www.nature.com/scientificreports OPEN Non-uniform salinity in the root zone alleviates salt damage by increasing sodium, water and Received: 15 December 2016 nutrient transport genes expression Accepted: 25 April 2017 Published: xx xx xxxx in cotton Xiangqiang Kong, Zhen Luo, Hezhong Dong, Weijiang Li & Yizhen Chen Non-uniform salinity alleviates salt damage through sets of physiological adjustments in Na transport in leaf and water and nutrient uptake in the non-saline root side. However, little is known of how non-uniform salinity induces these adjustments. In this study, RNA sequencing (RNA-Seq) analysis shown that the expression of sodium transport and photosynthesis related genes in the non-uniform treatment were higher than that in the uniform treatment, which may be the reason for the increased photosynthetic (Pn) rate and decreased Na content in leaves of the non-uniform salinity treatment. Most of the water and nutrient transport related genes were up-regulated in the non-saline root side but down-regulated in roots of the high-saline side, which might be the key reason for the increased water and nutrient uptake in the non-saline root side. Furthermore, the expression pattern of most differentially expressed transcription factor and hormone related genes in the non-saline root side was similar to that in the high-saline side. The alleviated salt damage by non-uniform salinity was probably attributed to the increased expression of salt tolerance related genes in the leaf and that of water and nutrient uptake genes in the non-saline root side. It was estimated that 80 million hectares of the cultivated lands in the world were affected by soil salinity . Excessive soil salinity can cause ion toxicity, osmotic stress, water and nutrient deficiency and therefore rapid 2–5 reduction in growth of crops due to decreased photosynthesis . Maintaining ionic homeostasis, balancing root water uptake and leaf transpiration and increasing nutrient uptake are critical for plants to cope with saline envi- 4 + ronments . The extrusion of Na to the apoplast or external environment by salt overly sensitive (SOS) pathway + + proteins (SOS1, SOS2, and SOS3) or sequestration in vacuoles by vacuolar Na /H antiporters (NHX) are two + 4, 6, 7 efficient ways to protect cells from Na injury . Salinity induced water deficit is caused by the imbalance between root water uptake and leaf transpiration . Many studies suggested that plasma membrane intrinsic protein (PIP) aquaporins are involved in regulation of root hydraulic conductance (L ) under both osmotic and hydrostatic forces and therefore regulate whole root 9–11 water uptake . Under salt stress conditions, regulation of root water uptake is more crucial to overcome stress injury than that of leaf transpiration. The rate of root water uptake is ultimately regulated by aquaporin activ- ity and, to some extent, by suberin deposition . A decrease in L under saline conditions has frequently been 12–15 observed and the initial decrease in L upon salt exposure was correlated with a down-regulation of PIP genes . The decrease in L under salt stress might be a strategy to diminish water flow from roots to soil while the soil osmotic potential is lower than that of the roots . After few days of salt stress, a partial or total recovery of L alonged with accumulation of PIP proteins in roots has been reported in some species, which should be accom- 12, 15–17 panied by an osmotic adjustment of the root cells in order to avoid cell dehydration . Phytohormones play critical roles in regulating plant responses to stress . The contents of abscisic acid (ABA), ethylene (ET), jasmonic acid (JA), and cytokinins (CKs) as well as enzymes related to their biosynthesis exhibited Cotton Research Center, Shandong Key Lab for Cotton Culture and Physiology, Shandong Academy of Agricultural Sciences, Jinan, 250100, PR China. Correspondence and requests for materials should be addressed to H.D. (email: firstname.lastname@example.org) Scientific Repo R ts | 7: 2879 | DOI:10.1038/s41598-017-03302-x 1 www.nature.com/scientificreports/ Figure 1. Venn diagram of genes identified as up- (A) and down- (B) regulated in leaves and up- (C) and down- (D) regulated in roots under uniform and non-uniform salinity treatment. 18, 19 20 significant changes under salt stress . ABA is an important internal signal which can be induced by salt stress . Many ABA responsive transcription factors can be induced by ABA to promote expression of salt tolerant genes and therefore increase salt tolerance of plants . It is well known that ABA can increase root hydraulic properties 21–23 by increasing PIP expression and protein abundance . In most cases, ABA is correlated with the water poten- tial of leaf or soil, suggesting that salinity-induced increase in endogenous ABA is due to water dec fi it rather than specific salt effect . Like ABA and ET, JA biosynthesis have also been enhanced in plant under salt stress and 25–28 these activate many vital processes to cope with stress . On the contrary, salt stress decreased the expression of isopentenyltransferases (IPT) genes SlIPT3 and SlIPT4 in Tomato (Solanum lycopersicum L.) and overexpression of SlIPT3 increased salt tolerance of transgenic tomato . 