Physiological and transcriptome analysis reveal molecular mechanism in Salvia miltiorrhiza leaves of near-isogenic male fertile lines and male sterile lines

Background Our previous study finds that male sterility in Salvia miltiorrhiza could result in stunted growth and reduced biomass, but their molecular mechanisms have not yet been revealed. In this article, we investigate the underlying mechanism of male sterility and its impact on plant growth and metabolic yield by using physiological analysis and mRNA sequencing (RNA-Seq). Results In this study, transcriptomic and physiological analysis were performed to identify the mechanism of male sterility in mutants and its impact on plant growth and metabolic yield. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, it is found that the pathways are mainly enriched in processes including organ development, primary metabolic process and secondary metabolic process. Physiological analysis show that the chloroplast structure of male sterile mutants of S. miltiorrhiza is abnormally developed, which could result in decrease in leaf gas exchange (A, E and gs), chlorophyll fluorescence (Fv, Fm and Fv/Fm), and the chlorophyll content. Expression level of 7 differentially expressed genes involved in photosynthesis-related pathways is downregulated in male sterile lines of S. miltiorrhiza, which could explain the corresponding phenotypic changes in chlorophyll fluorescence, chlorophyll content and leaf gas exchange. Transcriptomic analysis establishes the role of disproportionating enzyme 1 (DPE1) as catalyzing the degradation of starch, and the role of sucrose synthase 3 (SUS3) and cytosolic invertase 2 (CINV2) as catalyzing the degradation of sucrose in the S. miltiorrhiza mutants. The results also confirm that phenylalanine ammonialyase (PAL) is involved in the biosynthesis of rosmarinic acid and salvianolic acid B, and flavone synthase (FLS) is an important enzyme catalyzing steps of flavonoid biosynthesis. Conclusions Our results from the physiological and transcriptome analysis reveal underlying mechanism of plant growth and metabolic yield in male sterile mutants, and provide insight into the crop yield of S. miltiorrhiza.


Background
Male sterility could be the result of abnormal development of stamens of owering plants, which cannot produce normal pollen, but their pistils can still develop normally and receive pollen and fertilize [1,2].As we know, the male sterile could be divided several types: cytoplasmic male sterility (CMS), genic male sterility (GMS) and thermo-photo sensitive genic male sterile (TPSGMS), which are found in crops such as Zea mays, Oryza sativa, Brassica napus and Triticum aestivum, etc. [1,[3][4][5][6].Shu et al. discovered a natural male sterile mutant of Salvia miltiorrhiza in 2002 [7].Through continuous backcross and testcross in 2006-2009, we established that this male sterile mutant of S. miltiorrhiza was GMS type [7,8].Male sterility systems have been widely used in crops such as maize, wheat, and rice [4,9,10].Male sterility plays an extremely important role in heterosis utilization and hybrid seed yield of crops and its use has successfully increased crop yield [11][12][13].S. miltiorrhiza is generally used as a medicinal plant and has been widely used to treat cardiovascular and cerebrovascular diseases, hyperlipidemia, and acute ischemic stroke [14][15][16][17][18].There are many traditional Chinese medicines such as Fufang Danshen Tablets and Danshen injections, etc., which contain active ingredients extracted from S. miltiorrhiza [19].Due to the increasing demand in recent years, its planting area has expanded rapidly.The S. miltiorrhiza cultivation is, however, affected by the factors such as climate and region, which lead to unstable quality of active ingredients.Since the discovery of the male sterile mutants of S. miltiorrhiza, we have created and developed near-isogenic male fertile lines (MF) and near-isogenic male sterile lines (MS) through multiple consecutive testcrosses and backcrosses [20].It promises possibility to achieve stable and controllable quality of active ingredients.
Since the discovery of the male sterile mutants of S. miltiorrhiza in 2002, our team has done some research work [7,20].Researchers have studied the development and biological properties of pollen.The markers tightly linked to drought stress genes have been identi ed and the genetic linkage map between male fertile lines and male sterile lines of S. miltiorrhiza has been constructed by ampli ed fragment length polymorphism (AFLP).The results also nd that male sterility in S. miltiorrhiza could result in stunted growth and reduced biomass.At present, the research on male sterility mainly focuses on the regulation and restoration of fertility, but little attention has been paid to the effect on plant growth and metabolic yield.The molecular mechanism of plant growth and metabolic yield in the male sterile mutants of S. miltiorrhiza has not yet been studied.
The revelation of underlying mechanisms of plant growth and metabolic yield is very important to help the crop improvement.Here, we analyse the primary metabolism in leaves and secondary metabolism (active ingredients) in leaves, owers and roots of S. miltiorrhiza between male fertile lines and male sterile lines.It is shown that primary metabolism in male sterile mutants is decreased but secondary metabolism is boosted.In order to elucidate the molecular mechanism, we performed the transcriptome and physiological analysis of the S. miltiorrhiza leaves of male fertile lines and male sterile lines.Our results from the physiological and transcriptomic analysis reveal underlying mechanism of plant growth and metabolic yield in the plant leaves, and provide insight into crop yield of S. miltiorrhiza.

