
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)11997-4
10.1016/j.heliyon.2024.e35966
e35966
Research Article
Physiological, transcriptomic, and metabolomic analyses reveal that Pantoea sp. YSD J2 inoculation improves the accumulation of flavonoids in Cyperus esculentus L. var. sativus
Wang Saisai abc1
Huang Yanna abc1
Sun Yu d
Wang Jinbin d
Tang Xueming xueming.tang@sjtu.edu.cn
abc⁎
a School of Agriculture and Biology, Shanghai Jiao Tong University, Dongchuan Road 800, Minhang District, Shanghai, 200240, PR China
b Key Laboratory of Urban Agriculture, Ministry of Agriculture and Rural Affairs, Dongchuan Road 800, Minhang District, Shanghai, 200240, PR China
c Shanghai Yangtze River Delta Eco-Environmental Change and Management Observation and Research Station, Ministry of Science and Technology, Ministry of Education, Dongchuan Road 800, Minhang District, Shanghai, 200240, PR China
d Biotechnology Research Institute Key Laboratory of Agricultural Genetics and Breeding, Shanghai Academy of Agricultural Sciences, Beidi Road 2901, Minhang District, Shanghai, 201106, PR China
⁎ Corresponding author. School of Agriculture and Biology, Shanghai Jiao Tong University, Dongchuan Road 800, Minhang District, Shanghai, 200240, PR China. xueming.tang@sjtu.edu.cn
1 Sai-Sai Wang and Yan-Na Huang contributed equally to this work.

08 8 2024
30 8 2024
08 8 2024
10 16 e3596628 2 2024
1 7 2024
7 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Plant growth-promoting microorganisms (PGPMs), such as Pantoea sp. YSD J2, promote plant development and stress resistance, while their role in flavonoids accumulation still needs to be further understood. To investigate the complex flavonoid biosynthesis pathway of Cyperus esculentus L. var. sativus (tigernut), we compared Pantoea sp. YSD J2 inoculation (YSD J2) and water inoculation (CK) groups. YSD J2 significantly elevated the content of indole-3-acetic acid (IAA) and orientin. Furthermore, when analyzing flavonoid metabolome, YSD J2 caused increased levels of uralenol, petunidin-3-O-glucoside-5-O-arabinoside, luteolin-7-O-glucuronide-(2 → 1)-glucuronide, kaempferol-3-O-neohesperidoside, cyanidin-3-O-(2″-O-glucosyl)glucoside, kaempferol-3-O-glucuronide-7-O-glucoside, quercetin-3-O-glucoside, luteolin-7-O-glucuronide-(2 → 1)-(2″-sinapoyl)glucuronide, and quercetin-4′-O-glucoside, which further enhanced antioxidant activity. We then performed RNA-seq and LC-MS/MS, aiming to validate key genes and related flavonoid metabolites under YSD J2 inoculation, and rebuild the gene-metabolites regulatory subnetworks. Furthermore, the expression patterns of the trans cinnamate 4-monooxygenase (CYP73A), flavonol-3-O-L-rhamnoside-7-O-glucosyltransferase (UGT73C6), shikimate O-hydroxycinnamoyltransferase (HCT), chalcone isomerase (CHI), flavonol synthase (FLS), and anthocyanidin synthase (ANS) genes were confirmed by qRT-PCR. Additionally, 4 transcription factors (TF) (especially bHLH34, Cluster-37505.3) under YSD J2 inoculation are also engaged in regulating flavonoid accumulation. Moreover, the current work sheds new light on studying the regulatory effect of Pantoea sp. YSD J2 on tigernut development and flavonoid biosynthesis.

Highlights

• Pantoea sp. YSD J2 inoculation significantly elevated the content of indole-3-acetic acid (IAA) and orientin.

• Pantoea sp. YSD J2 caused increased levels of nine differential metabolites, further enhancing antioxidant activity.

• The flavonoid synthesis-related structural genes and metabolites were validated.

