Identification of a DEAD-box RNA Helicase BnRH6 Reveals Its Involvement in Salt Stress Response in Rapeseed (Brassica napus)

Rapeseed (Brassica napus) is one of the most important vegetable oil crops worldwide. Abiotic stresses such as salinity are great challenges for its growth and productivity. DEAD-box RNA helicase 6 (RH6) is a subfamily member of superfamily 2 (SF2), which plays crucial roles in plant growth and development. However, no report is available on RH6 in regulating plant abiotic stress response. This study investigated the function and regulatory mechanism for BnRH6. BnRH6 was targeted to the nucleus and cytoplasmic processing body (P-body), constitutively expressed throughout the lifespan, and induced by salt stress. Transgenic overexpressing BnRH6 in Brassica and Arabidopsis displayed salt hypersensitivity, manifested by lagging seed germination (decreased to 55–85% of wild-type), growth stunt, leaf chlorosis, oxidative stress, and over-accumulation of Na ions with the K+/Na+ ratio being decreased by 18.3–28.6%. Given the undesirable quality of knockout Brassica plants, we utilized an Arabidopsis T-DNA insertion mutant rh6-1 to investigate downstream genes by transcriptomics. We constructed four libraries with three biological replicates to investigate global downstream genes by RNA sequencing. Genome-wide analysis of differentially expressed genes (DEGs) (2-fold, p < 0.05) showed that 41 genes were upregulated and 66 genes were downregulated in rh6-1 relative to wild-type under salt stress. Most of them are well-identified and involved in transcription factors, ABA-responsive genes, and detoxified components or antioxidants. Our research suggests that BnRH6 can regulate a group of salt-tolerance genes to negatively promote Brassica adaptation to salt stress.


Introduction
Agroecosystems are complex systems full of multi-environmental challenges, including biotic and abiotic stresses constantly encountered by sessile plants. Salinity stress is one of the most severe abiotic stresses for most terrestrial plants [1]. Plants grown in excess salt environments suffer from osmotic stress and ionic toxicity that disturb numerous metabolisms, antioxidative systems, and consequent growth and development [2][3][4]. To deal with the adverse environmental challenge, plants have developed diverse mechanisms, including the altered expression of massive stress responsive and tolerant genes [5,6]. The well-known transcription factors and kinases in signaling cascades, for example, are good cases in point [5,6].
RNA helicases are a class of ATPases functioning in RNA duplex unwinding and ribonucleoprotein (RNP) structure remodeling [7]. Based on the sequence, the RNA helicase can be classified into six superfamilies (SFs) [8]. SF2 is the major superfamily. Based on the amino acid sequence of conserved motif II, SF2 can be further divided into three subfamilies: ( DExD/H-box [9]. DEAD-box RNA helicases are the largest subgroup and participate in almost all RNA biological processes, such as RNA transcription, RNA transport, mRNA splicing, RNA silencing, and RNA decay [10]. They are also actively involved in plant growth, development, and plant-environment interactions [11,12]. To date, a bunch of DEAD-box RNA helicase genes has been shown to play roles in plant abiotic stress response regulation. For example, low expression of osmotically responsive genes 4 (LOS4, a DEAD-box RNA helicase gene) is required for the low temperature tolerance by promoting mRNA export in Arabidopsis [13]. OsBIRH1 is a DEAD-box RNA helicase gene in rice; heterologous overexpression of OsBIRH1 in Arabidopsis increased oxidative stress tolerance by elevating the expression of antioxidative genes [14]. In Arabidopsis, RNA helicase 3 (AtRH3) confers salt stress tolerance by splicing mRNA in chloroplast under salt stress [15]. Nevertheless, most DEAD-box RNA helicase members remain to be investigated, particularly the ones responding to abiotic stresses.
Brassica napus (Brassica) is an amphidiploid Brassica rapa and Brassica oleracea [16]. Brassica and Arabidopsis thaliana (Arabidopsis) belong to the same cruciferous family. As one of the main resources of vegetable oil, Brassica is cultivated on upland over the world and is vulnerable to a variety of adverse environmental stresses such as drought, salinity, and unfavorable temperature. Identifying the gene function related to abiotic stress tolerance would contribute to breeding varieties that ensure the vegetable plants are more productive under adverse environments. We previously isolated two DEAD-box RNA helicase genes in Brassica under environmental stress, both are homologs of DDX6 (DEAD-box RNA helicase 6) in animals and RH6 (RNA helicase 6) in Arabidopsis. Recent studies show that DDX6 and AtRH6 are involved in RNA turnover and miRNA biogenesis [17][18][19], but whether they function in plant salt stress response remains unknown. Here, we identified an uncharacterized locus encoding a DDX6 homolog gene BnRH6 in Brassica. We show that BnRH6 is localized to the nucleus and processing-body (P-body) in the cytoplasm, and can be upregulated under salt stress at the transcription level. Transgenic overexpressing BnRH6 in Brassica and Arabidopsis revealed hypersensitivity to salinity, suggesting a negative role of BnRH6 played in tolerance to salt stress. We took advantage of an AtRH6 T-DNA insertion mutant line rh6-1 to profile the genome-wide transcripts under salt stress and found significant changes in the expression of many salt-tolerant genes, including signaling components (phytohormones and transcription factors), and proteins or enzymes for metabolism and antioxidation. Our work supports the important role of BnRH6 in the negative regulation of the Brassica adaptation salinity environment and unveils its downstream genes with mechanistic pathways leading to the plant response to salt stress.