Plant materials
This work used S. miltiorrhiza leaves of near-isogenic male fertile lines and near-isogenic male sterile lines as experimental materials, which were collected from a botanical garden of S. miltiorrhiza at Northwest A&F University in Yangling, Shaanxi Province, China.The two plant materials (MF S. miltiorrhiza and MS S. miltiorrhiza) used in this paper had been grown under same growth conditions and for same growth years.The other phenotypes of tissues were presented in Additional le 1: Fig. S1A-B.
The leaves of MF and MS were processed under sterile conditions, immediately frozen and stored in liquid nitrogen at -80℃ before the RNA isolation.Leaf subcellular structures and features were observed by transmission electron microscopy (TEM).

Determination of primary metabolites
Polysaccharide, starch, sucrose and proteins were identi ed and estimated by an anthrone-sulfuric acid assay, a perchloric acid hydrolysis, a resorcinol-based assay and a Coomassie Brilliant Blue G-250-based assay.

Determination of secondary metabolites
In this study, we collected 5 whole plants, deactivated their enzymes at 105°C for 15 min, and then dried them to constant weight at 45°C.The dried leaves of S. miltiorrhisa were weighed about 0.1 g, and 4 ml 70% methanol aqueous solution was added.The material stood overnight in the methanol aqueous solution, and ultrasonic extraction was applied for 45 min and the extract was separated by centrifugation.The supernatant was taken for determination of total phenolic, total avonoid and phenolic acids.
Total phenolic was determined using a modi ed method [21].Test samples (1.0 mL) were mixed with 1 mL Folin-Ciocalteu and incubated at room temperature for 3 min.After the addition of 4.0 mL of 7.5% Na 2 CO 3 and 4.0 mL distilled water, the reaction tubes were incubated for 1 h at 40℃ and the absorbance readings were taken at 765 nm.The measurements were done with a calibration curve plotted against gallic acid to establish the content of total phenolic in extracts, whose results are shown as milligrams of gallic acid equivalents per gram of dry extract.The curve equation Y = 0.0128 X + 0.0001 (R 2 = 0.9994) is used, where Y is absorbance and X total phenolic content.
Total avonoid content was measured using a modi ed aluminums chloride colorimetric assay [22].Test samples (1.0 mL) were mixed with 6.4 mL 30% ethanol, 0.3 mL 0.5 M NaNO 2 and 0.3 mL 0.3 M AlCl 3 and kept in the dark for 5 minutes at room temperature.After the addition of 2.0 mL of 1 M NaOH, the absorbance readings were taken at 506 nm.The same measurements and calculations were done to establish total avonoid content.And the gallic acid curve equation is shown as Y = 0.0007 X + 0.0071 (R 2 = 0.9991).
The extraction of phenolic acids was done according to the protocol for total phenolic and total avonoid.The quantitative and qualitative analysis of the phenolic acids were carried out according to reference procedures [23].All chromatographic isolation was performed by using HPLC system equipped with an autoinjector, a UV detector, and LC-solution software (Waters, UK).The column type, column temperature, mobile phase ow rate, mobile phase solvent ratio and program settings are set according to the reference.The injection volume was 10 µl.Spectral data were collected at 280 nm in the whole run.Sample and mobile phase ltration were done at a 0.22 µm lter before a HPLC injection.Analysis of each extract was conducted in triplicate.
RNA extraction, library preparation and mRNA-seq Total RNA was extracted with an RNA pure Plant kit (Tiangen Biotech Co., Ltd., Beijing, China).The total RNA concentration, RIN value, 28S/18S and fragment size were determined using an Agilent 2100 Bioanalyser (Agilent Technologies Co. Ltd., Santa Clara, CA, USA) and an Agilent RNA 6000 Nano Kit.The purity measurement of the samples was made using an ultraviolet spectrophotometer NanoDrop TM .After the isolation and fragmentation of the total RNA, the eukaryotic mRNA was extracted by using Oligo (dT) coupled to magnetic beads.The preparation of cDNA libraries was based on the PCR method described in the following procedure.The rst strand cDNA was manufactured from the fully spliced mRNA as a template, and then a second strand reaction system was used to make double-stranded cDNA.The second strand cDNA was synthesized and puri ed using a system kit.When the cDNA ends were repaired, an 'A' nucleobase hybridized to the 3 ends of the cDNA.Then, the cDNA fragments of different sizes were selected and then ampli ed.The qualities of the ampli ed cDNA libraries were tested.Finally, the sequencing was run on an Illumina HiSeq™ 2500 platform.

