553	62.418	7.03	TBC	108	497	−0.457	Nuc	Nuc	Chl
SlRabGAP21a	Solyc12g009610	12	2864130	2875362	656	75.17	5.32	TBC	351	589	−0.49	Nuc	Cyt	Nuc
SlRabGAP21b	Solyc07g008000	7	2697439	2709902	678	78.042	5.78	TBC	374	612	−0.518	Nuc	Cyt	Nuc
SlRabGAP22	Solyc03g082590	3	52516500	52523765	559	62.937	5.84	TBC	115	495	−0.425	Nuc	ER	Chl
SlRabGAP23a	Solyc10g006440	10	1046336	1054965	450	51.287	6.09	TBC	68	359	−0.483	Cyt	Cyt	Nuc
SlRabGAP23b	Solyc07g064230	7	66456796	66462000	425	48.5	8.57	TBC	60	333	−0.409	Cyt	Cyt	Cyt
SpeRab3GAP1	Sopen07g032940	7	77919830	77932192	674	74.805	5.04	Rab3-GTPase_cat	359	514	−0.48	Nuc	Cyt	Nuc
SpeRab3GAP2	Sopen06g033580	6	59015664	59042332	942	106.478	4.85	Rab3-GTPase_cat	555	720	0.394	Cyt	Cyt	Nuc
SpeRabGAP1a	Sopen05g032190	5	75703069	75712722	483	53.886	5.56	TBC	252	442	−0.367	Nuc	Cyt	Nuc
SpeRabGAP1b	Sopen09g034940	9	82925996	82937126	493	55.01	5.76	TBC	269	459	−0.357	Nuc	Cyt	Nuc
SpeRabGAP2a	Sopen07g004770	7	4263306	4270907	822	90.576	4.91	TBC	106	355	−0.504	Nuc	Cyt	Nuc
SpeRabGAP2b	Sopen02g003110	2	7156270	7165642	763	84.747	5.52	TBC	15	299	−0.5	Nuc	Cyt	Chl
SpeRabGAP3	Sopen06g003110	6	2485987	2500959	432	49.464	8.34	TBC	177	323	−0.367	Cyt	Cyt	Cyt
SpeRabGAP4	Sopen11g001030	11	25457	41033	447	51.461	5.34	TBC	147	400	−0.372	Nuc	Cyt	Nuc
SpeRabGAP5	Sopen09g002030	9	807911	827341	798	90.531	4.98	TBC	289	449	−0.613	Cyt	Cyt	Cyt
SpeRabGAP6	Sopen04g004720	4	3140714	3154812	829	93.647	4.71	TBC	245	480	−0.577	Cyt	Cyt	Chl
SpeRabGAP7	Sopen03g012120	3	18722571	18792333	692	78.416	6.14	TBC	375	525	−0.455	Nuc	Cyt	Nuc
SpeRabGAP9a	Sopen07g024980	7	70500266	70507267	395	44.936	7.42	TBC	108	322	−0.154	Cyt	Cyt	Nuc
SpeRabGAP9b	Sopen12g001880	12	657246	665470	395	44.947	6.75	TBC	108	322	−0.17	Cyt	Cyt	Cyt
SpeRabGAP10	Sopen07g030690	7	76219201	76225527	456	50.618	7.34	Rab3GAP2_N	28	416	−0.063	PM	Cyt	Chl
SpeRabGAP14	Sopen02g020470	2	44244421	44247921	414	48.169	5.36	TBC	123	403	−0.454	Nuc	Cyt	Cyt
SpeRabGAP15	Sopen12g001200	12	187739	195817	438	50.174	7.08	TBC	174	333	−0.36	Cyt	Nuc	Nuc
SpeRabGAP16	Sopen09g028140	9	76268366	76277038	468	53.047	5.22	TBC	203	362	−0.499	Nuc	Cyt	Nuc
SpeRabGAP18	Sopen01g044540	1	101408643	101416953	352	40.937	8.28	TBC	80	294	−0.169	Cyt	Cyt	Cyt
SpeRabGAP20	Sopen06g018250	6	45454478	45463520	553	62.472	6.91	TBC	354	470	−0.453	Nuc	Chl	Chl
SpeRabGAP21a	Sopen12g004600	12	3139864	3156889	656	75.112	5.22	TBC	351	586	−0.48	Nuc	Cyt	Nuc