BnRH6 Is Transcriptionally Upregulated under Salt Stress
Two homologues, BnRH6-1 (DEAD-box RNA helicase 24, BnaA04g26450D, named BnRH6 in this article) and BnRH6-2 (DEAD-box RNA helicase 100, BnaC04g50480D), were identified in Brassica ( Figure S1) [16,20]. Alignment of amino acids of BnRH6 revealed that there are typical DEAD-box RNA helicase domains with high similarity within Brassica and Arabidopsis ( Figure S1B). RH6s have two homologs, RH8s and RH12s ( Figure S1C), which is very similar to those of the Arabidopsis species, indicating that BnRH6s are highly conserved in Brassicaceae plants including Brassica and Arabidopsis.
BnRH6 was transcriptionally expressed in various tissues or organs throughout the lifespan. In young seedlings, BnRH6 is dominantly expressed in euphylla and hypocotyl and moderately in other tissues such as root and cotyledon ( Figure 1A). At early developmental stages, BnRH6 is evenly expressed in all tissues. During flowering and seed developing stage, BnRH6 showed a higher expression in stems, flowers, and siliques but a relatively lower expression in leaves ( Figure 1A). To examine whether BnRH6 was induced by salt stress, two-week-old seedlings were treated with NaCl at 100, 200, and 300 mM. When plants were exposed to 100 mM NaCl for 6 h, the BnRH6 transcript levels in roots and shoots were increased by 2.5-and 2.6-fold higher than those of the control, respectively ( Figure 1B). At 200 mM NaCl, BnRH6 expression declined but remained at a significantly higher level over the control. These results indicated that BnRH6 is transcriptionally induced by salt stress. relatively lower expression in leaves ( Figure 1A). To examine whether BnRH6 was induced by salt stress, two-week-old seedlings were treated with NaCl at 100, 200, and 300 mM. When plants were exposed to 100 mM NaCl for 6 h, the BnRH6 transcript levels in roots and shoots were increased by 2.5-and 2.6-fold higher than those of the control, respectively ( Figure 1B). At 200 mM NaCl, BnRH6 expression declined but remained at a significantly higher level over the control. These results indicated that BnRH6 is transcriptionally induced by salt stress.

BnRH6 Is Localized to the Nucleus and Cytoplasm
We further examined the subcellular localization of the BnRH6 protein. The BnRH6 coding sequence was amplified and fused to the green fluorescent protein (GFP) by linking the N-or C-terminal of BnRH6. The fusion and GFP alone were further ligated to a vector with a 35S promoter to drive its expression. In the meantime, two fluorescent marker proteins including MADS3 targeted to the nucleus and AtDCP1 to the cytoplasm were obtained. The vectors were transferred into the leaf cells of tobacco. GFP alone served as a technical control ( Figure 1C). The BnRH6-GFP fusions with green signals and marker proteins with red signals were dominantly expressed in the same regions of the nucleus and cytoplasm. The overlapping colors merged to create yellow ones ( Figure 1D,E), indicating that BnRH6-GFP can be targeted on both sides. Since AtDCP1 was reported as a P-body marker [21], there is a likelihood that BnRH6 is associated with P-body.

