Transcriptomic Analysis Reveals Salt Tolerance Mechanisms Present in Date-Plum Persimmon Rootstock (Diospyros Lotus)

Background Drought and salinity are two of the main challenges in agriculture. In many areas, crop production needs solutions to adapt the grown species to the increasing salinity. Research on physiological and molecular responses activated by salinity in plants is needed to elucidate mechanisms of salinity tolerance. Transcriptome proling (RNA-Seq) is a powerful tool to study the transcriptomic prole of genotypes under stress conditions. In temperate fruit tree species, tree grafting on salinity tolerant rootstocks is a common method to compensate for the cultivar saline sensitivity. Persimmon species have different levels of tolerance to salinity, knowledge of this variability provides the basics for development of salt tolerant rootstocks. Results In this study, we conducted a physiological and transcriptomic proling of roots and leaves in tolerant and sensitive plants of persimmon rootstock, Diospyros lotus, grown under saline and control conditions. Results from characterization of the physiological responses along with gene expression changes in roots and leaves allowed identifying several salt-tolerance mechanisms related to Ion transport and thermospermine synthesis. Differences were observed in putative H + /ATPases that allow transmembrane ionic transport and Chloride channel protein-like genes. Furthermore, an overexpression of thermospermine synthase found in the roots of tolerant plants may indicate that alterations in root architecture could act as an additional mechanism of response to salt stress. Conclusions Results indicate that D. lotus presents a genetic variability for salt tolerance trait related to the regulation of chloride transport, transmembrane electrochemical potential and thermospermine root synthesis. The study provides knowledge on mechanism of salt stress tolerance in persimmon for further breeding of tolerant persimmon rootstocks.


Abstract
Background Drought and salinity are two of the main challenges in agriculture. In many areas, crop production needs solutions to adapt the grown species to the increasing salinity. Research on physiological and molecular responses activated by salinity in plants is needed to elucidate mechanisms of salinity tolerance. Transcriptome pro ling (RNA-Seq) is a powerful tool to study the transcriptomic pro le of genotypes under stress conditions. In temperate fruit tree species, tree grafting on salinity tolerant rootstocks is a common method to compensate for the cultivar saline sensitivity. Persimmon species have different levels of tolerance to salinity, knowledge of this variability provides the basics for development of salt tolerant rootstocks.
Results In this study, we conducted a physiological and transcriptomic pro ling of roots and leaves in tolerant and sensitive plants of persimmon rootstock, Diospyros lotus, grown under saline and control conditions. Results from characterization of the physiological responses along with gene expression changes in roots and leaves allowed identifying several salt-tolerance mechanisms related to Ion transport and thermospermine synthesis. Differences were observed in putative H + /ATPases that allow transmembrane ionic transport and Chloride channel protein-like genes. Furthermore, an overexpression of thermospermine synthase found in the roots of tolerant plants may indicate that alterations in root architecture could act as an additional mechanism of response to salt stress.
Conclusions Results indicate that D. lotus presents a genetic variability for salt tolerance trait related to the regulation of chloride transport, transmembrane electrochemical potential and thermospermine root synthesis. The study provides knowledge on mechanism of salt stress tolerance in persimmon for further breeding of tolerant persimmon rootstocks.