SpeRabGAP21b	Sopen07g003880	7	2957429	2975101	678	78.098	5.69	TBC	374	609	−0.53	Nuc	Cyt	Nuc
SpeRabGAP22	Sopen03g022350	3	53888278	53898904	540	61.034	5.93	TBC	337	455	−0.454	Nuc	Mit	Chl
SpeRabGAP23a	Sopen10g002390	10	1110898	1124727	442	50.427	6.09	TBC	60	322	−0.398	Mit	Cyt	Cyt
SpeRabGAP23b	Sopen07g032380	7	77586099	77593750	439	50.223	8.65	TBC	60	322	−0.398	Mit	Cyt	Cyt
SpiRab3GAP1	Sopim07g064740	7	−	−	673	74.853	5.14	Rab3-GTPase_cat	359	514	−0.478	Nuc	Cyt	Nuc
SpiRab3GAP2	Sopim06g082200	6	−	−	907	102.787	4.83	Rab3-GTPase_cat	555	720	−0.391	Cyt	Cyt	Nuc
SpiRabGAP1a	Sopim05g053710	5	−	−	393	43.78	5.38	TBC	145	380	−0.487	Nuc	Cyt	Nuc
SpiRabGAP1b	Sopim09g092070	9	−	−	418	47.161	5.98	TBC	194	384	−0.283	PM	Cyt	Cyt
SpiRabGAP2a	Sopim07g008840	7	−	−	822	90.686	4.98	TBC	106	355	−0.491	Nuc	Cyt	Nuc
SpiRabGAP2b	Sopim02g014450	2	−	−	726	80.876	5.31	TBC	15	252	−0.544	Nuc	Cyt	Chl
SpiRabGAP3	Sopim06g008250	6	−	−	432	49.448	8.53	TBC	177	323	−0.352	Cyt	Cyt	Cyt
SpiRabGAP4	Sopim11g005010	11	−	−	447	51.432	5.4	TBC	147	400	−0.364	Nuc	Cyt	Nuc
SpiRabGAP5	Sopim09g007160	9	−	−	797	90.48	5.08	TBC	289	449	−0.625	Cyt	Cyt	Cyt
SpiRabGAP6	Sopim04g009560	4	−	−	828	93.555	4.79	TBC	244	479	−0.581	Cyt	Cyt	Chl
SpiRabGAP7	Sopim03g058470	3	−	−	571	64.49	5.58	TBC	254	404	−0.46	Nuc	Cyt	Chl
SpiRabGAP9a	Sopim07g049580	7	−	−	394	44.936	8.38	TBC	108	322	−0.154	Cyt	Cyt	Nuc
SpiRabGAP9b	Sopim12g005930	12	−	−	394	44.931	7.21	TBC	108	322	−0.163	Cyt	Cyt	Cyt
SpiRabGAP10	Sopim07g062450	7	−	−	455	50.645	8.59	Rab3GAP2_N	28	416	−0.067	PM	Cyt	Chl
SpiRabGAP14	Sopim02g071420	2	−	−	459	53.267	5.93	TBC	123	410	−0.376	Nuc	Cyt	Cyt
SpiRabGAP15	Sopim12g005220	12	−	−	438	50.259	7.99	TBC	174	323	−0.387	Cyt	Nuc	Nuc
SpiRabGAP16	Sopim09g066420	9	−	−	485	54.757	5.61	TBC	220	379	−0.513	Nuc	Cyt	Nuc
SpiRabGAP18	Sopim01g101090	1	−	−	352	40.937	8.97	TBC	80	294	−0.169	Cyt	Cyt	Cyt
SpiRabGAP20	Sopim06g053190	6	−	−	553	62.418	7.03	TBC	354	470	−0.457	Cyt	Nuc	Chl
SpiRabGAP21a	Sopim12g009610	12	−	−	656	75.17	5.32	TBC	351	586	−0.49	Nuc	Cyt	Nuc
SpiRabGAP21b	Sopim07g008000	7	−	−	720	78.042	5.56	TBC	374	609	−0.518	Nuc	Cyt	Nuc
SpiRabGAP22	Sopim03g082590	3	−	−	559	62.937	5.84	TBC	357	475	−0.425	Nuc	ER	Chl
SpiRabGAP23a	Sopim10g006440	10	−	−	450	51.287	6.09	TBC	184	342	−0.483	Cyt	Cyt	Nuc
SpiRabGAP23b	Sopim07g064230	7	−	−	425	48.5	8.57	TBC	173	308	−0.409	Cyt	Cyt	Cyt