BnRH6 Overexpressed Brassica and Arabidopsis Display Salt Sensitivity
To evaluate the role of BnRH6 under salinity stress, we generated BnRH6-overexpressing Brassica (BnOE) and Arabidopsis (AtOE) lines ( Figure S2A-D). Three transformants of each BnOE and AtOE plant (T3 Homozygotes) were randomly selected, genotyped at DNA levels ( Figure S2A,C) and determined at transcriptional levels ( Figure S2B,D). No obvious differences in phenotypes were observed between wild-type (WT) and BnOE lines without NaCl treatment (Figure 2A,B). When exposed to 100, 125, and 150 mM NaCl, however, the BnOE plants were more sensitive to salt stress than WT (Figure 2A,B). Consistently, the seed germination ratio between WT and BnOE plants remained unchanged in the absence of NaCl, while following a three-day exposure to 100, 125, and 150 mM NaCl, the seed germination ratio of BnOE lines decreased to 55-85% of the WT, respectively ( Figure 2C). Similar responses were observed for the cotyledon greening rate ( Figure 2D). Nearly all WT and BnOE seeds exhibited cotyledon greening on the 3rd day without NaCl, while more leaf chlorosis symptoms were observed in BnOE plants than in WT under NaCl exposure ( Figure 2D). Furthermore, the growth of root and shoot and fresh biomass of BnOE seedlings was more severely inhibited under salt stress compared with that of WT ( Figure 2E-G). With regard to AtOE, similar responses concerning seed germination, cotyledon greening, root elongation, and biomass were observed ( Figures S3 and S4). These results suggest that BnRH6-overexpression repressed seed germination under salt stress.
We conducted an additional study with 10-day-old BnOE and its WT plants treated with NaCl. While both varieties showed no evident growth difference under normal conditions, the BnOE plants displayed compromised growth status relative to WT ( Figure 3A,B). The root length and shoot height of BnOE plants were reduced by 8.14-18.79% and 11.97-19.34% under NaCl treatment, and the dry weight of root and shoot of BnOE seedlings dropped by 14.47-23.47% and 15.05-29.69%, respectively ( Figure 3C-F). Likewise, the AtOE lines displayed more growth defects in root elongation and shoot growth than WT plants ( Figure S4). These results pointed out that overexpression of BnRH6 could make the plant vulnerable to salt stress.
Chlorophyll and malondialdehyde (MDA) concentrations are typical physiological indicators reflecting plant growth status and stress-induced generation of peroxides under salinity stress, respectively [22,23]. As shown in ( Figure 4A), when BnOE and WT were exposed to NaCl, the leaf of BnOE lines became much more chlorotic, particularly at higher degrees of salt stress ( Figure 4A). The leaf phenotype in AtOE plants with salt treatment showed a similar chlorosis symptom ( Figure 4B). Assessments of chlorophyll revealed that the chlorophyll concentrations in BnOE and AtOE plants significantly decreased as compared with those of wild-type ( Figure 4C,D). Meanwhile, measurements of MDA showed that the MDA concentrations were significantly increased in the transgenic plants ( Figure 4E,F). These results suggest that BnRH6 overexpression could also impair the physiological response under salt stress.

BnRH6 Overexpression Promotes Na + Accumulation in Plants
To identify whether the BnRH6-mediated compromised growth and physiological response is associated with the accumulation of Na + in plants, concentrations of Na ions in roots and shoots were separately determined in the transgenic and wild-type plants. An ICP-MS analysis showed that Na + levels were significantly higher in both roots and shoots of the BnOE than in WT plants, especially at the high level of NaCl (200 mM) ( Figure 5A,B). The enhanced Na + levels were also detected in roots and shoots of the AtOE lines, where the Na + concentrations increased by 61.5-73.1% and 14.3-33.3% compared with wild-type, respectively ( Figure S5A,B). Since the ratio of K + /Na + is commonly used as one of the important physiological responses for plant tolerance to salt stress [24], we simultaneously measured the K ions in the plants. The K + concentrations were comparable between the overexpression lines and wild-type ( Figures 5C,D and S5C,D). However, calculations of the K + /Na + ratio showed that the ratio declined in the BnOE and AtOE plants compared with the wild-type plants. The significant differences between the transgenic plants and wildtype occurred at 150-200 mM of NaCl ( Figures 5E-G and S5E,F). In the shoot of the BnOE plants, the K + /Na + ratio decreased by 18.3-28.6% compared with wild-type ( Figure 5G). The results suggest that overexpression of BnRH6 results in the over-accumulation of Na ions.

Suppression of RH6 Enhances Plant Tolerance to Salt Stress by Upregulating the Expression of Salt Stress-Tolerant Genes in Arabidopsis
To figure out the mechanisms or pathways responsible for plant tolerance to salinity regulated by RH6, we performed a genome-wide identification of transcriptomes by RNA sequencing. Due to the lack of desirable knockout lines of Brassica, we took an advantage of an Arabidopsis knockout mutant rh6-1. The rh6-1 mutant with a T-DNA insertion in an exon near 5 -UTR was identified by PCR ( Figure S2E-G). We applied the atrh6-1 for the study because both BnRH6 and AtRH6 are highly conserved, by which the amino acid sequences between BnRH6 and AtRH6 share an 85.72% similarity, and the key domains, DEAD domain (ATPase domain including Q, I, Ia, Ib, II and III motif) and HELIC domain (RNA binding domain including III, IV and VI motif) between BnRH6 and AtRH6 share similarities of 98.98% and 97.56%, respectively ( Figure S1B). Furthermore, the rh6-1 mutant displays an enhanced salt-tolerance phenotype with better growth (longer root length and greater biomass) compared with the wild-type (Col-0) ( Figure S6), confirming that both AtRH6 and BnRH6 should be negative regulators of plant response to salt stress.
Four RNA libraries regarding rh6-1 and WT connecting −Na or +Na were constructed in terms of WT(−Na), WT(+Na), rh6-1(−Na), and rh6-1(+Na) with three biological replicates (4 × 3 samples) and sequenced by high-throughput RNA-sequencing using Illumina technology. The quality of the sequencing datasets was statistically evaluated, and it turned out to be reliable (Table S1 and Table S2; Figure S7). To validate the expression pattern of the genes determined by RNA-seq, a total of 21 genes were selected for qRT-PCR validation. Expression of all genes showed a pattern identical to the RNA-seq ( Figure S8).