Background
Drought and salinity are two of the main challenges in agriculture. Reduction of water availability and increase of salinity in soils and water reservoirs can limit crop production due to severe inhibition of plant growth and development via osmotic and ionic stresses (1,2). The negative effect of salinity in photosynthesis is caused either by a reduction in available water, by the toxic accumulation of ions in the cell, or both. Furthermore an attenuated photosynthesis leads to an increase in Reactive Oxygen Species (ROS) and a consequent oxidative damage of the cells (3). The cultivated surface affected by either stresses increases year after year as a consequence of more frequent drought episodes associated to climate change. Among the affected areas, the Mediterranean basin registered a signi cant increase in salinity in soils and water reservoirs in the last 10 years (4). Therefore, there is a pressing need to nd solutions to alleviate the negative impact on plant production in an area dominated by fruit tree crops.
Controlled crosses and selection (plant breeding) has proven an e cient method to develop new cultivars with improved characteristics such as salinity tolerance in several species (5)(6)(7)(8). In the case of temperate fruit trees, salinity stress can also be overcome by means of tree grafting on tolerant rootstocks (9). This method provides a solution for dealing with the two components of salinity stress: the osmotic effect, or water balance loss, through decreasing the area of stomatal aperture via phytohormonal root signalling (10,11) and ionic toxicity, by maintaining the ion homeostasis in the cell of the aerial parts (12).
Furthermore, grafting of existing varieties on rootstocks contributes to avoid losing the current genetic diversity.
Previous studies have contributed knowledge about the molecular mechanisms that regulate plant´s physiological responses to salinity in both model and economically important crops (13)(14)(15). Those mechanisms involve changes in gene expression associated to several biological processes such as ionic transport and exchange mechanisms (16), photosynthesis (17), avonoid biosynthesis (18), ROS scavenging and detoxi cation (19), ethylene production (20), signalling networks related to ABA (Abscisic Acid) (21) and protein refolding (22). Expression of different functional genes related to osmoregulation and cell protection as dehydrins and aquaporins are involved as well in salinity response (22)(23)(24).
One of the most prompt physiological response following exposure to saline environments is the differential regulation of growth water balance and uptake, and the activation of different cascades of molecular networks that result in the expression changes of speci c stress-related genes, and their encoded metabolites (25,26). Studies focused on model plants revealed the involvement of several transduction pathways in salt stress response, including SOS (Saline Overly Sensitive) (27), ABA (11) and ethylene signaling pathways (28). Additional cell wall changes result in differential growth responses required to adapt to saline environments (29). Signi cant amount of data concerns the role of plant mitogen-activated protein kinases (MAPKs) in osmoregulation. MAPK cascades are now recognized as major signal transduction mechanisms to regulate gene expression, multiple cell activities and protein functions in various developmental and adaptive processes (30), including salt stress (31). Ion toxicity caused by Na + and Cl − accumulation also affects plant photosynthesis, which can lead to leaf and stem necrosis. Mechanisms to limit Na + uptake and translocation are therefore linked to salinity tolerance.
Several genes have been identi ed in Arabidopsis, such as the Na + /H + antiporter responsible for the exclusion of sodium from the cytosol (32) in the SOS response pathway. In Arabidopsis thaliana, the serine/threonine type protein kinase SOS2 (CBL-interacting serine/threonine-protein kinase 24) interacts with the calcium sensor SOS3 (Calcineurin B-like protein 4) to regulate the Na + /H + antiporter SOS1, thereby conferring salt tolerance (33,34). Na + /K + homeostasis in poplar is regulated by CIPK (CBLinteracting serine/threonine-protein kinase) genes which interact with CBL1 (Calcineurin B-like protein 1) (35). The CIPK genes from wild barley and maize are also known to be implicated in salt tolerance responses (36,37). Furthermore, in tolerant persimmon species such as Diospyros virginiana, salt stress tolerance is believed to be caused by a high a nity potassium transporter HKT-1-like gene in roots (38), which was previously described in other species as key factor in salinity tolerance (39)(40)(41)(42). Although Na + ion translocation and regulation mechanisms have previously been reported in several species, little is known about Cl − transport and regulation. It has been suggested that Cl − uptake occurs mainly through passive water intake (2), although chloride channels are also suggested to be able to sequestrate Cl − ions (43). Chloride accumulation effect is also especially important in persimmon where it causes a decrease in fruit quality and a shorter post-harvest life (44).
In this study, the main objective is aimed at unravelling the mechanism of salinity tolerance in persimmon rootstock. Persimmon species have different levels of tolerance to salinity, making them an appropriate model to study the mechanisms of salinity tolerance, and to provide knowledge on persimmon speciesṕ otential as salt tolerant rootstock. Transcriptome pro ling (RNA-Seq) using next-generation sequencing technologies has proven a powerful tool to detect changes in gene expression associated to physiological responses to salt stress (45)(46)(47)(48)(49). In this study, we conducted physiological and RNA-Seq analyses of roots and leaves in persimmon rootstocks (Diospyros lotus) grown under saline and control conditions.