29, 30 Plant response to salt stress varies greatly with soil environmental conditions . Soil salinity is oen h ft eter - ogeneous in saline fields, and many studies have shown that crops grow better in heterogeneous (non-uniform 29–34 salinity) conditions than in uniform salinity conditions . Non-uniform salinity has been simulated with a split-root system in a greenhouse or growth chamber, in which the root system was divided into two or more 30, 31 equal portions and each portion irrigated with varied concentrations of NaCl solution . Non-uniform salinity alleviated plant salt damage by decreasing Na concentration and osmotic stress in leaf, and increasing water and nutrient uptake by roots in the low-saline side and enhancing Na efflux from the low salinity root side via 33–36 SOS1 . However, the underlying molecular mechanism of the increased water and nutrient uptake in the low-salinity root side leading to alleviation of salt damage is far from clear. In the present study, using a split-root system to simulate non-uniform root zone salinity, we performed RNA-Seq on leaf and root samples of cotton plants under uniform- and non-uniform salinity treatments, and analyzed the global changes in the leaf and root of different treatments. The objectives were to investigate, (i) the mechanism of the improved plant growth and decreased leaf Na content under non-uniform salinity by analyz- ing the expression patterns of the sodium transport and photosynthesis related genes in leaves; (ii) the mechanism of increased water and nutrient uptake by roots in the non-saline root side by analyzing the expression patterns of the water and nutrient uptake related genes in roots and (iii) the expression patterns of the hormone related genes and transcription factor genes in the roots. Results RNA-Seq analysis and identification of differentially expressed genes. RNA-Seq analysis was per- formed on the leaves and roots of NaCl-free, uniform salinity and non-uniform salinity treatments at 6 h after salt stress (HAS). We generated more than 9.1 million raw tags in each library. Aer fi ft ltering out the low quality tags, we obtained clean tags ranging from 8.8 to 11.6 million per library (SRA submission number: SRP068502). The gene sequences of G . hirsutum genome were used as reference to align and identify the sequencing reads. This allowed for the mapping of approximately 80% of the distinct clean tags that passed our filters, representing more than 7.1 million reads per library with about 94% of them mapped unique reference genes (Supplemental Table S1). Putative differentially expressed genes were finally selected depending on the expression profiles and whether: (a) the average fold change between two treatment genes was more than or equal to two folds, and (b) the false discovery rate (FDR) was less than 0.001. Accordingly, 506 differentially expressed genes (DEGs) were identified in leaves under uniform salinity treatment, whereas only 131 DEGs were identified in non-uniform salinity treat- ment compared with NaCl-free control (Fig. 1A,B). There were 12 common up-regulated genes and 62 common Scientific Repo R ts | 7: 2879 | DOI:10.1038/s41598-017-03302-x 2 www.nature.com/scientificreports/ down-regulated genes in leaves of uniform and non-uniform salinity treatments (Fig. 1A,B). 474, 378 and 2725 DEGs were identified in roots under uniform salinity, non- and high-saline root sides (Fig. 1C,D). Functional classification of differentially expressed genes in leaves. Gene ontology (GO) analysis was performed by mapping each DEG into the records of the GO database (http://www.geneontology.org/). The GO annotation of DEGs in leaf showed that 24 groups, such as response to salt stress, chlorophyll catabolic pro- cess and response to ABA stimulus etc, were identified in uniform salinity treatment, whereas only 15 groups like photosynthesis, response to salt stress and ABA stimulus, etc. were identified in non-uniform salinity treatment (Supplemental Fig. S1). As shown in Supplemental Fig. S1, many groups related to response to oxidative stress, response to salt stress and response to ABA stimulus were identified in both non-uniform and uniform salin- ity treatments and these gene expression patterns were similar, though the gene numbers in each group under non-uniform treatment was lower than that under uniform salinity treatment. Interestingly, 3 DEGs related to photosynthesis and light harvesting were all up-regulated under non-uniform treatment, but most of genes related to catabolic process of light-harvesting complex II and chlorophyll were up-regulated under uniform treatment (Supplemental Fig. S1). Expression analysis of some important genes and Pn rate and ion contents in leaves under non-uniform and uniform salinity treatments. Twenty five DEGs were up-regulated under non-uniform treatment but down-regulated under uniform treatment (Table 1). The expression of the 3 photosynthesis-elated genes, Lhcb8, PsbA1 and PsbA2 increased in the leaves under non-uniform salinity but decreased in those under uniform salinity (Fig. 2A–C). The leaf Pn under non-uniform salinity was higher than under uniform salinity at 1 day after treatment (DAT) although the Pn significantly decreased under both uni- form and non-uniform salinity (Fig. 2D). To determine if sodium transport related genes in leaves of the non-uniform and uniform salinity treatments were up-regulated as described in RNA-Seq data and check their temporal expression patterns, the expression patterns of SOS1, SOS2, plasma membrane H ATPase (PMA1, PMA2), NHX1 and NHX6 were analyzed by real-time PCR at 3, 6, 9 and 24 HAS. The expression of these genes in both non-uniform and uniform leaves increased gradually after salt stress and most of them peaked at 6 HAS (Fig. 3). The expression of SOS1, SOS2, PMA2 and NHX2 in the non-uniform leaf was higher than that in the uniform leaf aer ft salt stress (Fig. 3A,B,D and F). The expression of PMA1 in the non-uniform leaf was higher than that in the uniform leaf at 6, 9 and 24 HAS (Fig. 3C). The expression of NHX1 in the non-uniform leaf was higher than that in the uniform leaf at 24 HAS (Fig. 3E). The leaf Na content under non-uniform salinity was significantly lower than that under uniform salinity though salt stress increased the Na content under both uniform and non-uniform salinity at 1 DAT (Fig. 3G). In contrast, the leaf K content under non-uniform salinity was significantly higher than that under uniform salinity (Fig. 3H). Functional classification of differentially expressed genes in root. e GO a Th nnotation of DEGs in root is presented in Fig. 4. The main functional groups related to salt stress, oxidative stress, water deprivation, etc. were up-regulated in high-saline root side of non-uniform salinity treatment (Fig. 4A). Most of the genes related to ABA, ET and JA mediated signaling pathway and response to ABA, ET and JA