• Four transcription factors under YSD J2 inoculation are engaged in regulating flavonoid accumulation.

• The interaction of host plants-PGPMs is a new strategy to increase flavonoids production.

Keywords

Tigernut
Plant growth-promoting microorganisms
Flavonoid biosynthesis
Metabolome
Transcriptome
Abbreviations

ANOVA One-way analysis of variance

ANR anthocyanidin reductase

ANS anthocyanidin synthase

BZ1 anthocyanidin 3-O-glucosyltransferase

C. esculentus L. Cyperus esculentus L, var. sativus

CCoAOMT coffee acyl coenzyme A-O-methyltransferase

CHI chalcone isomerase

CHS chalcone synthase

CHR chalcone reductase

CYP71D9 flavonoid 6-hydroxylase

CYP73A trans cinnamate 4-monooxygenase

CYP75A flavonoid 3′,5′-hydroxylase

CYP81E isoflavone/4′-methoxyisoflavone 2′-hydroxylase

CYP93G1 flavone synthase II

C12RT1 flavanone 7-O-glucoside 2″-O-beta-L-rhamnosyltransferase

DEGs differentially expressed genes

DEMs differential metabolites

DFR bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase

F3H naringenin 3-dioxygenase

FPKM fragments per kilobase of transcript per million mapped reads

FLS flavonol synthase

GT1 anthocyanidin 5,3-O-glucosyltransferase

HCT shikimate O-hydroxycinnamoyltransferase

HIDH 2-hydroxyisoflavanone dehydratase

IAA indole-3-acetic acid

IF7MAT isoflavone 7-O-glucoside-6″-O-malonyltransferase

LAR leucoanthocyanidin reductase

MDA malondialdehyde

MRM multiple reaction monitoring

MSEA metabolite sets enrichment analysis

OPLS-DA orthogonal partial least squaresdiscriminant analysis

PAL Phenylalanine ammonia-lyase

PGPMs Plant growth-promoting microorganisms

PTS pterocarpan synthas

POD peroxidase

qRT-PCR quantitative real-time polymerase chain reaction

RNA-seq RNA sequencing

SOD superoxide dismutase

TIC Total ion chromatography

TF Transcription factor

UPLC-MS/MS ultra-high performance liquid chromatography coupled with tandem mass spectrometry

UGT73C6 flavonol-3-O-L-rhamnoside-7-O-glucosyltransferase

VIP variable importance in projection

5MaT1 anthocyanin 5-O-glucoside-6‴-O-malonyltransferase
==== Body
pmc1 Introduction

Flavonoids, derived from phenylpropanoid compounds, belong to secondary metabolites extensively discovered from plants [1]. According to the degree of oxidation of heterocycles and hydroxyl or methyl group number on benzene ring, flavonoids are categorized as the following 12 groups, including chalcones, stilbenes, aurones, flavanones, flavones, isoflavones, phlobaphenes, dihydroflavonols, flavonols, leucoanthocyanidins, proanthocyanidins, and anthocyanins [2]. Flavonoids have enormous potential in food and medicine fields due to their extensive biological activities, including antioxidant [3], anti-inflammation [4], anti-virus [5], anti-cancer [6], liver protection [7], anti diabetes [8], antidepressant [9], neuroprotective [10], and cardiovascular protection [11]. Furthermore, flavonoids have extensive distribution and vital impacts in plant growth, adaptation, signaling, as well as biotic and abiotic stress responses [12].

Cyperus esculentus L. var. sativus (tigernut) is a kind of traditional perennial C4 plant of the Cyperaceae family [13]. Tigernut contains three parts: aboveground leaves (stems), underground tubers, and roots. Previous studies have indicated that its valuable components in stems, such as lactones, flavonoids, and coumarins, along with the corresponding steroids, glycosides, cardiac glycosides, and triterpenoid [[14], [15], [16]], have pharmaceutical effects such as antioxidation and mitigation of cerebral ischemia injury and are expected to be new drug candidates for treating ischemic stroke [17,18]. Study of tigernut flavonoids is still in the early stages of development. Till the present, efforts to detect the flavonoid compositions of tigernut stems have increased significantly. Nevertheless, only a handful of flavonoid components (such as orientin, isoorientin, and luteolin, etc.) have been successfully detected and isolated [19,20]. Furthermore, such limitations may probably hinder the growth of tigernut utilization in the food industry and medicine field. Therefore, there is a need to obtain more information about flavonoids of tigernut to improve its utilization efficiency.