In respect to the classification of RabGAP proteins, the phylogenetic analysis evidenced that they are grouped in seven subclasses (I, II, III, IV, V, VI-Rab3GAP, and VIII), according to their homologs in A. thaliana, shown in Figure 1 [15]. This analysis revealed that the subclass with the most members was subclass I and those with the fewest members were subclasses III and V. In addition, subclasses II, V, and VIII of S. lycopersicum had fewer members than those of A. thaliana, while subclasses III and IV had more.

Figure 1

Phylogenetic tree of tomato RabGAPs. Seven subclasses (I, II, III, IV, V, VI, and VIII) were identified. 26 RabGAP of A. thaliana [15] are represented in green circles. In red, 24 RabGAP of S. lycopersicum; in light blue, 24 RabGAP of S. pimpinellifolium; and in dark blue, 24 RabGAP of S. pennellii.

3.2. Events of Segmental Duplication Have Kept Constant the Number of Members of the RabGAP Family

To evidence the genomic organization and the role of duplication events in the evolutionary history of RabGAP family genes, synteny mapping, intron-exon structure identification and selection pressure analysis were performed, see in Figure 2. First, the genomic analysis revealed that the SlRabGAP genes were differentially distributed on 11 chromosomes of S. lycopersicum: One gene each on Sl-Chr1, -4, -5, -10, and -11; two genes each on Sl-Chr2 and Sl-Chr3; three genes each on Sl-Chr6, -9, and -12; and six genes on Sl-Chr7. Then, the comparative analysis between the exon-intron structures and their chromosome locations allowed us to identify seven events of segmental duplication (SlRabGAP20-SlRabGAP22, SlRabGAP21a-SlRabGAP21b, SlRabGAP1a-SlRabGAP1b, SlRabGAP9a-SlRabGAP9b, SlRabGAP5-SlRabGAP6, SlRabGAP2a-SlRabGAP2b, and SlRabGAP3-SlRabGAP16) and one triplication event between SlRabGAP23a, -23b, and -15. Along with the above, in all these cases the ratios of synonymous and non-synonymous nucleotide substitutions (Ka/Ks) were less than 1, indicating that the function of the genes has not diverged and has been maintained due to a stabilizing selection force. Finally, the estimated time in which these duplications occurred was 15 to 28 mya, see in Table 2.

Figure 2

Syntenic relationships among RabGAP. Paralogy and orthology relations between RabGAP from S. lycopersicum and Arabidopsis thaliana. Orthologous and paralogous genes are connected by red and gray lines, respectively.

Table 2

Evolutionary parameters for duplicated RabGAP genes. Table shows Ka/Ks ratios, duplication and selection types and time of divergence.