stimulus and ethylene biosynthetic process were also up-regulated in the high-saline root side of the non-uniform salinity treatment (Fig. 4A). However, the main functional groups of down-regulated genes were related to response to nitrate, nitrate transport, cellular response to iron ion starvation, iron ion transport and water transport (Fig. 4A). Many up-regulated genes in the high-saline root side, such as response to ABA and ET stimulus, were still up-regulated in the uniform salinity root and non-saline root side (Fig. 4). Interestingly, most of the genes related to nitrate transport, iron transport and water transport were down-regulated in both root sides of uniform salinity treatment and the high-saline root side of non-uniform salinity treatment, but most of these were up-regulated in the non-saline root side under non-uniform salinity (Fig. 4). Surprisingly, the DEGs related to cellular responses to nitric oxide, ET stimulus, iron, zinc ion trans- membrane transport, oxidative phosphorylation, hydrogen peroxide transmembrane transport, ET biosynthetic process, response to phosphate starvation and ABA mediated signaling pathway were all up-regulated in the non-saline root side (Fig. 4C). Differential expression of nutrient transport genes and nutrient uptake in roots under non-uniform and uniform salinity treatments. Analysis of expression level of nutrient transport genes in roots of uniform and non-uniform salinity showed 13 nitrate, 7 potassium and 10 phosphate transport-related genes in the RNA-Seq data. Ten of the 13 nitrate transport-related genes were down-regulated in the high-saline root side and uniform salinity root (Table 2). Interestingly, 7 nitrate transport-related genes which were down-regulated in the high-saline root side were up-regulated in the non-saline side root except the other 3 up-regulated nitrate transport-related genes in all treatment roots (Table 2). The 7 potassium transport-related genes were all down-regulated in the high-saline root side and uniform salinity root and the expression of these genes were all lower than that in non-saline root side (Table 2). There were 10 phosphate transport-related genes which were up-regulated in all treatment roots (Table 2). A net NO influx was observed in cotton roots under both NaCl-free and salt stress conditions, but the net influx in the high-saline root side and the uniform salinity root were significantly lower than that in the NaCl-free control (Fig. 5A). However, the net NO influx in the non-saline root side was significantly higher than that in the NaCl-free control (Fig. 5A). A net NH influx in roots of the NaCl-free and non-saline root side was also observed but the net influx in the non-saline side was higher than that in the NaCl-free control (Fig. 5B). The net NH flux were reversed to efflux in either root side under uniform salinity and in the high-saline root side under Scientific Repo R ts | 7: 2879 | DOI:10.1038/s41598-017-03302-x 3 www.nature.com/scientificreports/ Gene ID (Cotton_D_gene_) Log [(100/100)/(0/0)] Log [(0/200)/(0/0)] Gene annotation 2 2 10022083 −2.383 1.220 Fasciclin-like arabinogalactan protein 19 [Gossypium hirsutum] 10014653 −1.620 1.488 Fasciclin-like arabinogalactan protein 19 [Gossypium hirsutum] GDSL-like Lipase/Acylhydrolase superfamily protein [e Th obroma 10031440 −1.072 1.174 cacao] GDSL-like Lipase/Acylhydrolase superfamily protein [e Th obroma 10031813 −0.986 1.067 cacao] 10008000 −2.239 1.306 Li-tolerant lipase 1 isoform 1 [Theobroma cacao ] 10015446 −1.824 0.774 Cu-predoxin superfamily protein [Theobroma cacao ] 10010133 −1.669 1.509 Proline-rich protein [Gossypium hirsutum] 10005614 −1.557 1.737 HCO -transporter family isoform 1 [Theobroma cacao ] 10033947 −1.023 1.429 SKU5 similar 5 isoform 1 [Theobroma cacao ] 10023571 −1.375 1.142 SKU5 similar 5 isoform 1 [Theobroma cacao ] 10010899 −1.683 0.818 Predicted protein [Populus trichocarpa] 10033759 −1.191 1.034 Uncharacterized protein TCM_029927 [Theobroma cacao ] 10040515 −1.314 1.047 Uncharacterized protein TCM_000740 [Theobroma cacao ] 10021939 −1.127 1.137 Polygalacturonase 2 [Theobroma cacao ] 10001543 −1.297 0.769 Beta-tubulin 1 [Gossypium hirsutum] 10032154 −1.139 0.708 Beta-tubulin 2 [Gossypium hirsutum] 10039398 −1.112 0.901 Alpha-tubulin [Gossypium hirsutum] 10001239 −0.479 1.003 Light-harvesting complex II protein Lhcb8 [Theobroma cacao ] 10014103 −0.752 1.653 PsbA1 [Cardiandra alternifolia] 10015895 −0.578 1.471 PsbA2 [Dianthus versicolor] 10021012 −0.639 1.853 Xyloglucan endotransglucosylase/hydrolase 16 [Theobroma cacao ] 10022459 −0.552 1.332 Xyloglucan endotransglucosylase/hydrolase [Gossypium hirsutum] 10001483 −1.019 0.678 Xyloglucan endotransglucosylase/hydrolase [Gossypium hirsutum] 10025800 −1.5 0.671 Cytochrome P450, putative [Theobroma cacao ] 10036871 −2.854 2.819 Gibberellin-regulated family protein, putative [Theobroma cacao ] Table 1. Summary of differentially expressed genes in leaves which were significantly up-regulated in non- uniform treatment but down-regulated in uniform treatment or vice-versa. non-uniform salinity (Fig. 5B). A net K influx was observed in the non-saline root side under non-uniform salinity, but net K flux under the NaCl-free treatment, in either root side under uniform salinity, and the high-saline root side under non-uniform salinity was reversed to efflux (Fig. 5C). Differentially expressed aquaporin genes and L in roots under non-uniform and uniform salin- ity treatments. There were 27 die ff rentially expressed aquaporin genes, of which 24 were down-regulated in the high-saline root side and 21 down-regulated in the uniform salinity root compared with NaCl-free control (Table 3). Unlike the high-saline root side, most of the aquaporin genes (18) were up-regulated in the non-saline root side (Table 3). The expression levels of the 24 genes down-regulated in the high-saline root side were higher in the non-saline root side than that in the high-saline and uniform salinity roots, with 15 of the genes up-regulated in the non-saline root side (Table 3). The 3 up-regulated