Flavonoid Biosynthesis Pathway Participating in Salt Resistance in a Landrace Sweet Sorghum Revealed by RNA-Sequencing Comparison With Grain Sorghum

Abiotic stresses affect crop productivity worldwide. Plants have developed defense mechanisms against environmental stresses by altering the gene expression pattern which leads to regulation of certain metabolic and defensive pathways. Sorghum [Sorghum bicolor (L.) Moench] is an important crop in those regions irrigated by salty water. Sweet sorghum is a variant of common grain sorghum and is relatively more adapted to marginal growing conditions. Here, we compared the different response to salt stress of sweet and grain sorghum. We investigated six traits related with seed germination under salt-stress and normal conditions, conducted a genome-wide research on the salt effect on the gene expression of a landrace sweet and two grain sorghum by RNA-sequencing at seedling stage. The results showed that salt stress had significant inhibition to sorghum seed germination capability, and the inhibition to grain sorghum was greater. By comparing sweet and grain sorghum and the KEGG pathway analysis based on the DEGs, six genes involved in flavonoid biosynthesis pathway to tannins and anthocyanins from phenylalanine were identified in the landrace sweet sorghum, which expression was significant different with that in grain sorghum. Quantitative real-time PCR (qRT-PCR) data were closely in accordance with the transcript patterns estimated from the RNA-seq data. Tannins accumulation changes were associated with the genes expression under salt stress and control. These suggested that flavonoid biosynthesis pathway was involved in the sorghum resistance to salt stress. The present results suggested that flavonoid biosynthesis plays an important role in the sweet sorghum capacity for salt tolerance.


Introduction
Salt stress is one of the main restrictions to crop production as it affects more than 800 million hectares (ha) of land worldwide (Farooq et al., 2017). Moreover, salinity stress is a major environmental threat to agriculture and its adverse impacts are getting more serious in those salt affected areas (Johnson et al., 1992;Zeng et al., 2001). Salinity stress involves a complex process such as osmotic stress, ionic imbalances, and sets in secondary stresses such as accumulation of toxins, nutritional imbalances and oxidative stress in plants, ultimately causing a reduction in plant growth and development (Munns & Tester, 2008;Türkan & Demiral, 2009). Defense strategies of plants against salinized conditions depend upon activation of cascades of molecular networks involved in stress sensing, signal transduction and the expression of specific stress-related genes and metabolites (Türkan & Demiral 2009;Deinlein et al., 2014;Wang et al., 2010). Understanding the molecular mechanisms of abiotic stress tolerance is therefore critical for developing stress tolerant crop plants and use of saline water in the future. bioenergy production (Carpita & McCann 2008;Rooney et al., 2007;Vermerris, 2011;Regassaand & Wortmann 2014). It is relatively more adapted to marginal growing conditions, such as salinity, alkalinity, water stress and other environmental stresses compared with grain sorghum and maize (Zhang et al., 2011;Michael et al., 2017;Regassa & Wortmann 2014). Its remarkable levels of salinity tolerance make it a good candidate to identify salt-resistance genes.
Many genes expression is induced or suppressed by salt stress, and the study on gene expression regulation has become a hot topic in recent years. The release of genome sequence of sorghum and next/third generation sequencing (NGS) technologies provide a favorable reference to investigate interaction mechanisms between environment stress and sorghum genes response (Paterson et al., 2009;Klára Kosová et al., 2011;Liu et al., 2012;Martin et al., 2011;Suja et al., 2017). This study, therefore, compared sweet and grain sorghum seedlings responses to salt stress based on RNA-sequencing and found genes and pathways in sweet sorghum which may participate in salt stress response by regulation of flavonoid biosynthesis. Our results presented here help us elucidate the salinity stress effects and develop salinity management strategies aimed at enhancing the use of saline water in agriculture based on plant improvements.

