Transcriptomics Reveals Host-Dependent Differences of Polysaccharides Biosynthesis in Cynomorium songaricum

Cynomorium songaricum is a root holoparasitic herb that is mainly hosted in the roots of Nitraria roborowskii and Nitraria sibirica distributed in the arid desert and saline-alkaline regions. The stem of C. songaricum is widely used as a traditional Chinese medicine and applied in anti-viral, anti-obesity and anti-diabetes, which largely rely on the bioactive components including: polysaccharides, flavonoids and triterpenes. Although the differences in growth characteristics of C. songaricum between N. roborowskii and N. sibirica have been reported, the difference of the two hosts on growth and polysaccharides biosynthesis in C. songaricum as well as regulation mechanism are not limited. Here, the physiological characteristics and transcriptome of C. songaricum host in N. roborowskii (CR) and N. sibirica (CS) were conducted. The results showed that the fresh weight, soluble sugar content and antioxidant capacity on a per stem basis exhibited a 3.3-, 3.0- and 2.1-fold increase in CR compared to CS. A total of 16,921 differentially expressed genes (DEGs) were observed in CR versus CS, with 2573 characterized genes, 1725 up-regulated and 848 down-regulated. Based on biological functions, 50 DEGs were associated with polysaccharides and starch metabolism as well as their transport. The expression levels of the selected 37 genes were validated by qRT-PCR and almost consistent with their Reads Per kb per Million values. These findings would provide useful references for improving the yield and quality of C. songaricum.

The genus Nitraria L. is a perennial shrub and always used as a vital ecological protection plant for windbreak and sand fixation [17]. It contains 11 species in the world and 6 of them are in China [18]. C. songaricum is found to mainly host in four species including: N. roborowskii Kom., N. sibirica Pall., N. tangutorum Bobr. and N. sphaerocarpa Maxim [19,20]. Except the N. sphaerocarpa, the other three species mainly distribute in Qinghai, China [21]. Extensive surveys on habitat have found that N. roborowskii prefers locating in the margin of desert, N. sibirica in the salinized sand and drought hillslope and N. tangutorum is a transitional ecotype between N. roborowskii and N. sibirica [22,23]. Previous investigations into the differences in growth characteristics between N. roborowskii and N. sibirica have demonstrated that the growth indexes (e.g., seed weight, fruit weight and seedling height) of N. roborowskii are greater than N. sibirica [24]; while the salt tolerance, seed-setting rate, contents of nutritional components and trace elements of N. sibirica are higher than N. roborowskii [25][26][27][28][29][30].
C. songaricum is currently an endangered species, in large part because of an indiscriminate uprooting of wild plants to meet the increasing commercial demand of the pharmaceutical industry. As a holoparasitic herb, C. songaricum totally depends on the Nitraria L., for nutrients and water during the whole growth and development cycle [31]. C. songaricum is widely used as a traditional Chinese medicine and several pharmacological activities are largely relied on polysaccharides [10,15]; moreover, the growth differences in C. songaricum host in the two N. roborowskii and N. sibirica have been reported [22][23][24], the regulation mechanism of polysaccharides biosynthesis has not been revealed. Thus, it is urgent and necessary to identify the optimization host to increase production of C. songaricum.
Up to now, studies on the effect of different hosts on growth and metabolite accumulation of C. songaricum have not been conducted. This study examines biomass, soluble sugar accumulation, antioxidant capacity and transcriptional alternations of stem between CR and CS.

Comparison of Growth Characteristics between CR and CS
As shown in Figure 1, significant differences in growth characteristics of stems between the CR and CS were observed, with FW of total stems, FW per stem, stem length and diameter of CR exhibiting a 5.1-, 3.3-, 1.4 and 1.3-fold increase compared to that of CS, respectively. and Cynomorium songaricum host in Nitraria sibirica (CS) (mean ± SD, n = 20). Images (A-D) represent FW of total stems, FW per stem, stem length and diameter, respectively. A t-test was applied for independent samples, the "*" is considered significant at p < 0.05 between CR and CS.

Comparison of Soluble Sugar Content and Antioxidant Capacity between CR and CS
As shown in Figure 2, significant differences in soluble sugar content and antioxidant capacity between the CR and CS were observed, with a 1.1-, 1.5-and 1.5-fold respective decrease of soluble sugar content, DPPH scavenging activity and FRAP value on an FW basis in stem of CR compared to that of CS (Figure 2A,C,E), while a 3.0-, 2.1-and 2.1-fold increase on a per stem basis ( Figure 2B,D,F).

Figure 2.
Soluble sugar content and antioxidant capacity in stems between the CR and CS (mean ± SD, n = 20). Images (A-D) as well as (E,F) represent soluble sugar content, DPPH scavenging activity as well as FRAP value on an FW and per stem basis, respectively. A t-test was applied for independent samples, the "*" is considered significant at p < 0.05 between CR and CS.