FPKM calculation and DESeq2 differential expression analysis
We used the alignment package Bowtie2 (v2.2.5) to align clean reads back to reference genomes [24].
Then the gene expression level of every sample was calculated by RSEM (v1.2.12), a software package designed to estimate gene and isoform expression levels from RNA-Seq data [25].Fragments per kilobase per million (FPKM) mapped reads values were calculated by mapping the reads to fragments, which could be used to quantify the abundance of the transcripts and so to analyse differential gene expression in the samples.Differential expression analysis was performed with DESeq2 package [26].DEGs were selected according to p-value (p ≤ 0.05) and fold change analysis (FC ≥ 2.00).

GO and KEGG enrichment analysis of DEGs
According to the o cial classi cation categories and based on the GO and KEGG annotation results [27], the DEGs are classi ed into functional and biological pathways, and the R function phyper is used for enrichment analysis.A p-value of 0.05 (p ≤ 0.05) implies that the accepted false discovery rate (FDR) is 5%.

RT-qPCR validation and DEGs analysis
To validate internal control genes for expression analysis, the ABI StepOnePlus TM RT-PCR System (Applied Biosystems, USA) was used to perform RT-qPCR and investigate differential expression of gene sets.GenScript Real-time PCR online software was adopted to build sequencing primers, shown in Additional le 2: Table S1.RT-qPCR were carried out using the PrimeScript TM RT reagent Kit and SYBR Green TM Premix Ex Taq TM to transcribe RNA into complementary DNA (cDNA) (Takara Biotech Co., Ltd., Japan) with β-actin used as an internal control.Three independent technical replicates and three biological replicates for each sample were used to validate the performance of RT-qPCR.

Determination of leaf photosynthesis and chlorophyll content
The photosynthetic parameters were determined between 08:00 am to 12:00 am by the portable photosynthesis system (LI-6400, LI-COR Inc., USA) [28].Fluorescence kinetic values of chlorophyll were determined by FluorCam uorometer acquisition system (PDA-100 Heinz Walz, Germany).Chlorophyll uorescence analysis was used to assess the photosynthetic performance of S. miltiorrhiza.Before the measurements, the leaves were kept in the dark for 30 min using cuvettes on ice.A 5 s light pulse at 400 μmol m -2 s -1 was employed.
In this study, about 0.1 g (fully expanded second leaf blade) of the fresh sample was extracted with 80% acetone, and then the chlorophyll content of the leaves was determined by an ultraviolet spectrophotometer.Absorbance of the chlorophyll extractions and blank (80% acetone) were measured at 663 nm and 645 nm.Finally, chlorophyll a, b and total chlorophyll were calculated by Arnon equations.

Statistical analysis
Each experiment was carried out in triplicates.The results were shown as means ± standard error and dealt with using SPSS (version 21.0).ANOVA and Tukey s Honestly Signi cant Difference were used to determine signi cant differences among the samples.A 0.05 signi cance level was considered.