Evaluation of Seed Germination Under Salt Stress
Three sorghum varieties were selected as materials; i.e., a landrace sweet sorghum variety, GZ, which has high tolerance to salt; two grain sorghum cultivars, TAx623B (genome sequenced) and HN16, which are relative susceptible to salt. Before germination experiment, the seeds were surface-sterilized using 15% bleach for 20 min and washed subsequently three times with sterile distilled water. 30 seeds from each cultivar were placed in Petri dishes on two layers of filter paper soaked with 10 mL NaCl solution. The filter paper was soaked with 10 mL distilled water in the control. Then the seeds were cultured in an illumination incubator at day/night temperature 30 °C/25 °C with 10-h light/14-h dark photoperiod under relative humidity of 80% for 10 days. Four different NaCl concentrations (40 (S1), 80 (S2), 120 (S3) and 160 mM (S4)) were set in order to compare the sorghum different response to stress and determine the most suitable salt concentration for further experiment. Each treatment was performed three biological replicates. Distilled water was used to supplement the daily evaporation to maintain NaCl concentration. Seeds germination index (GI), vigor index (VI), germination rate potential (GP), germination rate (GR) and inhibition rate (IR) were calculated.
The GR represented the germination percentage at 7 days (GR = Nt/N0 × 100%, Nt is the number of germinated seeds at day 7 and N0 is the total number of experimental seeds). GP was the germination percentage at 4 days after treatment. GI was calculated by the formula: GI = Σ(Gt/Tt) (Gt is the accumulated number of germinated seeds at day t and Tt is the time corresponding to Gt in t days). 10 germinated seeds were chosen to measure the average root length (RL)), root dry weight (RDW) and leaf dry weight (LDW) after 10 days. The seed vigor index (VI) was measured by the formula: VI = S × GI (S is the accumulated fresh root weight). The response to salt stress was evaluated with inhibition ratio (IR): IR = (1 − Ps/Pc), (Ps is mean performance of a cultivar under stress; Pc represents average performance of the same cultivar under control) (Wang et al., 2010).

Plant Culture and RNA Isolation
For salt stress treatments, three-leaves-old sorghum plants were irrigated with 160 mM NaCl. Whole plants were collected at 0, 48 and 72 h after treatment and stored at -80 °C for mRNA isolation (the treatment code was as shown in Table 1). Each collection was replicated three times (every sample consisted of 5 plants). Total sorghum RNA was extracted using Trizol plus RNA kit according to the manufacturer's instructions. The RNA from three replicates was mixed at 1:1:1 ratio. RNA quality and integrity were measured using Nanodrop TM 1000 Spectrophotometer (Thermo, MA, USA). Qualified RNA was used for subsequent RNA library construction and sequencing.

Preparation of RNA Library and Sequencing
Poly (A) mRNAs were purified from total RNA using oligo (dT) magnetic beads and cut into short fragments with fragmentation buffer. These fragments were used as templates for first-strand cDNA synthesis using random hexamer primers. The second strand cDNA was synthesized using Buffer, dNTPs, DNA polymerase I and RNase H. Double-stranded cDNA was purified by AMPure XP beads, and then end-repaired. Sequencing adaptors were ligated to the fragments and the desired size fragments were selected to be enriched by PCR amplification (Wang, et al., 2017). The amplified products were sequenced using an Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA.).

Identification of Differentially Expressed Genes (DEGs) and Gene Annotation
Raw data were quality-filtered with criteria of less than 0.1% low quality bases (Phred score < 30). Adapter sequences and primers were subsequently removed. The filtered reads were aligned to the sorghum reference genome using TopHat2 software (Love et al., 2014). The sequences were further annotated by GO, KOG, Swiss-Prot, KEEG, COG and NR databases with criteria of E-value equals 10 -5 . The GO terms and KEGG pathway analysis were conducted using Blast2Go. Fragments per kilo base of exon per million fragments mapped (FPKM) and DESeq (Anders & Huber, 2010)  (1)

Gene Expression Analysis by qRT-PCR
To evaluate the genes associated with salt stress response mined from the Illumina data, transcriptomes obtained from the comparison between sweet and grain sorghum were selected to validate the RNA-seq results by qRT-PCR with Actin (LOC110436378) as the endogenous control. Gene-specific primer sequences were designed using Primer 3 software and are listed in Table 2. RNA for qRT-PCR analysis was extracted from 0, 48 and 72 h NaCl stressed plants (RNA and cDNA were as same as RNA sequencing). Non-template controls were performed in every reaction. Relative transcription levels were calculated using the 2 -ΔΔCt method (Livak & Schmittgen, 2001). qRT-PCR data shown were averages of three technical replicates for each independent experiment.

Determination of Tannins Content
The seedlings were washed with fresh water, chopped into small pieces and oven-dried (samples were collected at the same time with RNA sequencing). The material was ground to fine powder using an electric grinder. Tannins were determined by the acid butanol assay (Terrill et al., 2010). Tannic acid was also used in this study as standard. Every sample was performed three biological replicates.
Total tannins content was expressed as milligrams tannic acid equivalents per gram of dry plant extract (mg /g) through the calibration curve of tannic acid, whose linearity range was from 10 to 100 mg/mL (R 2 > 0.99).