Gene Pairs Duplicated		Duplication Type	Ka	Ks	Ka/Ks	Selection	Time (mya)
SlRabGAP23a	SlRabGAP23b	Segmental	0.1	0.52	0.192	Purify	17.3
SlRabGAP23b	SlRabGAP15	Segmental	0.12	0.55	0.218	Purify	18.3
SlRabGAP20	SlRabGAP22	Segmental	0.18	0.62	0.290	Purify	20,6
SlRabGAP21a	SlRabGAP21b	Segmental	0.1	0.71	0.140	Purify	23.6
SlRabGAP1a	SlRabGAP1b	Segmental	0.11	0.69	0.159	Purify	23
SlRabGAP9a	SlRabGAP9b	Segmental	0.07	0.49	0.142	Purify	16.3
SlRabGAP5	SlRabGAP6	Segmental	0.09	0.86	0.171	Purify	28.6
SlRabGAP2a	SlRabGAP2b	Segmental	0.11	0.46	0.230	Purify	15.3
SlRabGAP3	SlRabGAP16	Segmental	0.13	0.63	0.206	Purify	21

Ka: number of nonsynonymous substitutions per non-synonymous site; Ks: number of synonymous substitutions per synonymous site. Mya: million years ago.

In relation to the characterization of the RabGAP gene structure, the results showed that the number of introns is very diverse, see in Figure 3, Figure 3A. On average, the RabGAP genes had 13 introns, of which RabGAP2a, -20, and -22 had the lowest number (four introns), whereas Rab3GAP2 had the highest number (21 introns). On the other hand, the RabGAP proteins were characterized by having between four or five conserved motifs associated with the “catalytic core” from the TBC domain, as shown in Figure 3, Figure 3B. Exceptionally, the catalytic domain of subclass VI, specifically associated with Rab3, was present in three proteins—Rab3GAP1, Rab3GAP2, and RabGAP10—as seen as in Table 1.

Figure 3

Identification of exon-intron structure and conserved motifs in RabGAP from S. lycopersicum. (A). Exon-intron structure of RabGAP genes from S. lycopersicum. In yellow, exons; in blue, the 3′ and 5′ UTR regions; and the gray lines are introns. (B) Conserved motif of RabGAP proteins. Ten motifs were identified with MEME database. The gray lines are the complete amino-acid sequence represented proportionally. Each color represents a different motif. Together, the pink, red, purple, orange, and light blue motifs represent the TBC domain. The consensus sequences are detailed in Table S2.

3.3. In Silico Analysis of Protein-Protein Interactions Suggests the Association of RabGAP Proteins with Specific Vesicular Trafficking Pathway

In order to suggest specific vesicular trafficking pathways in which the RabGAP proteins could participate, we identified their possible affinities or physical interactions with the RabGTPase subfamilies of S. lycopersicum using the STRING database, see in Table S3. The results showed that in the exocytic pathway, SlRabGAP5, -6, -9a, and -9b could be interacting with RabGTPases of the subfamilies-A and E. In the anterograde pathway, RabGAP4 could be interacting with the subfamily-D, whereas in the retrograde pathway, SlRabGAP2a, -2b, -20, -21a, -21b, -23a, and -5 could be related with subfamily-H. In the endocytic pathway, only RabGAP18 could be interacting with subfamily-F, whereas SlRabGAP3, -7, -15, -16, -20, -21a, -21b, and -22 could be relating with subfamily-G (see in Figure 4).

Figure 4

Identification of proposed protein-protein interactions between RABs and RabGAPs. The vesicular trafficking routes directed by the RabGTPase subfamilies between the different organelles and membranes are represented by arrows. RabGAP proteins were associated to each RabGTPase using the protein-protein interaction functional networks provided by the STRING database [41]. ER: Endoplasmic Reticulum. MVB: Multi-Vesicular Body. All proposed protein-protein interaction networks are detailed in Table S4.