genes in the high-saline root side occurred in the non-saline root side and uniform salinity roots (Table 3). Consistent with the decreased expression of aqua- porin genes, the L in either root side of the uniform salinity and high-saline root side of the non-uniform salinity treatment also decreased, but the L of the non-saline root side under non-uniform salinity increased by 116.4% compared with NaCl-free control (Fig. 5D). Differentially expressed hormone related and transcription factor genes in roots. Four IPT genes were significantly down-regulated in the high-saline root side and uniform salinity root and their expres- sion was lower than that in the non-saline root side (Supplemental Table S2). Four 9-cis-epoxycarotenoid diox- ygenase (NCED) genes were up-regulated in roots of uniform and non-uniform salinity treatment, and their expression was higher than that in the non-saline root side (Supplemental Table S2). In contrast, the other 3 ABA biosynthesis aldehyde oxidase (AAO) genes were all down-regulated in all uniform and non-uniform salinity roots and their expression in the high-saline root side and uniform salinity root was lower than in the non-saline root side (Supplemental Table S2). Surprisingly, the expression of 4 ABA catabolic genes CYP707A in the high-saline root side and uniform salinity root were increased, being higher than in the non-saline root side, although 2 CYP707A genes were also up-regulated in the non-saline root side. As for the 5 differentially expressed ethylene biosynthesis genes ACC oxidase (ACO), 4 of them were up-regulated in all uniform and non-uniform salinity roots and their expressions in the high-saline root side were higher than in non-saline root side (Supplemental Table S2). e Th re are a large number of transcription factors (TFs) in plants to perceive and mediate responses to environ - mental changes which act as the earliest and vital players during stresses. We found 47, 16 and 144 up-regulated Scientific Repo R ts | 7: 2879 | DOI:10.1038/s41598-017-03302-x 4 www.nature.com/scientificreports/ Figure 2. e exp Th ression patterns of Lhcb8 (A), PsbA1 (B) and PsbA2 (C) in main-stem leaves under uniform (100/100 mM NaCl) and non-uniform (0/200 mM NaCl) salinity treatments and net photosynthetic (Pn) in leaves at 1 day aer s ft alinity treatments. Data are means of six biological replicates (±SD). Different letters indicate a significant difference (P < 0.05) within each panel. TFs and 41, 6 and 105 down-regulated TFs in roots under uniform salinity, non- and high-saline root side (Supplemental Table S3). The expression pattern of the die ff rentially expressed NAC, WRKY, GRAS, MYB and Nuclear Y subunit TFs in the non-saline root side were similar to that in the high-saline root side and uniform salinity root (Supplemental Table S4). There are only 4 ERF TFs which have similar expression pattern in the non- and high-saline root sides though 9 differentially expressed ERF TFs were found (Supplemental Table S4). Confirmation of Solexa Expression Patterns by RT-PCR Analysis. To validate the results of the gene expression analysis obtained by RNA-Seq, RT-PCR analysis was performed for a subset of 9 genes in leaf and 11 genes in root as identified by RNA-Seq. The results showed that 48 of the 51 gene expression data had similar expression profiles as the original RNA-Seq (Supplemental Table S5). This indicates that the original data of RNA-seq was validated in 94.1% of the cases. This was not the case for the other gene presumably because the RNA used for RNA-seq and RT-PCR was extracted from different plants. The expression patterns of the 20 genes were highly consistent with the RNA-seq ratios, with a relative R of 0.8215 (Supplemental Fig. S2, Supplemental Table S5). Discussion Salt stress caused ion toxicity, osmotic stress and nutrient deficiency and thus affected plant growth by up- or 4, 5, 37, 38 down-regulating many salt-related genes in cotton . In our study, 506 DEGs were identified in the leaf under uniform salinity, whereas only 131 DEGs were identified under non-uniform salinity. The results sug- gested that plants under non-uniform salinity suffer less salt stress than those under uniform salinity. Salt stress 34, decreased leaf photosynthesis, and many genes involved in the photosynthesis pathway were down-regulated 37–39 . It was reported that total energy gain and plant growth decreased with greater salinity stress by decreasing photosynthetic rate following induced damage to cellular and photosynthetic machinery . Our data showed that many genes involved in photosynthesis were down-regulated in leaves under both uniform and non-uniform salinity treatments, but the number of down-regulated genes under non-uniform treatment was lower than that under uniform treatment. These results may explain the increased plant growth under non-uniform. Most of chlorophyll and light-harvesting complex II catabolic process related genes which have negative function on photosynthesis were up-regulated in leaves under uniform salinity treatment. However, the positive genes Lhcb8, PsbA1 and PsbA2 on photosynthesis were up-regulated in the non-uniform salinity treatment, which may be the reason for the increased Pn relative to the uniform salinity treatment (Fig. 2; Table 1). Scientific Repo R ts | 7: 2879 | DOI:10.1038/s41598-017-03302-x 5 www.nature.com/scientificreports/ Figure 3. e exp Th ression patterns of SOS1 (A), SOS2 (B), PMA1 (C), PMA2 (D), NHX1 (E) and NHX2 (F) in leaves under uniform (100/100 mM NaCl) and non-uniform (0/200 mM NaCl) salinity treatments and Na (G) and K (H) contents in leaves at 1 day aer s ft alinity treatments. Data are means of six biological replicates (±SD). Different letters indicate a significant difference (P < 0.05) within each panel. Salt stress induced Fasciclin-like arabinogalactan, Li-tolerant lipase, Xyloglucan endotransglucosylase/hydro- lase and Cytochrome P450 genes play important roles in plant salt tolerance. Their overexpression increased the 41–45 salt tolerance of transgenic plants . In this study, 25 