Male sterility inhibiting primary metabolism
Since the discovery of the natural male sterile mutants of S. miltiorrhiza in 2002, a lot of laboratory work has been done to create and develop near-isogenic lines.After many generations of testcrossing, backcrossing and genotyping, our team obtained genetically stable near-isogenic male fertile (MF) and near-isogenic male sterile (MS) lines.Previous results showed that the height of MF S. miltiorrhiza was 1.71 times higher than that of MS ones.In this study, we measured the content of primary metabolites including polysaccharide, sucrose, starch, and protein (Fig. 1A).The results showed very signi cant differences in the content of the primary metabolites in MF and MS S. miltiorrhiza leaves, and the MF S. miltiorrhiza leaves had higher content of primary metabolites than those in MS S. miltiorrhiza leaves.Speci cally, the content of polysaccharide, sucrose, starch and proteins were respectively 27.30%, 14.91%, 15.72% and 18.90% higher in MF plants than in MS plants.

Male sterility promoting secondary metabolites
In previous studies, some phenolic acids have been perceived as the major active ingredients of the aqueous extracts of S. miltiorrhiza, including rosmarinic acid and salvianolic acids B. In all analysed extracts, salvianolic acid B and rosmarinic acid were most plentiful, both recognized as antioxidants.These compounds are cause of the antioxidant activity in both root and leaf extracts, indicating that they are present in the whole plant of S. miltiorrhiza, regardless of their tissue type and location.The avonoids and polyphenols could also play important roles in antioxidant activity, since they are particularly rich in S. miltiorrhiza leaves [29].As a result, the S. miltiorrhiza leaf extract can be used as a powerful herbal antioxidant activity [30].As we know, the leaf biomass of S. miltiorrhiza makes up a signi cant fraction of the whole plant.Nevertheless, during the harvest, the leaves of S. miltiorrhiza are disposed as wastes.The results indicated that the leaves of S. miltiorrhiza could make a natural material for drug, nutritious foods, cosmetics and other industries.
In this paper, we identi ed some of the above-mentioned active ingredients in MF and MS S. miltiorrhiza leaves (Fig. 1B).The results show that the content of these secondary metabolites was higher in MS leaves than in MF leaves.In Fig. 1B, we have found that the relative content of salvianolic acid B, rosmarinic acid, total phenolic and total avonoid are respectively 12.62%, 23.59%, 18.55%, and 23.06% higher in MS leaves than in MF leaves.We have also measured the active ingredients of salvianolic acid B and rosmarinic acid in other tissues (Additional le 1: Fig. S1 C-D).The results suggest that the content of salvianolic acid B and rosmarinic acid are higher in MS owers and roots than in MF owers and roots.In owers, the content of salvianolic acid B and rosmarinic acid in male sterile lines is 17.96% and 21.18% higher than in male fertile lines, respectively.And in the roots, the content of salvianolic acid B and rosmarinic acid in male sterile lines is 18.71% and 32.81% higher than in male fertile lines, respectively.In this article, we have determined the compositions of the tanshinones (including tanshinone I, tanshinone II-A, dihydrotanshinone and cryptotanshinone) in the owers, leaves and roots of S. miltiorrhiza.It has been found that tanshinones are detected only in the roots (Additional le 1: Fig. S1-E), while no tanshinones are detected in the owers and leaves, indicating that tanshinones are tissue speci c.In the roots, the content of tanshinone I, tanshinone II-A, dihydrotanshinone and cryptotanshinone in male sterile lines are 82.58%,82.06%, 72.58% and 110.97% higher than in male fertile lines, respectively.The results indicate that the active ingredients in the MS tissues are more abundant than in the MF tissues.In all, we infer that male sterile mutants have the primary metabolism inhibited but have the secondary metabolism promoted in leaves.However, the underlying mechanisms of plant growth and metabolic yield in the male sterile mutants are still unclear.

The analysis of RNA-seq data
To explore the molecular mechanisms of plant growth and metabolic yield in male sterile S. miltiorrhiza leaves, we used transcriptomics for further research.Six libraries (male fertility: F1, F2 and F3; male sterility: S1, S2 and S3) were prepared, and the preparation of the library for each sample was repeated 3 times.We used the Illumina HiSeq™ 2500 platform to sequence the libraries.The transcriptomic pro le of ltration, reads, ribosomal alignment, and genes coverage are shown in Additional le 3-7: Table S2, Table S3, Table S4, Fig. S2 and Fig. S3, respectively.In Additional le 3: Table S2, we nd that the clean reads for each sample are more than 3.0 Gb.The results in Additional le 4: Table S3 show that the Q20 > 90%, Q30 > 90% and N < 5% after ltering.In Additional le 5: Table S4, the statistics show less than 5% ribosomal RNA sequences of each sample are aligned.The results in Additional le 6-7: Fig. S2 and S3 nd that the majority of genes coverage is in the range of 80% and 100% and most raw reads was classi ed as clean reads.These results demonstrate that the transcriptional pro ling data are reliable for further analysis.After data ltering, reads were aligned to the reference genome and the statistical results are shown in Table 1.The ratio of mapped reads to the reference genome were 95.04%, 91.35%, 92.71%, 91.3%, 94.66%, and 90.84%, respectively.