We also observed that some RabGAPs could form protein complexes. Homodimers are formed in the case of SlRabGAP1a, -1b, -3, -4, -9a, -9b, -14, -16, -21a, -21b, or -23a; heterodimers between SlRabGAP10, -18 or -23a; protein complexes between vacuolar protein sorting (VPS) and SlRabGAP3, -7, -15, -20, -21a, -21b, -22, -23a, or -23b. The complexes between proteins related to cell division checkpoint processes, such as SlRabGAP5, -6, -7, -9a, or -9b, and complexes with proteins that respond to hormones involved in stress, such as SlRabGAP5 or -6, were also identified (see Table S2).

3.4. Differential Expression of RabGAP Genes in Different Tissues and Development Stages of Cultivated and Wild Tomato Species

To explore patterns in the transcriptional regulation of the RabGAP genes, we analyzed the expression profiles in different organs and development stages of S. lycopersicum and S. pennellii (see Figure 5, File S1). We evidenced that the expression patterns of RabGAP genes in flowers, shoots, seedling shoots, seedling roots, and mature fruit of S. pennellii were inverse to that of S. lycopersicum. Furthermore, in almost all organs of S. lycopersicum the expression patterns were positive, while in S. pennellii, they were only positive in leaves and vegetative meristems. At the subclass level, no obvious differences were observed. However, we highlight that the most up-regulated RabGAP genes in S. lycopersicum were SlRabGAP23a in flowers, leaves, and vegetative meristems, SlRabGAP18 in stems and developing fruit, SlRabGAP2a in seedling shoots, and RabGAP15 in seedling roots. While in S. pennellii were SpRabGAP18 in seedling shoots, seedling roots, flowers and stems. SpRabGAP23a in leave, SpRabGAP3 in vegetative meristems, SpRabGAP15 in mature fruit, and SpRabGAP2a in developing fruit. It is also interesting to mention that RabGAP18, the only RabGAP possibly associated with RabGTPase of the F subfamily, was ubiquitously expressed in all organs and stages of development of both wild and cultivated tomato.

Figure 5

Expression patterns of RabGAP genes in different tissues, organs and developmental stages of tomato. Induction of genes is represented in red and repression in blue. F: flowers, S: shoots, L: leave, MV: vegetative meristems, SS: seedling shoots, SR: seedling roots, MF: mature fruit, DF: developing fruit. All values are detailed in Table S5.

To evidence differential patterns of regulation between tomato and wild relatives in normal conditions, we analyzed the expression profiles of the RabGAP genes in leaves of S. pimpinellifolium, S. habrochaites, and S. pennellii, see in Figure 6. SpiRabGAP23a, ShaRabGAP3, and SpeRabGAP10 were the most up-regulated genes in each wild species, while SpiRab3GAP1, ShaRabGAP14, and SpeRabGAP16 were the most down-regulated. Studying an expression pattern associated with the degree of tolerance to abiotic stress of each wild species, we observed that RabGAP1a, -10, and -21a had positive correlations, whereas only RabGAP18 had a negative tendency. In addition, the analysis showed that both RabGAP2a and -22 represented about 35% of the number of transcripts of RabGAP family (see Table S6).

Figure 6

Expression profiling of RabGAP genes in wild tomato species. Transcript levels of RabGAP genes from wild tomato species relative to S. lycopersicum are represented. Higher or lower gene expressions are shown in red and dark blue, respectively. Nomenclature: SLY: S. lycopersicum, SPI: S. pimpinellifolium, SHA: S. habrochaites and SPE: S. pennellii. All values are detailed in Table S6.