down-regulated genes under uniform salinity treatment, which included the genes mentioned above, were up-regulated under non-uniform salinity. The increased expres- sion of these genes may contribute to the increased salt tolerance and hence decreased salt damage under the non-uniform salinity (Table 1). Maintaining ionic homeostasis is critical for plant to cope with saline environments. SOS pathway proteins + + and H -ATPase can be induced to transport Na out of the cytoplasm while NHXs can also be induced to seques- + 4, 7, 46–49 ter Na in the vacuole to reduce ionic toxicity in plant leaves under salt stress . The expression of SOS1, SOS2, PMA1, PMA2, NHX1 and NHX6 genes in leaves were all up-regulated under salt stress and the expression of most of these genes was higher under non-uniform than uniform salinity. This may be an important reason for the reduced leaf Na content and salt damage under non-uniform salinity (Fig. 3). The high expression of these sodium related genes in the non-uniform salinity may be ascribed to some signals originating from the high-saline root side, implying that the high-saline root can induce some important salt tolerant genes to increase the salt tolerance of cotton. Roots play a primary role in particular changes occurring in plants because they are directly in contact with 18, 50 the soil and absorb water and other essential nutrients from the soil . Root systems have important roles in improving crop salt tolerance through increasing water and nutrients uptake and limiting salt acquisition, although salt stress limits water and nutrient uptake by roots . Aquaporin proteins, which regulate a large propor- tion of water transport across membranes, are rapidly influenced both transcriptionally and post-translationally 52 53–55 by salt . Moreover, many studies have shown that the uptake of water by roots is mainly mediated by PIPs . Fetter et al. found that co-expression of PIP1s and PIP2s in Xenopus laevis oocytes led to an increase in the osmotic water permeability coefficient (Pf ) and the increased Pf was attributable to the formation of tetramers Scientific Repo R ts | 7: 2879 | DOI:10.1038/s41598-017-03302-x 6 www.nature.com/scientificreports/ Figure 4. GO analysis of differentially expressed genes in roots of the high-saline root side (0/200-200) (A), uniform-salinity treatment (100/100-100) (B) and non-saline root side (0/200-0) (C) obtained from RNA sequencing. The abscissa of the bar plot represents the gene count within each GO category. All processes listed had enrichment p values < 0.05. by PIP1 and PIP2 proteins. In the present study, most water transport related genes were down-regulated in uniform-salinity root and the high-saline root side but most of these were up-regulated in non-saline root side, which was parallel to our previous study that water uptake decreased from the uniform- and high-saline root side but increased in the non-saline root side (Fig. 4). As shown in Table 3, 24 of the 27 differentially expressed aquaporin genes in the high-saline root side were down-regulated, whereas most of these were up-regulated in the non-saline root side. The root L under uniform salinity and high-saline side decreased but that in the non-saline side increased (Fig. 5D). These results suggested that the increased water uptake may be due to the increased L as measured by increased expression of aquaporin genes in the non-saline root side. The increased water uptake in the non-saline root side may decrease osmotic stress and then alleviate salt damage under non-uniform salinity. Plant growth can be adversely ae ff cted by salinity-induced nutrient imbalance through changes in nutrient availability, competitive uptake, transport or partitioning within the plant . Nutrient uptake by active transport through the roots is the first major step to enhance nutrient use in any plant. Many studies have shown that salin- ity can directly ae ff ct nutrient uptake, such as reducing N, P and K uptake and decreasing the expression of high 4, 57–59 affinity nitrate transporters, AtNRT2.1 and AtNRT2.2 . Our previous study has shown that the non-saline root side uptakes more nutrients than the high-saline root side under non-uniform salinity . In this study, most of the differentially expressed genes related to nitrate, potassium and phosphate transport were up-regulated in the non-saline root side, but most were down-regulated in the high-saline root side and uniform salinity treatment (Fig. 4; Table 2). The net NO influx in the non-saline side root was significantly higher than in the high-saline root side under non-uniform salinity and in either root side under uniform salinity. Similarly, the + + net NH and K influx in roots of the non-saline root side were higher than in the high-saline side and the + + uniform salinity root because the net NH and K flux were reversed to efflux in the uniform salinity root and − + + high-saline side root aer s ft alt stress (Fig. 5A–C). The increased NO , NH and K influx in the non-saline side 3 4 root may be due to the increased expression of nutrient transport related genes, which possibly contributed to the increased nutrient uptake in the non-saline root side under non-uniform salinity. The increased nutrient uptake in the non-saline root side under non-uniform salinity mitigated nutrient deficiency and thus alleviated salinity damage. Scientific Repo R ts | 7: 2879 | DOI:10.1038/s41598-017-03302-x 7 www.nature.com/scientificreports/ Gene ID Log [(100/100- Log [(0/200- Log [(0/200- 2 2 2 Nutrition (Cotton_D_gene_) 100)/(0/0-0)] 0)/(0/0-0)] 200)/(0/0-0)] Gene annotation 10000609 −1.748128 0.430718 −2.86098 Nitrate transporter 1.1 [Theobroma cacao ] 10006958 −1.808451 −0.6115 −4.71918 Nitrate transporter 2:1 [Theobroma cacao ] 10008863 1.1008006 0.291231 3.14439 Nitrate transporter 1:2 [Theobroma cacao ] 10009751 −1.359804 0.25331 −4.77721 Nitrate transporter 1.1 [Theobroma cacao ] 10009753 −0.933707 0.737786 −2.98314 Nitrate transporter 1.1 [Theobroma cacao ] 10014248 −0.156504 0.114736 −2.19127 Nitrate transporter 1.7 [Theobroma cacao ] Nitrate 10019505 −0.6206 0.235432 −2.13213 Nitrate transporter [Arabidopsis thaliana] 10024700 1.1300605 0.442005 3.169925 Nitrate transporter 1.5 [Theobroma cacao ] 10032251 −1.007627 −0.4728 −2.90436 Nitrate excretion transporter 1 [Theobroma cacao ] 10032252 −1.430634 −0.47038 −3.09622 Nitrate excretion transporter 1 [Theobroma cacao ] 10033454 1.4188291 0.074001 0.185032 Nitrate transporter 1:2 [Theobroma cacao ] 10022762 −0.269833 0.468746 −1.6487 Nitrate transporters [Theobroma cacao ] 10037760 −0.859288 0.073784 −6.15915 Nitrate transporter 1.5 [Theobroma cacao ] 10008417 −0.1256 0.23249 −1.2006 Potassium uptake transporter 3 [Theobroma cacao ] 10016252 −0.75473 −0.60145 −2.31438 Potassium transporter 2 [Theobroma cacao ] 10016708 −0.520023 0.055607 −2.1062 Potassium transporter 2 [Theobroma cacao ] 10018786 −0.295198 0.095238 −2.91544 Potassium transporter [Theobroma cacao ] Potassium Potassium transporter family protein 10027906 −1.236198 −0.42094 −1.87055 [Theobroma cacao ] 10033349 −0.321 0.7571 −2.5697 High affinity K + transporter 5 [Theobroma cacao ] 10026743 −0.573044 0.099487 −1.38229 Potassium channel in 3 [Theobroma cacao ] 10022858 4.9166667 1.916667 13.9 Phosphate transporter 3,1 [Theobroma cacao ] 10021985 0.7352941 1.303922 0.235294 Phosphate transporter 2,1 [Theobroma cacao ] 10021898 1.1921397 0.633188 1.864629 Phosphate transporter 1,4 [Theobroma cacao ] 10024110 0.7725118 0.808057 0.279621 Phosphate transporter 4,3 [Theobroma cacao ] 10010804 0.4825397 0.76 0.777778 Phosphate transporter 3,1 [Theobroma cacao ] Phosphate 10036742 0.7368421 0.763158 0.052632 Phosphate transporter 1,9 [Theobroma cacao ] 10002982 −0.1568 0.1263 −2.83636 Phosphate transporter 1,7 [Theobroma cacao ] 10014884 1.2432432 0.297297 22.27027 EXS family protein [Theobroma cacao ] 10022222 0.5428571 0.819048 0.2 EXS family protein [Theobroma cacao ] 10040038 0.677792 0.782413 0.273427 Phosphate 1 [Theobroma cacao ] Table 2. e exp Th ression pattern of nitrate, potassium and phosphate transport genes in roots under uniform- and non-uniform salinity treatments. It is well known that ABA modifies root hydraulic properties by increasing L , PIP aquaporin expression and 21–23 protein abundance . The increased expression of NCED genes and decreased expression of ABA catabolic genes CYP707A may increase ABA content in the non-saline root side, which may be used as an important pos- itive signal to increase PIP aquaporin expression and then increase water uptake from the non-saline root side (Supplemental Table S2). Transcription factors are known to play vital roles in abiotic stress signaling in plants. Genome-wide tran- scriptome analysis revealed that a number of TFs were induced or repressed in response to abiotic stresses in 38, 60–62 cotton . In this study, 144 up-regulated and 105 down-regulated TFs were identified in the high-saline root side and 16 up-regulated and 5 down-regulated TFs were identified in the non-saline root side. Most of the TFs in NAC, ERF and WRKY families were up-regulated in the high-saline root side and 6 NAC, 4 ERF and 1 WRKY genes were induced in the non-saline root side. The results suggested that these genes may play important roles in cotton salt tolerance as reported previously . Plant roots are the first tissues to encounter salt or other osmotic stresses. Alterations in root hormones like CK, ABA, ET and IAA, could mediate root-to-shoot signaling in regulating shoot growth and physiology, and 64, 65 ultimately agricultural productivity . Root-specific induction of IPT gene could enhance root-to shoot cyto- 64, 65 kinin signaling, and thus delayed leaf senescence and improved plant growth . Plant root plasticity to fluctu- ating environments is a primary mechanism for optimizing water and nutrient acquisition through enhanced uptake/assimilation systems, and proliferation specifically in nutrient-rich zones depends on the integration of local and systemic signaling . Studies in systemic N signaling using a split-root system showed that root growth 66 67 under low N condition is controlled by the signal from the shoot . Recently, Suzuki et al. reported that tempo- ral–spatial interaction between reactive oxygen species (ROS) and ABA could regulate rapid SAA to heat stress in plants. These results suggests that systemic acquired acclimation (SAA) plays a key role in plant survival during stress. In our study, the increased expression of water and nutrient transport and hormone related genes, such as IPT, NCED and ACC in the non-saline root side may serve as positive signals from root to enhance cotton growth under the non-uniform salinity treatment. On the one hand, the enhanced expression of sodium related genes Scientific Repo R ts | 7: 2879 | DOI:10.1038/s41598-017-03302-x 8 www.nature.com/scientificreports/ Figure 5. Effects of non-uniform (0/200 mM) and uniform (0/0 and 100/100) root zone salinity on net NO + + (A), NH (B) and K (C) fluxes and Hydraulic conductance (L ) (D) in roots of cotton at 6 h aer t ft reatment. 4 p − + + e d Th ata are main fluxes of NO , NH and K within the measuring periods (15 min). Data are means of six 3 4 biological replicates (±SD). Bars with different letters (a, b, and c) differ significantly at P < 0.05. in the shoot may be induced by the high-saline root side; On the other hand, the increased expression of water and nutrient transport genes, TF and hormone related genes may be induced by some signals from the shoot. It seemed that both root-to-shoot and shoot-to-root signals were required to explain the decreased salt damage under non-uniform salinity. Materials and Methods Plant material preparation and salinity treatment. A commercial cotton (Gossypium hirsutum L.) cultivar, K836 developed by the Cotton Research Center, Shandong Academy of Agricultural Sciences, Jinan, was used in the experiment. Acid-delinted seeds were sown at ~3 cm depth in plastic boxes (60 cm × 45 cm × 15 cm) containing sterilized wet sand. The boxes were placed in growth chambers with light/dark regimes of 16/8 h, light −2 −1 intensity of 400 μmol m s PAR, and