Suppression of RH6 Enhances Plant Tolerance to Salt Stress by Upregulating the Expression of Salt Stress-Tolerant Genes in Arabidopsis
To figure out the mechanisms or pathways responsible for plant tolerance to salinity regulated by RH6, we performed a genome-wide identification of transcriptomes by RNA sequencing. Due to the lack of desirable knockout lines of Brassica, we took an advantage of an Arabidopsis knockout mutant rh6-1. The rh6-1 mutant with a T-DNA insertion in an exon near 5′-UTR was identified by PCR ( Figure S2E-G). We applied the atrh6-1 for the study because both BnRH6 and AtRH6 are highly conserved, by which the amino acid sequences between BnRH6 and AtRH6 share an 85.72% similarity, and the key domains, DEAD domain (ATPase domain including Q, I, Ia, Ib, II and III motif) and HELIC domain  We then performed Gene Ontology (GO) analysis and placed the DEGs into some functional categories related to salt stress response. While under the −Na condition, many DEGs in the rh6-1 mutant filled into the development of the biological process, and the DEGs of rh6-1(+Na) vs. rh6-1(−Na) were found with enrichment in the categories such as stimuli, chemical stress, and hormone response ( Figure S9). It was important to find that in the dataset of rh6-1(+Na)/WT(+Na), the DEGs were also classified into stress responses and different kinds of metabolic processes ( Figure S9). These results indicate that under salt stress the rh6-1 function most likely would shift from the developmental state to the stress responsive model by post-transcriptional regulation. The Kyoto Encyclopedia of Genes and Genomes (KEGG) refers to the genes categorized into specific pathways. The DEGs in the dataset rh6-1(−Na)/WT(−Na) were concentrated in the pathways of RNA degradation, MAPK and hormone-signaling pathway, metabolite biosynthesis, and plantpathogen interaction ( Figure S10). Similar results were found in the rh6-1(+Na)/WT(+Na) and rh6-1(+Na)/rh6-1(−Na) datasets, in which some DEGs participated in the stress signal, metabolite biogenesis, and RNA metabolism pathways ( Figure S10). These results suggest that mutation of RH6 led to many stress-responsive genes being involved in the salt resilience process.