Flavonoids have broad application prospects due to their various biological activities. However, currently the acquisition of flavonoids is still limited to direct plant extraction and chemical synthesis [21,22]. Traditional chemical synthesis and plant extraction in flavonoid production have been impeded due to a slow dearth of fuel, acute scarcity of land, heightened environmental protection awareness, and rising production expenses. Despite the microbial approach to flavonoid biosynthesis being straightforward and safe, its minimal yield necessitates additional refinement and enhancement. Recently, many studies have been conducted to explore how plant growth-promoting microorganisms (PGPMs) affect the accumulation of active ingredients [23,24]. Long-term host plant-PGPMs interactions and coevolution typically boost plant growth, enhance resistance to pathogens, persistently accelerate plant active ingredient accumulation, and improve plant quality and productivity [25,26]. It has been shown that inoculating PGPMs leads to changes in plant physicochemical characteristics, such as weight, height, antioxidant enzyme activities, and various levels of functional components [[27], [28], [29], [30]]. This is exemplified by using Pantoea sp. YSD J2, a PGPMs isolated in tigernut stem, which improved greengrocery growth arrtitubes and antioxidant activity [31]. Several studies have found that PGPMs inoculation results in the accumulation of flavonoids [32,33], and PGPMs-mediated flavonoids mechanisms were also studied [34]. Despite previous research efforts, but different interactions can occur in different plants, the interaction between tigernut and PGPMs to promote the accumulation of flavonoids and their regulatory mechanisms are yet to be explored.

Therefore, to systematically study the pathways, species, and differential genes of PGPMs-mediated secondary metabolite accumulation in medicinal plants, a PGPM, Pantoea sp. YSD J2, were selected for this study. Pantoea sp. YSD J2 showed good PGP characteristics and growth and metabolism-related abilities [31]. In the current work, flavonoid content and antioxidant activity were analyzed under Pantoea sp. YSD J2 inoculation. Then transcriptome combined with metabolome data were used to (1) explore the mechanism underlying gene and metabolite levels in response to Pantoea sp. YSD J2 inoculation; (2) screen critical regulatory genes associated with flavonoid biosynthesis pathways in response to Pantoea sp. YSD J2 inoculation; and (3) construct a regulatory network based on flavonoid biosynthesis as well as associated gene levels. To some extent, the obtained findings offer the experimental and theoretical foundation for investigating the Pantoea sp. YSD J2-mediated regulation on the growth and flavonoid biosynthesis of tigernut.

2 Materials and methods

2.1 Plant materials and treatments

Tigernut used in the present study is a round grain type variety (Zhongyousha I). Tigernut seeds were purchased in Baoding, Hebei Province of China (115°3′11″E, 38°21′9″N). Seeds that had been sterilized (soaked in 70 % ethanol for 10 min and 5 % NaClO for 5 min) were sprouted in a clean germination container lined with cotton and filter paper. Once the seedlings sprouted their third leaf, carefully select seedlings of the same size and transplant them back into a greenhouse irrigated with tap water without any fertilizer. Tigernut samples were collected every 7 days (five for each timing). During the sampling process, the seedlings of tigernut were carefully dug out, followed by repeated washing of the roots with distilled water. The height of each plant was measured separately. All samples were cut into 5–10 cm fragments and immersed in liquid nitrogen before being sent to the laboratory, followed by preservation under −80 °C.