Results
Plant physiological responses associated to salt stress tolerance Plants exposed to saline treatment showed signi cant reduction in growth, increased leaf damage and defoliation, and altered ion concentration. Among the treated plants, those physiological responses were especially pronounced in a subset of plants which we hereafter categorized as sensitive to salinity ( Table   1). As compared to tolerant plants the sensitive ones showed a signi cantly higher Clconcentration.
Interestingly, no signi cant differences were observed for Na + accumulation, neither were differences signi cant for Na + /K + and Na + /Ca 2+ ratios. Moreover, P accumulation was more pronounced in tolerant plants. The overall reduction in carbon xation capacity and water potential due to salinity were signi cantly more pronounced in the sensitive plants.
The morphological differences between tolerant and sensitive plants were signi cant for the Relative Growth Rate (RGR) (Figure 1). The sensitive plants showed less RGR than the tolerant ones at the end of the saline treatment (day 60).

Gene expression changes in response to salinity
To visualise the gene expression pro ling results, a principal component analysis (PCA) was conducted using the transcriptomic data for all samples. In this analysis, a clear separation was observed between roots and leaves. Leaves showed little variability between the three categories ( Figure 2). However, the percentage of variability explained between control and treated roots was of the same order of magnitude as that between leaves and roots. Furthermore, the separation between tolerant and sensitive plants was also signi cant based on root transcriptomic data as shown in Figure 2. This separation was consistent with the phenotypic data presented above.
The comparison between expression in tolerant vs sensitive plants revealed 2901 differentially expressed genes. These genes were further ltered using the BLAST results and annotations to look for putative genes related to salinity tolerance. Additionally, an orthogonal design analysis was conducted to be able to differentiate gene expression differences speci c to roots or to leaves. The orthogonal analysis identi ed 1277 genes differentially expressed between tolerant and sensitive leaves (Supplementary gure 1). Also, 1395 genes were differentially expressed between tolerant vs sensitive roots (Supplementary gure 2).
Differentially expressed genes were divided according to several salt-tolerance mechanisms: ion transport, photosynthesis and respiration systems, and ROS detoxi cation. No water transport related genes were found differentially expressed. Ion transport related genes were divided according to each ion type ( Table 2). Among Ca 2+ transport related genes, a calcium ATPase-like gene was down-regulated in the leaves of sensitive plants as compared to those in tolerant plants, whereas the opposite was true for the roots. Furthermore, a calcium uniporter protein-like gene was found to be upregulated in the leaves of sensitive plants. Potassium channels SKOR and KAT3-like genes were downregulated in the leaves of sensitive plants, whereas a probable potassium transporter was upregulated in the leaves of sensitive plants. With regard to ionic channels, vacuolar cation/proton exchangers were upregulated in the leaves of sensitive plants. Other cation/proton antiporters did not show a clear response to salinity stress, as some isoforms had different expression patterns. On the other hand, chloride channel protein-like genes were upregulated in both leaves and roots of sensitive plants and downregulated in tolerant leaves. Phosphate, magnesium and boron transport-related genes were upregulated in tolerant roots. Phosphate, zinc and ammonium transport-related genes were also upregulated in tolerant leaves. Regarding ATPases, Plasma membrane ATPase-like genes were strongly upregulated in the roots of tolerant plants and downregulated in sensitive leaves. Furthermore, a V-type proton ATPase subunit G-like gene was strongly upregulated in leaves of tolerant plants as compared to the sensitive ones.
Include here Table 2 Regarding photosynthesis and respiration genes, all genes were downregulated in roots from all plants in comparison to the leaf expression levels (Table 3). Differentially expressed genes in leaves were all upregulated in the tolerant plants as compared to the sensitive ones, while the expression levels of the tolerant plants were similar to the control plants.
Log2 FC and adjusted p-value for genes associated to photosynthesis and respiration which are differentially expressed in tolerant vs sensitive plants. Colors represent relative differences in expression according to the read counts: red color is assigned to the lowest count value and blue color is assigned to the highest count value. Leaves and roots are represented by the columns with letters "L" and "R". Brown columns correspond to the relative mean expression in control plants, blue tolerant plants and red sensitive plants. Tissue column indicates tissue speci city of the tolerant vs sensitive expression Concerning the ROS detoxi cation related genes (Table 4), all except Thermospermine synthase ACAULIS5-like presented a higher expression in the leaves. Peroxiredoxin-like and peroxidase-like genes were upregulated in the tolerant plants except for one peroxidase-like that was upregulated in sensitive plants. Thermospermine synthase ACAULIS5-like was strongly upregulated in the roots of the tolerant plants and strongly downregulated in the leaves of the sensitive plants.
Log2 FC and adjusted p-value for genes associated to photosynthesis and respiration which are differentially expressed in tolerant vs sensitive plants. Colors represent relative differences in expression according to the read counts: red color is assigned to the lowest count value and blue color is assigned to the highest count value. Leaves and roots are represented by the columns with letters "L" and "R". Brown columns correspond to the relative mean expression in control plants, blue tolerant plants and red sensitive plants. Tissue column indicates tissue speci city of the tolerant vs sensitive expression. In our study, the total content of Na + and Clwas analysed and therefore a possible vacuolar compartmentalization could not be detected. However, we observed higher expression of all the analysed H + -ATPases in tolerant plants. This suggest the involvement of these genes in the compartmentalization mechanism of cell protection from Na + toxicity. In spite of that the responsible proteins of the removal of sodium ions from the cytoplasm into the apoplast or vacuole are the speci c plasma membrane Na + /H + antiporters, the activity of this protein depends on the electrochemical proton gradient between membranes. The H + -ATPases are the only proteins that are able to generate the adequate electrochemical proton gradient that allows these antiporters to perform the Na + extrusion (50). Furthermore, the upregulation of this protein under salt conditions has been previously reported (51-54) and its critical role on the Na + extrusion has been con rmed using transgenic plants (55,56).