Expression of BnRH6 Compromises Growth of Plants under Salt Stress
In this study, we report that BnRH6 is a new regulator in salt stress response by downregulating salt-tolerant genes. BnRH6 was transcriptionally expressed in various tissues or organs all lifelong, indicating a potential role in all stages ( Figure 1A). This wide expression pattern is in agreement with its RH6 in Arabidopsis and other RNA helicases [25][26][27][28]. Moreover, BnRH6 was localized in the nucleus and cytoplasm (most likely in P-body) ( Figure 1C-E), suggesting potential functions on both sides. This is reminiscent of RH6 in Arabidopsis, which plays roles in both the nucleus and cytoplasm [19,25]. There is a likelihood that BnRH6 is associated with P-body, similar to the report on AtRH6 [25].
The transcript levels were increased drastically in the shoot and root of Brassica with NaCl exposure, implying a possible role in salt stress response ( Figure 1B). Several reports have indicated that the expression of RNA helicases correlated with salt stress response can be regulated by salinity stress in multiple species including Arabidopsis, rice, cabbage, tomato, barley, and so on [28][29][30][31][32]. However, not all salt stress response-related RNA helicases can be induced at the transcript level. For instance, Arabidopsis AtRH17 is a positive regulator to plant salt stress tolerance, whereas the AtRH17 transcript level was not affected under salinity stress [33]. Furthermore, a certain number of RHs participate in more than one kind of abiotic stress [28][29][30]32,34]. Whether BnRH6 expression can be altered by other stresses needs to be further investigated.
Genetic and functional investigation revealed that BnRH6 overexpression in both Brassica and Arabidopsis led to plant sensitivity to salt stress, in terms of a decreased germination ratio and cotyledon greening rate, weakened growing status, and more sensitive physiological reactions. This scenario is consistent with our RNA-seq data. BGLU31 and BGLU32 encode β-glucosidases as positive factors for seed germination [35] and were found to be upregulated in rh6-1 under salt stress ( Figure 6D). Notably, the gene SSP negatively mediating seed germination was found to be downregulated in rh6-1 plants ( Figure 6D,E) [36]. Several classes of growth-regulating genes, such as ELIP1 and ELIP2 encoding chlorophyll a/b binding family protein genes and ARR18 encoding response regulator genes, were all upregulated in the rh6-1 mutant lines ( Figure 6E). Thus, BnRH6 affects the salt stress response likely via indirect control of the expression pattern of those genes.
Overload of NaCl into plants initially triggers osmotic stress and later ion imbalance and toxicity [37,38]. Proline is an osmotic regulator induced by salt stress. One gene encoding an enzyme (P5CS2) in the proline synthesis was specifically upregulated in saltexposed rh6-1 plants ( Figure 6E). Our data showed that the Na + accumulation was increased in shoots and roots of BnRH6 overexpression lines. Even though the K + accumulation was not altered in overexpression lines, a relatively lower K + /Na + ratio was generated ( Figure 5F,G). The Na + accumulation and K + /Na + ratio was barely discussed in RNA helicase gene mutants or overexpression plants involved in the salt-stress response. Similar changes have been evident in other salt-stress regulator mutants or overexpression lines. For instance, AtDIF1 overexpression Arabidopsis conferred plant tolerance to salt stress by reducing the content of Na + and loss of K + [39]. SERF1 is a positive regulator for salt stress tolerance in rice. Loss-of-function or the knock-down mutant of SERF1 showed an increased Na + accumulation and a higher Na + /K + ratio [40].
Our RNA seq data showed that one ion channel gene, CAX3 was induced in rh6-1 under salt stress ( Figure 6D,E). A previous study showed that CAX3 can be induced by Na + treatment. CAX3 is a Ca 2+/ H + vacuolar antiporter for plant tolerance to salt under Na + stress. The cax3 mutant confers decreased P-ATPase activity, which further decreased Na + efflux [41]. On the contrary, an enhanced expression of CAX3 may promote Na + efflux and benefit the maintenance of a high K + /Na + ratio. Apart from the notorious detriment caused by Na + , the accompanied Cl − poisoning is also fatal [42,43]. SLAH3 is a Cl − efflux transporter [44]. Transcriptional SLAH3 was downregulated by NaCl treatment [45]. An increased expression of SLAH3 in rh6-1 under NaCl exposure was recovered from our RNA-seq data, which may decrease the Cl − accumulation. These results suggest that CAX3 and SLAH3 could play critical roles in ion homeostasis under salt stress.