3.5. RabGAP Genes Associated with Endocytic and Pre-Vacuolar Trafficking Are Up-Regulated in Roots Subjected to Salt Stress

To obtain more information about the role of RabGAP genes under abiotic stress conditions, we analyzed their expression profiles in response to heat, drought and salt stress in S. lycopersicum and compared them with those results obtained in wild relatives. In this case, the only data available for the RabGAP3, -4, -18, -20, -21a, and -21b were found in different databases (see Figure 7). During heat stress, only RabGAP4 and -20 were as strongly induced in the tolerant species as RabGAP4, -21a, and -21b were in the S. lycopersicum. Interestingly, under conditions of drought stress, unlike heat stress, RabGAP3, -4, -18, -20, and -21b of tolerant species versus RabGAP21a and -21b of S. lycopersicum, were up-regulated. Finally, under salt stress, similar expression profiles were observed in the sensitive and tolerant species. In this case, RabGAP21a and -21b were the most up-regulated genes in both species.

Figure 7

Transcriptomic profiles of RabGAP genes from tomato species with different degrees of abiotic stress tolerance. Expression patterns of RabGAP genes in S. lycopersicum (sensitive species) and S. pimpinellifolium or S. habrochaites (tolerant species) in saline, drought and heat stress are represented. Higher or lower gene expressions versus the control, without stress, are shown in red and dark blue, respectively. Saline stress: S. lycopersicum versus S. pimpinellifolium (GEO accession GSE16401). RNA extraction from leaves of 6-week-old plants treated with NaCl 200 mM for 5 h [46]. Stress by drought: S. lycopersicum versus S. habrochaites (GEO accession GSE22304). Extraction of RNA from leaves of four-week-old plants, previously without irrigation for 14 days. Heat stress: S. lycopersicum versus S. habrochaites (GEO accession GSE22304). RNA extraction from leaves of four-week-old plants treated at 40 °C [47]. All values are detailed in Table S7.

Considering the low number of genes previously analyzed, and to learn more about the stress response and determine the possible biological effect of RabGAP genes of S. lycopersicum during salt stress, 20 RabGAP genes representing at least one gene of each subclass were selected for expression analysis by qRT-PCR (see Figure 8). Along with the above, we used S. chilense as the wild and halophyte species to contrast the analyses, as shown in Figure 9. The results showed that, in both leaves and roots of S. lycopersicum, four genes were up-regulated (SlRabGAP2b, -7, -9b, and -20), while 10 genes were induced in leaves, and three only in roots. At early times of salt stress, RabGAP2b and -22 in roots and RabGAP9b and -20 in leaves were the most up-regulated. Similarly, at late times, RabGAP3 and -22 in roots and SlRabGAP1a, -4, -10, and -18 in leaves had the highest expression levels. Interestingly, RabGAP3, -16, and -22 in roots and RabGAP1b in leaves managed to maintain a strong and stable induction throughout the experiment. In this context, it should be also noted that pairs of paralogous genes (RabGAP1a, -1b, -2a, -2b, -9a, -9b, -21a, and -21b) showed different expression patterns.

Figure 8

Spatio-temporal expression patterns of RabGAP genes of S. lycopersicum exposed to salt stress. Relative levels of SlRabGAP gene transcripts in roots (orange bar) and leaves (green bar) of 10–12-week-old S. lycopersicum plants were determined at 0, 3, 6, 12, 24, 48, and 72 h after stress initiation with 300 mM NaCl. The relative levels of the salt-stress marker gene (SlAREB1) were measured. In SlRabGAP14 and -23b, transcripts were not detected. Columns and error bars represent the mean and standard deviation for three biological and three technical replicates. * p-value < 0.05.

Figure 9

Spatio-temporal expression patterns of RabGAP genes of S. chilense exposed to salt stress. Relative levels of RabGAP gene transcripts in roots (orange bar) and leaves (green bar) of 10–12 weeks old S. chilense plants were determined at 0, 3, 6, 12, 24, 48, and 72 h after stress initiation with 300 mM NaCl. The relative levels of the salt-stress marker gene (SchAREB1) were measured. In SlRabGAP14, transcripts were not detected. Columns and error bars represent the mean and standard deviation for three biological and three technical replicates. * p-value < 0.05.