Global Gene Analysis
To reveal the differences of carbohydrate metabolism between the CR and CS, comparison of the transcripts were performed. A robust data was collected, 51.2 and 46.8 million high-quality reads were obtained after data filtering, and 42.5 and 39.5 million unique reads as well as 1.6 and 1.4 million multiple reads were mapped from the CR and CS, respectively ( Figure 3; Table S1). Total 95,126 unigenes were annotated on KEGG (10,274), KOG (17,550), Nr (40,427) and Swissprot (16,181) databases (Figure 4), and the top 10 species distribution against Nr includes: Cajanus cajan, Vitis vinifera, Cephalotus follicularis, Theobroma cacao, Nicotiana attenuata, Juglans regia, Corchorus capsularis, Brassica napus, Brassica rapa and Medicago truncatula ( Figure 5).   A total of 16,921 DEGs were identified in the CR compared with CS, with 6580 genes up-regulated (UR) and 10,341 genes down-regulated (DR) ( Figure 6). Of these 16,921 DEGs, 2684 genes were identified to match with the databases ( Figure 7A). Among the 2684 genes, 2573 genes with known functions were partitioned into 1725 UR and 848 DR ( Figure 7B,C).

DEGs Involved in Starch Metabolism
Five DEGs, presenting two UR and three DR in the CR compared with CS, directly participate in starch metabolism including: At2g31390, DSP4, NANA, SBE2.2 and SS2 (Table 1). These genes were validated by qRT-PCR, and their RELs were consistent with the RPKM values, with UR 3.5-and 6.8-fold for the At2g31390 and SS2, and DR 0.6-, 0.9-and 0.6-fold for the DSP4, NANA and SBE2.2, respectively ( Figure 9).

Discussion
Although differences in growth characteristics and nutritional components of C. songaricum among the host species, especially in N. roborowskii and N. sibirica, have been observed in previous studies [25][26][27][28][29][30], the mechanism responsible for host-dependent growth and bioactive compound biosynthesis has not been dissected. Here, we found that there is a greater biomass, soluble sugar content and antioxidant capacity on a per stem basis in the CR than the CS (Figures 1 and 2). By transcriptomics analysis in the CR compared with CS, a total of 2573 characterized genes differentially expressed with 1725 UR and 848 DR ( Figure 7). By grouping genes based on biological functions, 50 genes (32 UR and 18 DR) were associated with carbohydrate metabolism and transport (Figure 7; Table 1).
Carbohydrates, one of the most abundant and widespread biomolecules in nature, not only plays an important role in plant growth and development, but also represents a treasure trove of untapped potential for pharmaceutical applications [32,33]. In this study, 37 genes were found to be involved in carbohydrate metabolism including polysaccharides (glucose, galactose, mannose, fucose, trehalose and fructose) and starch (Table 1). Among the 37 genes, 23 genes (62%) presenting up-regulated and 14 genes (38%) down-regulated suggest that the level of carbohydrate metabolism is greater in the CR than CS, which is in accordance with the higher content of soluble sugar on a per stem basis in the CR (Figure 2A,B).
For the starch metabolism, five genes associated with starch metabolic process include: At2g31390 involved in maintaining the flux of carbon towards starch formation [34]; DSP4 controlling the starch accumulation and acting as a major regulator of the initial steps of starch degradation at the granule surface [50]; NANA regulating endogenous sugar levels (e.g., sucrose, glucose and fructose) by modulating starch accumulation and remobilization [51]; SBE2.2 involved in starch biosynthesis and catalyzing the formation of the alpha-1, 6-glucosidic linkages in starch [52]; and SS2 participating in the pathway starch biosynthesis [53].
Transport plays critical roles in distribution and storage of carbohydrate from leaves to roots or other organs that required nutrition [54]. In this study, 13 genes were involved in carbohydrate transport with nine genes (69%) up-regulated and four genes (31%) downregulated, suggesting that the ability of carbohydrate transport is stronger in the CR than the CS (Table 1). Specially, the 13 genes include: At1g67300 participating in the efflux of glucose towards the cytosol [55]; ERD6 participating in sugar transport [56]; MST1 mediating active uptake of hexoses [57]; STP1, STP5, STP12 and STP13 participating in transporting glucose, 3-O-methylglucose, fructose, xylose, mannose, galactose, fucose, 2-deoxyglucose and arabinose [58]; SWEETs is a unique new family of sugar transporters that lead to many elusive transport steps including nectar secretion, phloem loading and post-phloem unloading as well as novel vacuolar transporters [59]. Here, four SWEETs genes SWEET5, SWEET12, SWEET14 and SWEET15 participate in phloem loading by mediating export from parenchyma cells feeding H + -coupled import into the sieve element/companion cell complex [59,60]; and UXT2 and UXT3 participate in transporting UDP-xylose and UMP [61].