Mutation of rh6-1 Confers Plant Salt Tolerance by Regulating Salt-Resilient Genes
To identify the downstream genes and possible regulation pathways governed by RH6 under salt stress, we performed a genome-wide analysis of transcriptome across the rh6-1 and Col-0 plants. While a large number of DEGs were expressed in the rh6-1 and wild-type plants under salt stress, there were many DEGs across the two cultivars exposed to NaCl (>2.0-fold change, p < 0.05). The largest group of specific DEGs was associated with genes encoding transcription factors (TFs), whose expression patterns were altered in the rh6-1 mutant. Nine MYB genes were found to be upregulated in rh6-1 mutant after salt treatment. MYB proteins play a fundamental role in numerous aspects of plant growth, development, and stress responses [46,47]. MYB80, MYB113, and MYB114 were upregulated in rh6-1 plants relative to WT only under salt stress ( Figure 6G, Table S7). MYB113 and MYB114 have been reported to promote anthocyanin synthesis in Arabidopsis and pear for defense against abiotic and biotic stresses [48,49]. MYB112 was strongly expressed in rh6-1 under salt stress ( Figure 6D) and is also required for anthocyanin accumulation under salinity and high light stress [50]. Several other gene members including MYB37, MYB62, MYB90, and MYB120 were also induced in rh6-1 under NaCl stress ( Figure 6E). Some of them are also involved in abiotic stress responses [51][52][53].
Several NAC (NAM, ATAF1/2 and CUC2) genes were detected in rh6-1 plants under salt stress including NAC019, NAC032 and NAC071 ( Figure 6D) and NAC044, NAC061, and NAC096 ( Figure 6E), and most of them have been reported to involve salt and other abiotic stress response [54,55]. Arabidopsis harboring an overexpressed GmNAC019 increased drought and salinity tolerance [56]. NAC061 is induced by AtHSFA7b (a heat shock transcription factor in Arabidopsis) and positively regulates salt tolerance [57]. In addition to MYB and NAC, several other TF family genes such as WRKY45 and CBF4 identified in the study are also reported to regulate plant response to salt stress [58,59]. Whether BnRH6 directly or indirectly interacts with TFs remains to be investigated.
Plant hormones are the major regulators of abiotic stress responses [60]. ABA has been long considered to be a crucial driver of plant abiotic stress, especially salinity and dehydration stress responses [61,62]. In this study, seven ABA signaling pathway genes for NCED9, ABI1, HAIs, AHG1, MAPKKK18, and CYP707A1 were identified as saltresponsive DEGs. Arabidopsis NCED9 (9-cis-epoxycarotenoid dioxygenase 9) is essential for ABA biosynthesis [63,64]. Expression of NCED9 is induced by salt, drought, and cold in Brassica oleracea [65]. Several DEGs encode ABI1, AHG1, HAI1, HAI2, and HAI3, and these proteins are phosphatases 2Cs (PP2Cs) responsible for ABA signal transduction [66,67]. CYP707A1 and MAPKKK18 were also involved in ABA-related stress responses [68][69][70]. This is reminiscent of some recent reports that other DEAD-box RNA helicases such as BnRH37 improved abiotic stresses of plants by inducing the expression of ABA biosynthesisrelated genes or ABA-dependent stress-response genes [33,71,72], but no report was published for BnRH6.
It is well known that salinity stress is usually accompanied by oxidative stress [73,74]. Under salt stress, some anti-oxidative enzymes such as glutaredoxins and thioredoxins actively participate in the removal of salt stress-induced ROS and toxicity [75]. Five genes encoding ROS scavengers were upregulated in rh6-1 plants. For example, GRXS4/ROXY13 and TH8 were transcriptionally upregulated in rh6-1 mutants under NaCl stress. GRXS4 belongs to the CC-type glutaredoxin (ROXY) protein family, and was reported to be induced by salinity and drought stresses [76,77]. Both TRX5 (thioredoxin H-type 5) and TH8 (thioredoxin H-type 8) encode thioredoxins and can be induced by multiple biotic and abiotic stresses [78]. GSTU4 and GSTU6 are tau class glutathione S-transferases; GSTU6 was reported to contribute to Cd stress tolerance by regulating intracellular ROS homeostasis in rice [79]. Overexpressing GmGSTU4 displays salt tolerance in tobacco through peroxidase activity and detoxification mechanisms [80,81]. In addition, genes indirectly participating in ROS scavenging were screened. For example, PtrLEA7, a gene encoding a Late embryogenesis-abundant (LEA) protein, can be slightly induced by salt stress; overexpressing PtrLEA7 in tobacco and Poncirus trifoliata positively regulates drought tolerance through enhancing antioxidant capacity [82]. Similarly, an RNA helicase VviDEADRH25 in grapevine plays a role as a negative regulator to drought stress tolerance. Overexpressing VviDEADRH25 in Arabidopsis decreased the expression of genes encoding ROS scavenging enzymes [83]. These results suggest that rh6-1-mediated plant tolerance to salt stress would be likely through these homolog genes.
Apart from the subsets above, we also detected some other protein family genes involved in the RH6-mediated pathways under salt stress. LEA proteins belong to a large hydrophilic protein family with regulatory roles in plant stress responses [84]. We found that LEA4-5, LEA7, LEA14, and LEA18 were significantly upregulated in rh6 under salt stress. LEA14 was previously reported to confer salt stress tolerance by stabilizing an E3 ligase in Arabidopsis [85]. LEA3 was induced only in rh6 but not in WT under Na stress, suggesting that LEA3 was most likely involved in the salt stress response. Supportive evidence comes from the rice LEA3 because the OsLEA3-overexpression line endowed drought resistance in the plant [86] These results suggest that the RH6 is likely a master regulator of RNA biosynthesis and metabolism under salt stress. Based on those results, we proposed a model of salt stress response in WT and BnOE Brassica (Figure 7). Our research found a new function of BnRH6, and it may be a promising molecular tool for engineering salt-tolerant Brassica and enriching Brassica varieties.
genes encoding ROS scavenging enzymes [83]. These results suggest that rh6-1-mediated plant tolerance to salt stress would be likely through these homolog genes.
Apart from the subsets above, we also detected some other protein family genes involved in the RH6-mediated pathways under salt stress. LEA proteins belong to a large hydrophilic protein family with regulatory roles in plant stress responses [84]. We found that LEA4-5, LEA7, LEA14, and LEA18 were significantly upregulated in rh6 under salt stress. LEA14 was previously reported to confer salt stress tolerance by stabilizing an E3 ligase in Arabidopsis [85]. LEA3 was induced only in rh6 but not in WT under Na stress, suggesting that LEA3 was most likely involved in the salt stress response. Supportive evidence comes from the rice LEA3 because the OsLEA3-overexpression line endowed drought resistance in the plant [86] These results suggest that the RH6 is likely a master regulator of RNA biosynthesis and metabolism under salt stress. Based on those results, we proposed a model of salt stress response in WT and BnOE Brassica (Figure 7). Our research found a new function of BnRH6, and it may be a promising molecular tool for engineering salt-tolerant Brassica and enriching Brassica varieties.