2.2 Orientin content measurement

Orientin was extracted according to previous description by Wang et al. [35]. Briefly, orientin content in tigernut stem was determined using a high-performance liquid chromatography (HPLC) (Agilent Company, USA). Chromatographic column was the Agilent Zorbax SB-C18 column (particle size, 5 μm; 4.6 mm × 250 mm). Solvent A (0.5 % acetic acid) and solvent B were the mobile phases (acetonitrile, Merck, Germany). Gradient elution: 0～35 min, A-B (85:15, V·V−1) to A-B (45:55, V·V−1). A 10 μL injection volume was injected at the 1.0 mL min−1 rate and the 340 nm UV detection wavelength.

2.3 Pot experiments and treatments using Pantoea sp. YSD J2

Strain YSD J2 is an endophytic bacterium isolated from the stem of tigernut in 2019, identified as Pantea sp. [31], and the Gen Bank entry number of the 16s rDNA gene is OK090942. After the surface sterilization treatment, the seeds of tigernut were immersed into distilled water for five days under ambient temperature and later positioned in the culture dish covered with filter paper for germination. Seven days later, seedlings exhibiting identical growth patterns were chosen for the pot experiment. In every pot, five seedlings received irrigation using 20 mL of bacterial suspensions at a concentration of 1.25 × 108 CFU/mL, maintained at an average temperature of 28.0 °C and 14 h/10 h light/dark cycle. Water was added every 2–3 d. Moreover, at the end of the experiment (sampling at 102 d), plants were carefully removed from each pot, indole-3-acetic acid (IAA) content was identified. IAA detection was done in line with procedure as previously described [12]. Antioxidant abilities including malondialdehyde (MDA) level, superoxide dismutase (SOD), and peroxidase (POD) activities were identified based on procedure according to the previous description [31,36].

2.4 Metabolite profiling by UPLC-MS/MS

The flavonoid metabolites of seedlings inocubated with YSD J2 and control group (CK) were measured. In this study, the ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) system (UPLC, Shim-pack SHIMADZU Nexera X2, SHIMADZU, Kyoto, Japan; MS/MS, Applied Biosystems 4500 Q TRAP, AB SCIEX, Foster City, CA, USA) was adopted for analyzing flavonoid extracts as follows: UPLC column, Agilent SB-C18 1.8 μm, 2.1 mm × 100 mm; solvent system, ultrapure water (supplemented with 0.1 % formic acid) as phase A, whereas acetonitrile (that contained 0.1 % formic acid) as phase B; gradient program, 95:5 V/V phase B at 0.00 min, 5:95 V/V at 9.00 min, 5:95 V/V at 10.0 min, 95:5 V/V at 11.1 min, and 95:5 V/V at 14 min; temperature, 40 °C; flow rate, 0.35 mL/min; injection volume: 4 μL. In addition, mass spectrometry (MS) data were analyzed as described by Zhou [37].

2.5 Metabolites qualitative and quantitative analyses

According to second-order spectral data, metabolite characterization was performed. We obtained metabolites using multiple reaction monitoring (MRM) methods following previous description [33]. After filtration, metabolite data were exposed to orthogonal partial least squares-discriminant analysis (OPLS-DA) using analyst 1.6.1 software. Subsequently, we used variable importance in the projection (VIP) parameter to check relative importance of different metabolites of OPLS-DA model. By applying R software (www.r-project.org), we analyzed metabolites from each sample using hierarchical cluster analysis. Metabolites under significant regulation in the two groups were analyzed according to the thresholds of |log2FC (fold change)| ≥ 1 and VIP ≥1. Subsequently, based on KEGG compound database, we annotated those detected metabolites and subsequently mapped then into KEGG pathway database. Later, metabolite set enrichment analysis (MSEA) of pathways mapped by significant metabolites was conducted, and p-values from hypergeometric test were determined in order to analyze significance.