The expression patterns of RabGAP genes in S. chilense revealed differences with those of S. lycopersicum, as seen as in Figure 9. In this species, 14 RabGAP genes significantly increased their relative expression in both leaves and roots during stress, whereas RabGAP15, -18, and -23a did it only in roots, and RabGAP21b only in leaves. We want to highlight the high and stable induction levels of RabGAP18 in roots of S. chilense throughout the salt stress, the inductions of RabGAP1a, -4, and -9a in roots at late times, and the large fluctuations in the expression of RabGAP2a in leaves. In addition, similar to S. lycopersicum, the expression levels of paralogous gene pairs also differed from the tissue in which they were expressed and their levels of induction.

4. Discussion

Within the Solanaceae family, the commercial tomato, S. lycopersicum, is the main cultivated vegetable in the world, but its sensitivity to abiotic stress negatively affects its productivity. On the other hand, wild species related to it such as S. pimpinellifolium, S. habrochaites, S. pennellii, and S. chilense are able to tolerate different degrees of abiotic stresses. A strategy of tolerance that S. chilense particularly uses could be associated with vesicular trafficking processes such as endocytosis, vacuolar compartmentalization of toxic ions, and damaged element recycling [21]. In this context, we have identified and characterized for the first time the family of RabGAP genes of different Solanaceaes through analyzing of the whole genome, mainly through transcriptional study. This allowed us to indicate the possible relationships between them and the components that direct the vesicular trafficking.

4.1. Overview of RabGAP Gene Family in the Tomato Genome

The RabGAP gene family encodes proteins capable of accelerating the hydrolysis of GTP to GDP bound to RabGTPase proteins, allowing them to direct a new round of vesicular trafficking [53]. In this work, 24 RabGAP genes were identified in each tomato species (S. lycopersicum, S. pennellii, and S. pimpinellifolium), a similar number of genes to that of other species such as rice or A. thaliana [15]. However, according to motifs and multiple alignment analyses, the results suggest that four proteins, including RabGAP23b, should not have RabGAP activity due to the absence of a conserved Arginine residue. In this regard, it has been evidenced that a point mutation on this residue in OsGAP1 (R450A) causes a severe loss of this activity against two RabGTPases—OsRab11 and OsRab8a—revealing its importance in catalytic activity [24].

Interestingly, the subclasses I and II of the RabGAP family are composed of six and two RabGAP genes, respectively. In addition, both families are potentially exclusive to plants and it is predicted that their evolution is an adaptation mechanism due to the loss of function related to the RasGAP family [15]. From the above, possible coevolution mechanisms between RabGTPases and RabGAPs, which have sculpted the endomembrane system, have been suggested [54]. On the other hand, the equivalent number of members of the RabGAP family between tomato and evolutionarily distant species, such as A. thaliana or rice, is an important question to raise. In this sense, it has been suggested that duplication and diversification events from five ancestral RabGAP genes have been the mechanisms that have kept the number of genes constant [15,28,55]. The exon-intron structure also provides relevant information to understand the evolution process of the RabGAP family. In this analysis, we found paralogous genes from subclasses I (RabGAP23b and -15), III (RabGAP21b and -21b), and VIII (RabGAP9a and -9b) located on chromosomes 7 and 12. The arrangements and size of exons and introns of these genes supports the hypothesis of an important segmental duplication event between both chromosomes, which has also been reported for members of the B-BOX, LEA, and GRAS families of S. lycopersicum [56,57,58]. Together, these data suggest that events of segmental duplications have played an important role in the gene evolution of the RabGAP gene family.