Analysis of GO, KEGG classi cation and enrichment of DEGs
After parameter screening and optimization (p < 0.05, |log 2 FC| > 1), GO analysis identi ed a total of 1369 DEGs between MF and MS lines.Fig. 2A shows a total of 853 differentially expressed genes are upregulated and 516 downregulated.Through GO analysis of DEGs, 30 signi cantly represented terms are displayed in Fig. 2B.The results show that the metabolic process, cellular process and singleorganism process terms were the most represented GO terms in the biological process category.Cell, cell part, organelle, organelle part and macromolecular complex are the most shared terms in the cellular component category.Catalytic activity and binding are signi cantly represented terms in the molecular function category.Through the deep mining analysis of the data, we selected several GO enrichment pathways that were represented in the data, as shown in Fig. 3A.It has been found that these pathways were mainly enriched in processes related to organ development, primary metabolic process and secondary metabolic process.
According to the KEGG orthology classi cation, DEGs have been annotated into 90 pathways (Additional le 8: Table S5) and divided into ve categories (Additional le 9: Fig. S4).Assignment of DEGs to metabolic pathways provides insight into the biological functions and gene interactions and we nd that the most represented category was metabolism.The results show that the metabolism category is the most annotated, including carbohydrate metabolism, biosynthesis of other secondary metabolites, etc.The bubble map of the DEG pathway enrichment analysis (only the top 20 metabolic pathways shown in Fig. 3B) demonstrates that the metabolic pathways with signi cant enrichment are phenylpropanoid biosynthesis pathway, protein processing in endoplasmic reticulum pathway, avonoid biosynthesis pathway, tyrosine metabolism pathway and carbon xation in photosynthetic organisms pathway.In Fig. 3, the GO and KEGG analyses suggest that their pathway enrichment patterns are basically similar, and those pathways include avonoid biosynthesis and phenylpropanoid biosynthesis.
Abnormal development of chloroplast structure in male sterile mutants Phenotype observation shows that the leaves of the male sterile lines are signi cantly smaller than those of the male fertile lines (Fig. 4A-B).Previous studies in the laboratory have found that the leaves of the male fertile lines are 1.46 times as long and 1.38 times as wide as those of the male sterile lines (25 samples from each line).Differences in organ development have been found in the GO annotation results of the transcriptomic data.Furthermore, the subcellular structure of leaves was observed by transmission electron microscopy.Interestingly, we nd that the chloroplast structure of the two samples is signi cantly different, which is shown in Fig. 4C-D.The results show that the chloroplast structure of male fertile leaves is fully developed and the thylakoid structure can be clearly seen.However, thylakoid structures could not be observed in the leaves of male sterile mutants.In addition, there is no spindle-like chloroplast in the male sterile leaves.Therefore, the results suggest that the chloroplast development of male sterile leaves might be incomplete.

Reduction of leaf gas exchange characteristics, chlorophyll uorescence parameters and photosynthesis pigments in male sterile mutants
In this study, we have measured the photosynthetic indicators of S. miltiorrhiza leaves of the male fertile lines and male sterile lines, and found that all the values of the indicators including the net photosynthetic rate (A), transpiration rate (E) and stomatal conductance (g s ) of the male fertile lines are higher than those of the male sterile lines except intercellular CO 2 concentration (C i ) in S. miltiorrhiza (Fig. 5A).The results show that the abnormal chloroplast structure points to the reduced photosynthesis.The results also indicate that the net photosynthetic rate is positively correlated with the stomatal conductance.
Using chlorophyll uorescence, a natural probe emitted from plants, can detect a lot of information about the state of plant growth [31].By comparing the chlorophyll uorescence detected from male fertile and male sterile plant leaves, it is found that there are signi cantly variations in F m , F v and F v /F m between the two lines except F 0 (Fig. 5B), and they are positively correlated with their growth and photosynthesis.This indicates that a higher growth rate is closely related to higher PSII potential viability and higher light conversion e ciency.These studies have fully demonstrated that chlorophyll uorescence could sensitively re ect the growth and development of plants, so it might become one of the indicators for predicting plant growth potential.
Furthermore, we have determined the photosynthetic pigment content of the S. miltiorrhiza leaves of male fertile and sterile lines.Our results nd that the chlorophyll a content and chlorophyll a+b content of male fertile and sterile leaves are signi cantly different, but there is no signi cant difference found in chlorophyll b content in both leaves (Fig. 5C).