Plant Growth and Salt Treatments
Seeds of Brassica (genotype: Westar) were surface-sterilized in 75% ethanol alcohol for 5 min and 10% NaClO for 15 min with gentle shakes, followed by thoroughly rinse with sterile water. The clean seeds were placed on a plastic net floating on the 1/2 Hoagland solution. After germination at 25 • C in the dark for two days, seeds were grown under the condition of 22 • C, 200 µmol m −2 s −1 light intensity and 14 h/10 h(light/dark) photoperiod [87]. Plant tissues at different stages were harvested for BnRH6 expression pattern analysis. Two-week-old Brassica seedlings were exposed to 1/2 Hoagland solution (pH = 5.8) with different NaCl concentrations (0, 100, 200, and 300 mM) for 6 h based on previous reports [88]. Shoots and roots were harvested separately for expression analysis.
Seeds of Arabidopsis (Col-0) and a rh6-1 T-DNA insertion mutant line (SALK_ 205997C, Columbia background, obtained from the Arabidopsis Biological Resource Center) were sterilized and sowed on 1 2 Murashige and Skoog (MS) medium supplemented with 3% sucrose and 0.9% agar. After two-day vernalization at 4 • C in darkness, seeds were grown in a growth chamber with 22 • C, 150 µmol m −2 s −1 light intensity and 16/8 h (light/dark) photoperiod.

Vector Construction and Transgenic Plants
Full-length CDS sequence of BnRH6 (Bn04g26450D) was cloned using specific primers (Table S8) and inserted into the pCAMBIA1300 vector with CaMV35S as the promoter [89]. The sequence-confirmed construct was transformed into Agrobacterium tumefaciens strain EHA105 following the standard method of transformation [90]. The Agrobacteriummediated plant transformation was performed with Brassica hypocotyl sections [87] for Brassica plants and floral dip method for Arabidopsis plants [88,91]. Transformants were selected with 100 and 50 mg/L kanamycin, respectively. More than fifteen independent transgenic lines (T1 generation) were obtained and genotyped by PCR. Three of them were randomly selected for propagation. The homozygous lines (T4 generation) were used in this study. For cloning of BnRH6-GFP, full-length CDS sequence of BnRH6 without stop codon was fused into pCAMBIA1305 backbone through Xba/IBamH I digestion and T4 ligation.

Analysis of Transcripts by RT-PCR and qRT-PCR
Total RNA was extracted with Trizol (Invitrogen). Total RNA was treated with DNase I (Transgen, Beijing, China). The first-strand cDNA was generated by reverse transcription with EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China) by Oligo(dT) RTprimer (mRNAs) according to the manufacturer's instructions. For regular PCR reactions, PCR products were fractioned in 1% agarose gels. For qRT-PCR, the amplification systems were processed in a 20 µL solution with 10 µL TransStart Green qPCR SuperMix (Transgen, Beijing, China), 4 ng template, and 0.8 µL (10 µmol) primers (Table S8). The reactions were run at 95 • C for 10 min, followed by 40 cycles of denaturation at 95 • C for 10 s and annealing at 60 • C for 1 min in the 7500 Real-Time PCR System (Applied Biosystems) [16]. Brassica Actin2 serves as an internal control. All primers used are listed in Table S8 (Table S8). Primers used for genotyping were designed on http://signal.salk.edu/tdnaprimers.2.html (accessed on 14 December 2022). AtRH6-qRT primers were synthesized according to [25]. AtActin2 primers were synthesized according to [19]. BnActin2 primers were synthesized according to [92]. Other primers were designed with Primer Premier6.

Determination of Salt Stress Responses
For germination and cotyledon greening analysis, sterilized WT and BnOE Brassica seeds were placed on 1 2 MS medium containing 0, 100, 125, and 150 mM NaCl [93,94]. The germination proportion (emerged radicle) and cotyledon greening (expanded cotyledon turning green) were recorded from the 1st day (the day exposed to light) to the 7th day. Images were photographed on the 7th day. Sterilized WT and AtOE Arabidopsis seeds were placed on 1 2 MS medium containing 0, 50, 75, and 100 mM NaCl [95]. The germination proportion (emerged radicle) and cotyledon greening (expanded cotyledon turning green) were recorded and photographed on the 4th day and 7th day, respectively [96].
For root/shoot length and dry weight, ten-day-old Brassica plantlets were treated with 1 2 Hoagland solution containing 0, 100, 150, and 200 mM NaCl for 14 days. Sterilized WT and AtOE Arabidopsis were grown on 1 2 MS medium with 0, 50, 75, and 100 mM NaCl for 12 days. Sterilized WT and rh6-1 Arabidopsis seeds were placed on 1 2 MS medium containing 0, 45, and 70 mM NaCl for 14 days. The plates were vertically placed in a conditioned chamber. The root and shoot length were measured with a ruler, and the fresh mass and dry mass were weighted as described previously. [39,88,89,97].