temperature of 30 ± 2 °C. At full emergence, seedlings were thinned to 100 plants per box. When most seedlings reached the two-true leaf stage, uniform seedlings were carefully removed from the sand and washed with distilled water. Split-root systems were established by grafting with these seedlings as described in Kong et al. . Grae ft d seedlings were transferred to plastic pots containing aerated nutrient solution. e Th solu- tion consisted of (mM): 1.25 Ca(NO ) , 1.25 KNO , 0.5 MgSO , 0.25 NH H PO , 0.05 EDTA-FeNa; and (μM): 3 2 3 4 4 2 4 10 H BO , 0.5 ZnSO , 0.1 CuSO , 0.5 MnSO , 0.0025 (NH ) Mo O , and was adjusted to pH 6 with KOH. When 3 3 4 4 4 4 6 7 24 a new leaf emerged from the grae ft d seedling 2 weeks ae ft r grafting, the plastic bag and parafilm were removed. Grae ft d seedlings with two uniform split-root systems were transferred to the greenhouse to grow under a 14/10 h (light/dark) photoperiod at 30/26 °C and relative humidity of 60/80% for 30 d. Nutrient solutions were renewed daily during the period of growth. Healthy seedlings with uniform split roots were selected for further study. Large plastic boxes (26 cm × 16 cm × 15 cm) were used for the uniform and non-uniform salinity treatment. eir inn Th er space was divided into two equal parts by standing a plastic board in the middle of the boxes to pro- duce a split-root box. The water in one side of the split-root box cannot flow into the other. Healthy seedlings with uniform roots were selected and each root portion was put into one side of the boxes. u Th s the two root portions of each seedling were exposed to different NaCl concentrations at the same time. The two root portions under different salt (0 and 200 mM NaCl) treatment were denoted as non-uniform salinity treatment (0/200 mM). Treatment with the two root portions in 0 mM NaCl was denoted as NaCl-free control (0/0 mM NaCl) and treat- ment with the two root portions in 100 mM NaCl was denoted as uniform salinity treatment. In the non-uniform salinity treatment (0/200 mM), the NaCl-free side was considered the non-saline root side (0 or 0/200-0), while the 200 mM side was considered the high-saline root side (200 or 0/200-200); 0/0-0 and 100/100-100 denoted the root of NaCl-free and uniform salinity treatment. For each treatment, three biological replicates were designed; the samples of each biological replicate were pooled from 10 plants, the plants being randomly selected to avoid Scientific Repo R ts | 7: 2879 | DOI:10.1038/s41598-017-03302-x 9 www.nature.com/scientificreports/ Gene ID Log [(100/100- Log [(0/200- Log [(0/200- 2 2 2 (Cotton_D_gene_) 100)/(0/0-0)] 0)/(0/0-0)] 200)/(0/0-0)] Gene annotation 10009326 −0.33267 0.424425 −2.42742 PIP protein [Gossypium hirsutum] 10004201 −0.39015 0.379398 −3.7498 TPA: TPA_inf: aquaporin TIP1;4 [Gossypium hirsutum] 10018179 −2.21045 −0.06931 −1.42916 PIP protein [Gossypium hirsutum] 10006595 −0.07887 1.016425 −2.24595 Plasma membrane intrinsic protein 2,4 [Theobroma cacao ] 10038574 −0.76512 −0.16798 −1.7053 aquaporin PIP2;3 [Gossypium hirsutum] 10008252 −2.14325 0.392041 −2.23742 Tonoplast intrinsic protein 2,3 [Theobroma cacao ] 10020312 −1.24614 −0.34652 −1.91007 aquaporin SIP1;7, partial [Gossypium hirsutum] 10019562 −2.07288 −0.73534 −1.05041 Plasma membrane intrinsic protein 2A [Theobroma cacao ] 10015612 −0.30775 −0.19935 −1.13458 aquaporin TIP2;3 [Gossypium hirsutum] 10022627 −0.35396 0.049896 −2.02539 NOD26-like intrinsic protein 5,1 [Theobroma cacao ] 10032274 −0.25704 0.23041 −1.83799 Plasma membrane intrinsic protein 2,4 [Theobroma cacao ] 10009738 0.610744 0.871895 −1.49557 Tonoplast intrinsic protein 2,3 [Theobroma cacao ] 10032221 −0.0926 0.802992 −3.08491 Tonoplast intrinsic protein 1,3 [Theobroma cacao ] 10004979 −0.7341 0.819265 −2.10376 Tonoplast intrinsic protein 1,3 [Theobroma cacao ] 10036429 −2.88826 −0.65004 −2.97082 TPA: TPA_inf: aquaporin TIP4;1 [Gossypium hirsutum] 10024645 0.937983 0.460841 2.726458 PIP1 protein [Gossypium hirsutum] 10009325 0.219162 0.54999 −2.02131 PIP protein [Gossypium hirsutum] 10025289 −1.62286 −0.05523 −3.17146 aquaporin TIP1;7 [Gossypium hirsutum] 10004995 −1.77047 −0.45767 −1.64657 TPA: TPA_inf: aquaporin TIP1;5 [Gossypium hirsutum] 10001242 −0.27999 0.224264 −1.46605 aquaporin PIP1;11 [Gossypium hirsutum] 10009324 −0.28816 0.216449 −1.90118 PIP protein [Gossypium hirsutum] 10001728 −1.49766 −0.37941 −1.93193 tonoplast intrinsic protein [Gossypium hirsutum] 10014030 −0.36778 1.063372 −2.44511 Plasma membrane intrinsic protein 2,4 [Theobroma cacao ] putative plasma membrane intrinsic protein PIP family 10024472 −0.67203 0.873597 −2.03637 member 1 aquaporin [Pachira quinata] 10006930 −0.6799 0.72487 −1.47373 PIP protein [Gossypium hirsutum] 10031445 0.934751 0.169815 2.755901 TPA: TPA_inf: aquaporin NIP1;1 [Gossypium hirsutum] 10001135 0.356561 0.381873 1.700648 Aquaporin sip2.1, putative [Theobroma cacao ] Table 3. e exp Th ression patterns of aquaporin genes in roots under uniform and non-uniform salinity treatments, which were significantly up- or down-regulated in the high-saline root sides. any potential effects of position within the greenhouse. Aer 6 ft h of treatment, leaves in the NaCl-free, uniform and non-uniform treatment and roots of the NaCl-free (0/0-0), uniform treatment (100/100-100), non-saline (0/200-0) and high-saline root sides (0/200-200) of the non-uniform treatment were sampled, washed 3 times with distilled water, and then frozen in liquid nitrogen and stored at −80 °C for use. RNA Extraction, DGE sequencing and analysis. Total RNA was extracted using the TRIzol reagent (Invitrogen), and mRNA was isolated from total RNA using Dynabeads Oligo (dT) (Invitrogen Dynal), following the manufacturer’s instructions. For RNA-Seq, total RNA from 10 representative individual plants of each treat- ment was mixed into one biological replicate. Approximately, 8 μg of total RNA was used. Tag libraries were pre- pared using the Illumina Gene Expression Sample Prep Kit, following the manufacturer’s protocol, as described in 68 TM Luan et al. . The libraries were