DEGs related to photosynthesis and carbon xation in photosynthetic organisms
To understand the incomplete development of chloroplast in leaves of MS S. miltiorrhiza, we have analysed the DEGs which are related to photosynthesis and carbon xation in photosynthetic organisms (Fig. 6).We identify three photosynthesis-related genes, which include one PSI gene (PsaB), one PSII gene (PsbC) and one photosynthetic electron transport gene (PetF), have been differentially expressed between MF and MS lines.And we also identify other 6 genes, encoding 6 enzymes responsible for carbon xation, which include fructose-bisphosphate aldolase (FBA), ribose 5-phosphate isomerase A (RpiA), ribulose 1,5-bisphosphate carboxylase small subunit (rbcS), alanine transaminase (ALT), phosphoenolpyruvate carboxylase (PPC) and fructose-1,6-bisphosphatase (FBP), have signi cantly differential expression between the MF and MS lines.
In Fig. 6, most of these genes are downregulated in male fertile lines.For example, FBA, RpiA, rbcS, ALT, FBP, PsaB and PsbC are downregulated in male sterile lines of S. miltiorrhiza.However, two genes (i.e., PPC and PetF) are upregulated in male sterile lines of S. miltiorrhiza.Most of these enzymes are downregulated in male sterile lines of S. miltiorrhiza and these results are consistent with the phenotypic changes including net photosynthetic rate, chlorophyll uorescence, and chlorophyll content (Fig. 5).
DEGs involved in sucrose-starch metabolism pathway between male fertile lines and male sterile lines of S. miltiorrhiza In order to further explore the metabolic pathways, we used MapMan program to analyse the transcriptome data.MapMan is specialized in functional classi cation of genes in metabolic pathways and biological processes in organisms [32,33], which is excellent at visualizing gene function classi cation and gene expression data.Mapping les are obtained from Mercator Automated Sequence Annotation using nucleic acid sequences of S. miltiorrhiza (Additional le 10: Excel S1).We nd that there are signi cant differences (Fig. 7) in sucrose synthase 3 (SUS3), cytosolic invertase 2 (CINV2), alpha amylase (AMY) and disproportionating enzyme 1 (DPE1) in the sucrose-starch metabolism pathway.It is inferred that the relative content of sucrose and starch is lower in the male sterile mutants than in male fertile plants (Fig. 1A).In Fig. 7, the DEGs between the two lines are mainly involved in the degradation process of sucrose and starch.
DEGs involved in phenylpropanoid metabolism pathway between male fertile lines and male sterile lines of S. miltiorrhiza The phenylpropanoid metabolism pathway has important physiological signi cance in plants.And its intermediate products and its further products are closely related to physiological activities such as differentiation of cells in plant development, resistance to pathogen infection, and formation of pigmentation [34].We have also used MapMan program to analyse the phenylpropanoid metabolism pathway, and the results are shown in Fig. 8 and Additional le 11: Fig. S5.It could be seen that there are signi cant differences in the key enzymes of the phenylpropanoid metabolism pathway, which include phenylalanine ammonialyase (PAL) in general phenylpropanoid pathway, caffeic acid Omethyltransferase (COMT) and ferulic acid-5-hydroxylase (F5H) in lignin biosynthesis pathway, and avonoid 3 -hydroxylase (F3 H), avonol synthetase (FLS), dihydro avonol 4-reductase (DFR) and avonol 3-O-glucosyltransferase (F3oGT) in avonoid biosynthesis pathway.PAL, an enzyme in the phenylpropanoid pathway, is involved in physiological processes such as anthocyanin accumulation, ligni cation, avonoid synthesis, and pathogen defenses.We nd that the expression of PAL is signi cantly upregulated in the leaves of male sterile mutants and PAL has a signi cant positive correlation with rosmarinic acid and salvianolic acid B content.Therefore, the result con rms that PAL plays an important role in the synthesis of phenolic acid compounds in S. miltiorrhiza.Our results show that most of the genes in the male sterile mutants are signi cantly upregulated.Overexpression of FLS gene has effect on the accumulation of avonoids [35].Therefore, FLS gene could be a key enzyme encoding gene involved in the avonoid biosynthesis.To validate the reliability of the transcriptome sequencing data, the sequences of seven important DEGs related to the phenylpropanoids pathway and the sucrose-starch metabolism pathway were analysed with RT-qPCR.The results of the RT-qPCR analysis exhibit a close similarity to the RNA-Seq results, as shown in Additional le 12: Fig. S6.