Discussion
Another ion directly involved in damage caused by toxicity is Cl -, especially in the case of persimmon as previously shown by others (44,57). Previously, we detected differential chloride accumulation in the D. lotus population from the plants were obtained (58), however we could not link any previously measured parameter to these phenotype. In our study, we have observed a direct relationship between expression of chloride channel proteins and chlorine accumulation in leaf. Recently, chloride channels were proposed as key transporters of Clinto the higher parts of the plant (43). In persimmon, our results suggest that these channels might be playing a key role in avoiding high Clleaf accumulation in tolerant plants.

Photosynthesis, respiration systems and ROS signalling
A decreased metabolism in sensitive plants is supported both by the downregulation of photosynthetic and respiratory genes, and by a signi cant reduction in carbon xation. This effect might be caused by a combination of reduced transpiration and cell damage caused by the accumulation of toxic ions causing leaf necrosis and defoliation (2). ROS inactivation is also higher in both control and tolerant leaves an effect that probably leads to higher leaf ROS content in the sensitive plants. Although ROS can cause toxic effect in the leaves, it is a part of a signalling mechanism that may not be key to explaining the salinity tolerance, as an e cient ROS detoxi cation is essential for plant survival under natural conditions (2). ROS are known to trigger programmed cell death (59) and tissue necrosis and defoliation, which are believed to be a mechanism for protecting the most photosynthetically active cells (60).

Root architecture involvement
Under stress conditions, thermospermine synthesis and K + uptake are linked to the inhibition of lateral root formation (61,62). Although root architecture was not characterized in this study, transcriptomic data revealed thermospermine synthesis and K + transport activation by salinity in roots of tolerant plants, which suggests that root architecture may also play a role in the mechanism of tolerance to salt stress. Although previous reports have pointed out the association between reduced lateral root growth and sensitivity to saline stress trough better maintenance of Na + /K + ratio by accumulating Na + in the secondary root stele (63), other studies have pointed that in some cases, such as in seedlings, accumulation of Na + in the stele can cause lateral root quiescence or even lateral root primordia damage (64) and therefore reduced root development. In this line, D. virginiana, which is considered a salt-tolerant persimmon rootstock the root system is characterized by a well-developed taproot (65). D. lotus, a saltsensitive rootstock, is characterized by a brous root system as compared to D. virginiana that presents a well-developed taproot (66), this morphological differences are believed to play an important role in the salinity tolerance in Diospyros rootstocks (67). Our expression results indicate a possible association between root architecture and tolerance to salinity warrants further studies.