Plant Materials
Stems of C. songaricum at vegetative growth stage, were host in the roots of N. roborowskii and N. sibirica ( Figure 11

Measurement of Growth Characteristics
Growth characteristics including fresh weight (FW) of total stems, FW per stem, and its length and diameter were immediately measured after the stems of C. songaricum were dug out and cleaned with running water and absorbent paper.

Extracts Preparation
Fresh stems (1.0 g) were ground into homogenate by adding ethanol (20 mL), agitated at 120 r/min and 22 • C for 72 h, then centrifuged at 5000 r/min and 4 • C for 10 min. The supernatant was increased 20 mL with ethanol and then kept at 4 • C for measurement.

Determination of Soluble Sugar Content
Soluble sugar content was determined by a phenol-sulfuric acid method [62,63]. Briefly, extracts (20 µL) were added in the reaction, absorbance reader was taken at 485 nm and soluble sugar content was calculated based on mg of sucrose.

Determination of Antioxidant Capacity
Antioxidant capacity was determined by DPPH and FRAP methods [64,65]. DPPH radical scavenging assay was determined according to the description of Nencini et al. [66] and Li et al. [63]. Briefly, extracts (5 µL) were added in the reaction, absorbance reader was taken at 515 nm and the capacity to scavenge DPPH radicals was calculated as following Equation (1): where "A 0 " and "A" were the absorbance of DPPH without and with sample, respectively. FRAP assay was determined according to the description of Benzie and Strain [67]. Briefly, extracts (20 µL) were added in the reaction, absorbance reader was taken at 593 nm and the FRAP value was calculated on the basic of (FeSO 4 ·7H 2 O, 500 µmol Fe (II)/g) as following Equation (2): where "A 0 " and "A" were the absorbance of FRAP without and with sample, respectively; A FeSO4·7H2O was the absorbance of FeSO 4 ·7H 2 O.

Total RNA Extraction, Illumina Sequencing, Sequence Filtration, Assembly, Unigene Expression Analysis and Basic Annotation
Total RNA samples of CR and CS with three biological replicates were extracted using an RNA kit (R6827, Omega Bio-Tek, Inc., Norcross, GA, USA). The processes of enrichment, fragmentation, reverse transcription, synthesis of the second-strand cDNA and purification of cDNA fragments was applied following previous protocols [68]. RNA-seq was performed by an Illumina HiSeqTM 4000 platform (Gene Denovo Biotechnology Co., Ltd., Guangzhou, China). Raw reads were filtered according to previous descriptions [68]. Clean reads were assembled using Trinity [69]. The expression level of each transcript was normalized to RPKM [70], and DEGs were analyzed according to a criterion of |log 2 (foldchange)| ≥ 1 and p ≤ 0.05 by DESeq2 software and the edgeR package [71,72]. Unigenes were annotated against the databases including: NR, Swiss-Prot, KEGG, KOG and GO [73].

qRT-PCR Validation
The primer sequence ( Table 2) was designed via a primer-blast in NCBI and synthesized by reverse transcription (Sangon Biotech Co., Ltd., Shanghai, China). First cDNA was synthesized using a RT Kit (KR116, Tiangen, China). PCR amplification was performed using a SuperReal PreMix (FP205, Tiangen, China). Melting curve was analyzed at 72 • C for 34 s. Actin gene was used as a reference control. The RELs of genes were calculated using a 2 −∆∆Ct method [74].

Statistical Analysis
All the measurements were performed using three biological replicates. A t-test was applied for independent samples, with p < 0.05 considered significant.

Conclusions
From the above observations, the stem biomass and polysaccharides accumulation of C. songaricum host in N. roborowskii are significantly greater than that of N. sibirica. A total of 1725 UR and 848 DR genes were observed in CR compared to CS, and 50 DEGs were involved in polysaccharides biosynthesis, which indicates that the polysaccharides biosynthesis in C. songaricum is host-dependent. The specific roles of candidate genes in regulating polysaccharides biosynthesis will require additional studies.
Supplementary Materials: The following are available online. Table supplemental legends: Table  S1: Summary of sequencing data for Cynomorium songaricum transcriptome; Table S2: Primary metabolism genes differentially expressed in CR and CS; Table S3: Transport genes differentially expressed in CR and CS; Table S4: Transcription genes factor differentially expressed in CR and CS; Table S5: Cell morphogenesis genes differentially expressed in CR and CS; Table S6: Bio-signaling genes differentially expressed in CR and CS; Table S7: Stress response genes differentially expressed in CR and CS; Table S8: Translation genes differentially expressed in CR and CS; Table S9: Secondary metabolism genes differentially expressed in CR and CS; Table S10: Photosynthesis and energy genes differentially expressed in CR and CS.