Discussion
Our results indicate that the male sterile mutants have reduced primary metabolism, but increased secondary metabolism in leaves.In order to understand the molecular mechanism for growth and metabolism in the male sterile mutants, we performed a comparative transcriptome and physiological analysis of the S. miltiorrhiza leaves between male fertile lines and male sterile lines.The height and biomass of male sterile lines are signi cantly less than that of male fertile lines, which might be responsible for signi cant change in the values of photosynthesis-related indicators, including leaf gas exchange (A, E and g s ), chlorophyll uorescence (F v , F m and F v /F m ) and chlorophyll content.Our ndings are consistent with those of previous studies that conclude photosynthesis is reduced in male sterile mutants [36].Additionally, we identify many DEGs related to photosynthesis, carbon xation in photosynthetic organisms, sucrose-starch metabolism and phenylpropanoid metabolism pathway through RNA-seq analysis between male fertile lines and male sterile lines.
In Fig. 4C, we nd structural abnormalities in the chloroplasts in male sterile mutants.Chloroplasts are highly specialized organelles in higher plants where photosynthesis including important metabolic pathways takes place [37].Photosynthesis is the basic source of plant biomass yield.The difference in photosynthetic characteristics between varieties is often one of the direct causes of difference in growth.
The analyses of leaf gas exchange characteristics, chlorophyll uorescence indicators and photosynthesis pigments demonstrate that the photosynthetic capacity is signi cantly reduced in the MS mutants compared with the MF S. miltiorrhiza (Fig. 5).Consistent with this observation, the DEGs were signi cantly enriched in the photosynthesis pathway.In Fig. 5A, we nd that all the values of the indicators except intercellular CO 2 concentration (C i ) suggested photosynthesis was reduced in the male sterile lines of S. miltiorrhiza.The research shows that net photosynthetic rate is 50% lower in male sterile mutants of Nicotiana sylvestris [36].Other researchers have also reported signi cant reduction of photosynthetic rate in other male sterile plants [38,39].Our results show that the chloroplast structure is abnormal and the performance of photosynthesis is consistent with other research results [40].The results also show that the net photosynthetic rate is positively correlated with the stomatal conductance, which agrees with the statistical analysis of other 14 species [41].90% -95% plants dry matter of the above-ground parts comes from photosynthesis.Therefore, the most important thing to increase yield is to increase the utilization e ciency of light energy, the photosynthetic rate of plants, and the yield per unit area.In Fig. 5B and 5C, we nd that the value of chlorophyll uorescence indicators (F m , F v and F v /F m ) and chlorophyll content are lower in the male sterile lines of S. miltiorrhiza.It is found that the chlorophyll content is correlated to the photosynthetic activity.The morphological structures and physiological functions of the plants are correlated closely, which can be proved by the fact that the photosynthetic processes are correlated to the morphological characteristics of the photosynthetic organs of the leaves.Therefore, the results indicate that one of the causes for the stunted growth, decreased biomass and decreased primary metabolism in the male sterile S. miltiorrhiza mutants could be due to the abnormal chloroplast structure, which signi cantly reduces the plant's physiological activities and performances such as gas exchange and chlorophyll uorescence and chlorophyll content.
In this study, we nd that the chloroplast structure in the male sterile mutants of S. miltiorrhiza is not fully developed.The abnormal development of chloroplast structure in male sterile mutants might be related to DEGs involved in photosynthesis-related processes.In Fig. 6, a detailed analysis of the RNA-seq data indicates that 1, 1, 1 and 6 genes, which are usually related to PSI, PSII, photosynthetic electron transport and carbon xation in photosynthetic organisms, respectively, are signi cantly differentially expressed between two lines.The most of these genes are downregulated in male sterile lines of S. miltiorrhiza.These results are consistent with the phenotypic changes including photosynthesis rate, chlorophyll uorescence, and chlorophyll content (Fig. 5).Photosynthesis is considered a major determinant of yield [42]. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities are associated with photosynthetic rate-limiting steps [43,44].A comprehensive analysis of our RNA-seq data reveals that the expression level of rbcS is sharply decreased in male sterile lines (Fig. 6).FBA is a key enzyme in plants that is involved in gluconeogenesis, glycolysis, and the Calvin cycle.FBA genes play signi cant roles in regulating growth and development [45].Reduction in sucrose content caused by downregulation of FBP results in tiller outgrowth cessation in rice mutants [46].Thus, the abnormal development of chloroplast structure in male sterile mutants might be related to the downregulated enzymes (i.g., rbcS, FBA and FBP) in photosynthesis-related processes.
In Fig. 7, the DEGs are mainly involved in the degradation process of sucrose and starch.Therefore, we select the genes as important genes which encode enzymes SUS3, CINV2 and DPE1 that are signi cantly upregulated in the sucrose-starch metabolism pathway.In most of higher plants, sucrose is a main sugar that moves from leaves through phloem to other tissues.Sucrose then needs to be hydrolyzed into hexoses by either INV or SUS [47], and our results just con rm this conclusion.Researchers have found that lack of DPE1 in plants causes slightly more accumulation of starch in the leaves than in the control plants [48], which agrees with our results.Therefore, the results suggest that one of the reasons for the decrease in sucrose and starch in male sterile mutants of S. miltiorrhiza might be due to fast degradation of starch, which is catalyzed by DPE1 and fast degradation of sucrose, which is catalyzed by SUS3 and CINV2.
In Fig. 8, we nd that there is a signi cant upregulation of PAL in S. miltiorrhiza leaves of the male sterile mutants.Researchers have found that PAL has a signi cant positive correlation with rosmarinic acid and salvianolic acid B content, and our results match this conclusion [49].Therefore, PAL plays an important role in the synthesis of phenolic acid compounds in S. miltiorrhiza.In this study, it is also found that male sterile mutants have higher relative content of total avonoid and total phenolic (Fig. 1B).Flavonoids and polyphenols are common secondary metabolites in higher plants and play an important role in diverse activities.For example, they can protect plants from pathogenic infection, provide owers with pigment to attract pollinators, and reduce the risk of oxidative damage to human health [50][51][52].In Fig. 8, we select the FLS gene, whose expression is signi cantly upregulated, as an important gene which is involved in the avonoid biosynthesis pathway.The expression of FLS gene requires activation of the MYB transcription factor [53]. Upregulated expression of FLS gene affects the accumulation of avonoids [35].Therefore, FLS gene could be a key enzyme encoding gene affecting the synthesis of avonoids.In addition, our research on the genes related to the metabolic pathways could provide useful information for metabolic engineering strategies to control the overall metabolic ux of potent antioxidants that have bene cial implications for human health.

Conclusions
In conclusion, our results show that the male sterile lines of S. miltiorrhiza have lower primary metabolism and more active secondary metabolism in leaves.In order to understand the molecular mechanism of plant growth and metabolic yield in the male sterile mutants, we performed the transcriptome and physiological analysis of the S. miltiorrhiza leaves of male fertile lines and male sterile lines.The differences in physiological processes and activities between the two line lie with the different development of their chloroplast structures.The abnormal chloroplast structure of male fertile leaves decreases the leaf gas exchange (A, E and g s ), chlorophyll uorescence (F v , F m and F v /F m ) and chlorophyll content.The results con rm that DPE1 catalyzes the degradation of starch, and SUS3 and CINV2 catalyzes the degradation of sucrose in S. miltiorrhiza.At the same time, the results suggest that PAL plays a vital role in the biosynthesis of rosmarinic acid and salvianolic acid B and FLS is essential for the biosynthesis of avonoids.Taken together, our results from the physiological and transcriptomic analysis reveal underlying mechanisms of plant growth and metabolic yield in the male sterile mutants.

Table 1
The statistics of reference genome alignment results