Conclusions
To sum up, in Diospyros lotus several mechanisms are present to overcome the saline stress. In this paper we could elucidate the ones that present variability within these species and therefore could be exploited for breeding new salt-tolerant rootstocks. At root level, thermospermine might play a role in root architecture, whose involvement in salt tolerance among Diospyros species has been previously pointed by other authors (67). On the other hand, chloride channel expression could facilitate Cl − transport inside the plant, and therefore cause higher Cl − accumulation in the higher parts of the plant. Finally, in the leaves, H + -ATPases seems to be crucial to be able to generate the electrochemical membrane gradient to allow Na + extrusion from the cytoplasm.

Plant material and RNA extraction
In this study we used a full-sib progeny of Diospyros lotus (DL) obtained from a cross between two D. lotus trees that belong to the Instituto Valenciano de Investigaciones Agrarias (IVIA) persimmon germplasm collection (accession number DK034). Identi cation of the plant material was made by the IVIA. Botany information and accessions are publicly available at https://www.gva.ivia.es. Seeds were strati ed in plastic bags lled with perlite in a cold chamber at 4 o C at the end of March. After 30 days of strati cation, seeds were transferred to peat-moss and perlite (4:1 ratio) and transferred to a greenhouse at 18-24ºC during two months (from April, 24, to June, 26, 2017).
One hundred and fty seedlings were transplanted into 1L pots lled with coarse sand. Plants were acclimatized in the greenhouse until apical meristem growth was observed. During this period, the plants were watered with a nutrient solution (3% Cristaljisa 18-18-18, soluble fertilizer with micronutrients). The plants were distributed randomly in the greenhouse to avoid possible biases. The plants were acclimated before exposition to salinity treatment. After acclimation, plants were exposed to saline stress treatment during 60 days. The saline stress consisted in adding 40 mM NaCl to the nutrient solution. Control plants remained watered with only nutrient solution.
Three of the plants were kept in control treatment, and six treated plants (three tolerant, three sensitive) seedlings were selected based on salt tolerance according to the visual and agro-morphological data, aspect of tolerant and sensitive plants are in supplementary gure 3.
The chosen plants were indeed extreme phenotypes from the same population analysis done in a previous work (58), therefore were chosen after an extensive population phenotyping.

Plant phenotyping
Plant phenotyping was carried out using the same measurement methods from a previous work (38). The morphological parameters total height (cm), leaves (no.), nodes (no.), internodes (cm) and defoliation (1no. leaves/no. nodes) were measured at days 0, 30, 45 and 60. Relative growth rate (RGR) was calculated as shown in the equation (1) Stem water potential (ψ H , MPa) was measured in fully expanded leaves using a Model 600 Schölander After saline treatment, three leaves from each plant were collected. Sample pre-treatments were performed as described previously (38). Na + , Ca 2+ , K + , Mg 2+ , P and S content was determined using a multiple-collector inductively coupled plasma mass spectrometry (MC-ICP MS, Thermo Finnigan Neptune). Chloride concentration was quanti ed by silver ion-titration (68) with a Corning 926 automatic chloridometer (Corning Ltd. Halstead Essex, UK).

RNA extraction
After 60 days of salt treatment young fully expanded leaves and root tip tissue were collected and immediately frozen and powdered using liquid nitrogen. Control samples from all populations were also collected and processed. The samples of each of the three individuals were mixed. The three biological replicates were made resampling and mixing new tissue samples. RNA was isolated according to Gambino et al. (2008). DNA was removed with the RNase-Free DNase Set (Qiagen, Valencia, CA, USA), using the RNeasy Plant Mini Kit (Qiagen). Puri ed RNA (500 ng) was reverse transcribed with PrimeScript RT Reagent Kit (Takara Bio, Otsu, Japan) in a total volume of 10 μl.

RNA Sequencing
RNA samples were sequenced by Novogene® using the following procedure: mRNA was enriched using oligo(dT) beads and fragmented randomly using a fragmentation buffer. cDNA was synthetized using random hexamers and reverse transcriptase. A second strand was generated using nick-translation with RNAse H and Escherichia coli Polymerase I with Illumina second strand synthesis buffer and dNTPs. AMPure XP beads were used to purify the cDNA and the rst strand was degraded with USER enzyme. Then, the resulting cDNA was puri ed, terminal repaired, A-tailed, ligated to the sequencing adapters, sizeselected and enriched with PCR. The resulting library was sequence using Illumina.