then sequenced using an Illumina HiSeq 2500 with 50-bp single-end (SE) reads each. The genome of G . hirsutum (ftp://ftp.ncbi.nih.gov/repository/unigene/Gossypium_hirsutum/Ghi.Seq.uniq. gz) was used as reference sequence to align and identify the sequencing reads. To map the reads to the reference, the alignments and the candidate gene identification procedure were conducted using the mapping and assembly with qualities software package . Clean tags mapping to reference sequences from multiple genes were filtered out, and the remaining clean tags were designated as unambiguous clean tags. For gene expression analysis, the number of unambiguous clean tags for each gene was calculated and then normalized to TPM (number of tran- 70, 71 scripts per million clean tags) . Identification of differentially expressed genes and Functional analysis. To identify DEGs across the 3 leaf and 4 root samples, pair wise comparisons among the 3 leaf and 4 root samples were performed using a rigorous algorithm method based on a previous method . The DEGs were obtained aer fi ft ltering using a thresh- old FDR of ≤0.001 and an absolute value of log Ratio ≥2. GO enrichment analysis was performed for functional categorization of DEGs using agriGO software and the P -values corrected by applying the FDR correction to control falsely rejected hypotheses during GO analysis . e Th pathway analysis was conducted using KEGG (www. genome.jp/kegg/). To study the difference between leaves under uniform and non-uniform salinity treatments, genes down-regulated under uniform salinity but up-regulated under non-uniform salinity treatments were selected manually. Genes related to nitrate, potassium, phosphate, zinc and iron transport were also selected manually. The Scientific Repo R ts | 7: 2879 | DOI:10.1038/s41598-017-03302-x 10 www.nature.com/scientificreports/ aquaporin genes and hormones, such as cytokinin, ABA and ethylene related genes which differentially expressed more than two folds in the non-saline (0/200-0) or high-saline root side (0/200-200) were also selected by hand. e t Th ranscription factor genes which differentially expressed in roots were also analyzed and classified. Real-time PCR (RT-PCR) analysis. Real-time PCR analysis was used to determine the expression of some important genes and validate the results of RNA-Seq. The samples used for RNA isolation in RT-PCR experi- ments were different from the samples used in RNA-Seq analysis. e Th samples from 15 representative individual plants of each line were harvested and every 5 samples were combined into one biological replicate and then extracted for total RNA using the TRIzol reagent (Invitrogen). cDNA fragment was synthesized from total RNA using Superscript II reverse transcriptase (Invitrogen). The specific primers for the selected genes and inter - nal control gene (actin) are listed in Supplemental Table S6. Samples were run in triplicate on a Bio-red IQ2 Sequence Detection System and Applied Biosystems software using 0.1 mL first-strand cDNAs and SYBR Green PCR Master Mix (Applied Biosystems). The results were normalized to the expression level of actin and relative to the NaCl-free control sample. The Pearson correlation coefficients of the expression patterns of selected genes between RT-PCR and RNA-Seq were calculated using the SAS software. Measurement of net Pn. Net Pn of the third fully expanded young leaf from the end on the main stem were measured between 09:00 h and 11:00 h on cloudless days when ambient photosynthetic photon flux density −2 −1 exceeded 1500 µM m s , using a LI-6400 portable photosynthesis system (Li-Cor, Lincoln, NE, USA). − + + − + + Measurements of net NO , NH and K flux with NMT. Net flux of NO , NH and K were meas- 3 4 3 4 ured by Non-Invasive Micro-Test Technology (NMT) [NMT system BIOIM; Younger USA, LLC.] as described in Kong et al. . Aer exp ft osure to the NaCl-free control and uniform and non-uniform salinity treatments for 6 h, root segments with 2–3 cm apices were sampled for ion flux measurements. Roots were rinsed with redistilled water and immediately incubated in the measuring solution to equilibrate for 30 min and then transferred to the measuring chamber containing 10–15 ml of fresh measuring solution. The measuring site was 5 mm from the − + root apex. The measuring solution for NO and NH consisted of (mM) 0.1 NH NO , 0.1 CaCl and 0.3 MES, 3 4 4 3 2 adjusted to pH 6.5 with choline and HCl. The measuring solution of K consisted of (mM) 0.1 KCl, 0.1 CaCl , 0.1 MgCl , 0.5 NaCl, 0.3 MES and 0.2 Na SO , adjusted to pH 6.5 with choline and HCl. Two-dimensional ionic 2 2 4 ux fl es were calculated using MageFlux developed by Yue Xu (http://xuyue.net/mageflux). Positive values in fig- + + − ures represent efflux for NH and K but influx for NO . 4 3 Root L . L of roots in both split-root compartments of the box was measured by pressurizing the roots in p p a pressure chamber (PMS 670, American) as described previously 24. 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Acknowledgements This work was supported by the National Natural Science Foundation of China (31501249; 31371573), the Natural Science Foundation of Shandong Province (ZR2015QZ03), the earmarked fund for China Agricultural Research System (CARS-18-21), the special found for Taishan Scholars (Tspd20150213), the Seed Improvement Funds from Shandong Province (2014-cotton), the Agricultural Scientific and Technological Innovation Project (CXGC2016C04) and Youth Scientific Research Foundation (2014QNZ01; 2015YQN20) from Shandong Academy of Agricultural Sciences. Author Contributions X.K. and H.D. conceived and designed the experiments; X.K. and Z.L. conducted the RNA-seq and physiological experiments; Z.L. and Y.C. confirmated the RNA-seq data by RT-PCR; X.K., and W.L. Analyzed the data; X.K. and H.D. wrote the manuscript. Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-03302-x Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 Scientific Repo R ts | 7: 2879 | DOI:10.1038/s41598-017-03302-x 13
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