Determination of Physiological Responses
For measuring seed germination, the surface-sterilized seeds of Brassica were placed on the solid 1/2 MS medium with the gradient NaCl concentrations at 0, 100, 125, and 150 mM. The germinated seeds (the seeds with emerged radicles) and the seeds with green cotyledons were counted every day from the 1st day to the 7th day. Surface-sterilized Arabidopsis seeds were sowed on solid 1/2 MS medium with the gradient NaCl concentrations at 0, 50, 75, and 100 mM. The germinated seeds (the seeds with emerged radicles) and the seeds with green cotyledons were counted on 4th day and 7th day, respectively. The calculation of seed germination ratio and cotyledon greening rate refers to [96].
For chlorophyll and MDA concentration measurements, Brassica seeds were hydroponically grown in 1 2 Hoagland solution for ten days and then grown in 1 2 Hoagland solution supplied with NaCl gradient from 0 to 100, 150, and 200 mM for 14 days. Arabidopsis seeds were potted in mixed soil (vermiculite and flower nutrient soil, 3:1) and placed in the chambers. Three-week-old Arabidopsis plants were irrigated with 0 and 200 mM NaCl solution for 12 days [98,99]. Total chlorophyll from Brassica or Arabidopsis was extracted with 80% (v/v) acetone and soaked for 36 h at room temperature in the dark until the leaves fully faded. The absorbance at 663 nm and 645 nm was recorded by a spectrophotometer [100]. The malondialdehyde (MDA) concentration was determined using a Lipid Peroxidation MDA Assay Kit (S0131, Beyotime, Shanghai, China) according to manufacturers' instructions.

Determination of Na + and K + Concentrations
Plant shoot and root tissues were separately harvested after NaCl treatment (the same as materials for chlorophyll and MDA measurements). The harvested tissues were immersed in 4 mM CaSO 4 solution for 5 min. After being washed with deionized water, the samples were exsiccated in an oven at 72 • C for 72 h. The samples were digested with nitric acid, and the Na + and K + concentrations were determined by ICP-MS (PerkinElmer, Waltham, MA, USA) as previously described [39].

Construction of Salt Treated cDNA Libraries of Arabidopsis rh6-1 and RNA-Sequencing
Arabidopsis seeds of rh6-1 and wild-type (WT, Col-0) were sowed on solid 1 2 MS medium for 14 days before being transferred to 1/2 Hoagland solution. The plants were then subjected to 200 mM NaCl treatment (+Na) with a 0 mM NaCl treatment (−Na) as a control (CK) for 6 h.
Four groups of samples were harvested and total RNA was isolated. The samples were prepared as RNA-seq libraries in terms of WT(−Na), WT(+Na), rh6-1(−Na), and rh6-1(+Na). For each library, three biological samples with a total of 12 samples were prepared for RNA sequencing according to the previous method [101]. The Illumina RNA sequencing platform (HiSeq 2500) was applied. Data analyses were performed as described previously [89].

Statistical Analysis
The study was set up in biological triplicate. Each result shown in the figures was the mean of three replicated treatments, and each treatment contained at least 15-18 seedlings.
With regard to the assessment of survival rates, 30 plants were employed. The significant difference between treatments was assessed through analyses of variance post hoc test (ANOVA, Tukey's test). The values of each assay, followed by an asterisk, are significantly different at p < 0.05. The data were analyzed using the statistical software package SPSS 22.0.

Conclusions
This study functionally characterized a DEAD-box RNA helicase family gene BnRH6 from Brassica. BnRH6 can be transcriptionally induced by salt stress. Overexpression of BnOE revealed plant hypersensitivity to salinity stress, in terms of reduced germination rate, cotyledon greening, root and shoot elongation, chlorophyll concentration, and increased MDA concentrations in plants. These results were well-confirmed by similar studies with transgenic Arabidopsis (AtOEs) expressing BnRH6. Furthermore, both the BnOE and AtOE lines accumulated more Na + than the wild-type. RNA-seq analysis revealed that a subset of downstream salt-tolerant genes was regulated by RH6, indicating that RH6 negatively regulates salt stress response by adjusting the downstream salt-tolerant genes. However, the detailed molecular mechanisms for how BnRH6 regulates its downstream genes or proteins remain to be investigated.

Conflicts of Interest:
The authors declare that they have no conflict of interest.