4.2. The New Subfamily VI and Rab3GAP Could Play an Important Role in Autophagy and Stress Tolerance

Regarding the characteristics of other identified genes, we found two new members of subclass VI with a Rab3GAP domain whose functional roles in plants have not yet been determined, see in Figure 1 and Table 1. However, homolog genes in animal cells participate in: (i) the deactivation of Rab3 [14], a homolog of RabD in plants, (ii) interaction with ATG8 in autophagy processes [59,60], (iii) or activation of a RabGTPase of subfamily C, acting as a guanine exchange factor in degradative pathways and macro-autophagy [61]. Intriguingly, a RabD of plants (RABD2A) localizes with proteins associated with salt-stress tolerance capacity, such as EHD1 or SYP41 [62,63]. In a similar way to expression patterns of Rab3GAP1, Rab3GAP2, and RabGAP10 of S. lycopersicum, the autophagy is a mechanism rapidly induced by salt stress [64]. These interesting characteristics suggest that the proteins of subclass VI can probably interact with RabD or ATG8 and play an important role during salt stress in tomato.

4.3. Endocytosis-Associated RabGAPs Could Be Mediating the Salt Stress Tolerance in Tomato

In general, the different RabGAP proteins of S. lycopersicum identified in this work were located in the nucleus or cytoplasm. Particularly, the predicted nuclear localization was consistent among RabGAP22 from S. lycopersicum and A. thaliana [65]. The patterns of expression of both genes are also similar. In both species, RabGAP22 is expressed in various tissues and stages of development, such as flowers, roots, or vegetative meristems. Along with this, RabGAP22 from A. thaliana can interact with peroxisome proteins such as AGT1 and be associated with photorespiration processes or adaptation mechanisms against abiotic stress [66]. According to this, the homolog of AtRabGAP22 in S. lycopersicum could have an important role in tomato roots when they are subjected to salt stress. On the other hand, the predicted subcellular location of RabGAP21a from S. lycopersicum, with respect to its rice homolog, OsGAP1 [23], is also coherent. In this case, the data suggest that SlRabGAP21a could be localized in the Trans-Golgi network and the pre-vacuolar compartments. Equally, it is interesting to observe that the expression patterns of OsGAP1 and RabGAP21a of S. lycopersicum are similar in leaves and roots when plants are subjected to salt stress [24]. These data suggest that RabGAP21 could also have an important role in a trafficking pathway that mobilizes transporters or proteins associated with the vacuole. Additionally, SlRabGAP21a could potentially interact with the TVP38 protein, which has been associated with the stability of the membranes [67]. The results for RabGAP18 are also consistent with the fact that the endocytosis mechanism is an inducible process against salt stress and regulated for RabGTPases of the subfamily-F [20,22]. To date, the RabGAP proteins that regulate this subfamily-F are unknown, however, the protein-protein interaction analysis suggested that RabGAP18 could be an exclusive regulator of this family. Interestingly, RabGAP18 is also strongly induced in the roots of S. chilense when they are exposed to salt stress in a similar manner to SchRabGDI1, whose overexpression in A. thaliana can increase its tolerance to salt stress [21]. In general, the various antecedents support the idea that RabGAP have a certain degree of affinity for RabGTPases, but the results equally show a high degree of promiscuity for some of them. In this sense, further analyses and experiments are needed to establish the vesicular trafficking routes in which the members of the RabGAP family of S. lycopersicum participate in order to finally understand their physiological functions during the abiotic stress.

5. Conclusions

In conclusion, we have identified 24 RabGAP genes in each S. lycopersicum, S. pennellii, and S. pimpinellifolium and classified them into seven subclasses. Two proteins with Rab3GAP domain were identified for the first time. All of them have the TBC domain characteristic of the RabGAP family. Evolutionarily segmental duplication and purifying selection have maintained constant the number and function of these genes in the S. lycopersicum genome. The results presented here show that the RabGAP proteins, such as RabGAP18 or RabGAP22, could be associating to different intracellular vesicular trafficking pathways and that the genes that encode them can change their expression depending on both endogenous and environmental stimuli. In this sense, the study of the regulation mechanisms of gene expressions, as well as the specific interactions with RabGTPase proteins could provide important information to understand their participation in tolerating abiotic stress in tomato species.