Data Analysis
Data were analyzed using EXCEL 2003 (Microsoft Corporation, WA, USA) and SPSS 17.0 (IBM SPSS, NY, USA.).

Salt Stress Effects on Seed Germination of Sweet and Grain Sorghum
GI, VI, GR, GP and IR were calculated for 3 sorghum cultivars. The seed germination capability significantly decreased as NaCl concentration increased. The inhibition of salt stress on GR, VI, RDW and RL was significantly different between the sweet and grain sorghum (p < 5%, Table 3, Figure 1). The inhibition of salt stress to the sweet sorghum seedlings was the least, which showed GZ has the best tolerance to salt ( Figure 2). The most inhibition was observed under 160 mM NaCl treatment. Therefore, 160 mM NaCl treatment was used to compare the different responses to salt stress between the sweet and grain sorghum. Whole plants were collected at 48 h and 72 h salt stress and control for RNA-sequencing.     Vol. 11, No. 6; sorghum in the salt stress inhibition. The analyses based on RNA-sequencing, 6 genes involved in flavonoid biosynthesis were found differential expressed in sweet sorghum GZ compared with grain sorghum. Swissprot annotation revealed the 4 up-regulated genes responsible for DFR, LDOX, ANR and F3H enzymes, were all involved in the biosynthetic pathway leading to the synthesis of anthocyanins and condensed tannins (proanthocyanidins). While, the 2 genes coding POD and CCR which are the key enzymes leading phenylalanine to lignin down regulated, which will help phenylalanine to synthesis of anthocyanins and condensed tannins. The anthocyanin biosynthetic pathway is well understood and is conserved among plants, and its key enzymes and genes have been characterized (Besseau et al., 2007;Hichri et al., 2011.). It is involved in the regulation of the environmental stress responses. Anthocyanin and tannins have high antioxidant capacity and help in the natural defense of plants against abiotic stress damage and pests and diseases (Dempsey et al., 2011).
In this study, the genes responsible for F3H, DFR, LDOX and ANR are key enzymes in anthocyanins and condensed tannins biosynthesis, the 2 genes coding POD and CCR were involved in the pathway from phenylalanine to lignin. Phenylalanine is the pre-material of flavonoid biosynthesis and synthesis of lignin. The changes of these genes expression will affect phenylalanine biosynthesis pathway directions and flavonoids accumulation. The RNA-sequencing data showed F3H, DFR, LDOX and ANR expressed significantly higher in sweet sorghum, while the 2 genes coding POD and CCR expressed lower than that in grain sorghum either under normal or in salt stress conditions. These changes of genes expression would result in the accumulation of anthocyanins or tannins. Meanwhile, the results also showed tannins content changes were positively related with the sorghum salt resistance. It is assumed that accumulation of tannins enhances the sorghum capacity for salt tolerance.
It has been documented that flavonoids have important physiological roles in plants, its accumulation is induced by abiotic stresses and is a hallmark of plant stress (Mori et al., 2005;Meng et al., 2015;Gu et al., 2015). For instance, flavonoid accumulation increases under chilling stress in maize, grape, red orange, apple and tobacco (Meng et al., 2015;Christie et al., 1994). In addition, it was observed that salt-tolerant species often accumulate more flavonoids than the salt-sensitive species, which suggested that there exist a relationship between flavonoids biosynthesis and salt stress resistance (Liu et al., 2012). The results of Meng et al. (2015) study in tobacco demonstrated that both chilling stress and SlF3HL (F3H-like protein gene) overexpression induced flavonoid accumulation in tobacco, implying that SlF3HL plays a key function in flavonoid biosynthesis or in regulating this biosynthetic pathway in response to chilling stress. The increased flavonoid contents may contribute to elevating the antioxidant activity of the plant tissues under stress (Piero et al., 2005;Ubi et al., 2006;López Climen et al., 2008). Our results implied flavonoid biosynthesis pathway played an important role in the high salt tolerance in the landrace sweet sorghum.
In conclusion, salt stress had significant inhibition to sorghum seed germination capability and seeding growth. Six genes involved in flavonoid biosynthesis were found based on RNA-Seq in the landrace sweet sorghum. The analysis results suggested that flavonoid biosynthesis pathway played an important funcion in the sweet sorghum high salt tolerance. This could provide a valuable reference for further understanding of sorghum response to saline stress.