2.6 RNA-seq analysis

Total RNAs were isolated from tigernut frozen stems, followed by the construction and sequencing of the mRNA library in every sample using the Illumina HiSeq 2000 platform. Later, adapter and low-quality sequences were eliminated with Fastp under the default parameters [38] to obtain clean reads, which were later assembled in expressed sequence tag clusters (contigs) and de novo assembly in transcripts with the trinity. Gene and transcript expression was explored by fragments per kilobase of transcript per million mapped reads (FPKM). In addition, we also detected differentially expressed genes (DEGs) through adopting binom test, and the DESeq function estimated size factors based on the thresholds of FC > 2 or < 0.5 and p < 0.05. The raw sequencing data were imported into NCBI BioProject database (project number, PRJNA1003814).

2.7 Quantitative real-time PCR assay

Eight flavonoid biosynthesis-related DEGs (CYP73A (Cluster-49537.1), CHI (Cluster-37365.0 and Cluster-50548.7), ANS (Cluster-49536.4 and Cluster-43695.0), FLS (Cluster-25595.0), HCT(Cluster-44099.0), and UGT73C6 (Cluster-51835.5)) were chosen for further verification using qRT-PCR. To this end, we extracted total RNA from tigernut stems following the instructions of the manufacturer (Magen kit, China). cDNA was prepared by total RNA by adopting Thermo Scientific Maxima RT reagent Kit (Thermo Scientific, MA, USA) through reverse transcription. qRT-PCR was conducted with 2X SG Fast qPCR Master Mix (High Rox, BBI, China) using the QuantStudio™ 1 Plus System (Thermo Scientific, MA, USA). In addition, three biological and three technical replicates were used. Table S1 displays the primers used in this study. To be specific, a 20 μL system was prepared for qRT-PCR, containing 10 μL SYBR qPCR Master Mix (2X), 0.4 μL of respective primers (10 μM each), 7.2 μL ddH2O and 2 μL cDNA template (5 ng/μL). PCR conditions were 3-min denaturation at 95 °C, 15-s amplification at 95 °C, and 30-s at 60 °C for 45 cycles. Then, melting curve measurements were carried out for 15s at 95 °C and 1min at 60 °C, using the 18S rRNA gene being the reference gene. Each sample was analyzed in triplicate. The 2–△△Ct approach was adopted for analyzing the quantitative results.

2.8 Statistical analysis

Data were subjected to calculated mean ± standard deviation. Origin 2022 was applied to draw curves and histograms. Statistical analyses were conducted using the SPSS 22.0 software (IBM, Chicago, IL, USA). Differences between samples were determined by one-way analysis of variance (ANOVA) and significant differences were calculated by Duncan's multiple range test at p < 0.05.

3 Results

3.1 Improved growth in tigernut inoculated with Pantoea sp. YSD J2

To explore how Pantoea sp.YSD J2 might influence flavonoid production in tigernut, both non-inoculated Pantoea sp.YSD J2 (CK) and inoculated Pantoea sp.YSD J2 (YSD J2) were chosen as experimental materials. Initially, the growth statement and flavonoid level of tigernut without YSD J2-inoculation (CK) was examined to determine the time point at which the maximum amount of flavonoid synthesized. The present study first analyzed plant growth attributes and orientin level of tigernut at different growth stages (Table S2). As a result, plant height, plant fresh weight, and orientin content of tigernut increased over time. At 102 d, the plant height and plant fresh weight reached 125.80 ± 4.98 cm and 131.40 ± 6.53 g, respectively, and orientin content was 11.07 ± 0.34 mg L−1. From 102 d to 127 d, the orientin content initially decreased and then increased with time. At 127 d, the orientin content reached 8.18 ± 1.20 mg L−1, and the plant height reached the maximum of 131.76 ± 16.33 cm. From 127 d onward, the two indicators remained over time. The plant fresh weight showed a stable upward trend after 102 days and reached the maximum of 145.00 ± 5.70 g at 151 d. Therefore, based on the results of three indicators, 102 nd day was finally determined as the sampling time points of tigernut for exploring how YSD J2 affected flavonoid synthesis of tigernut.

The IAA content, orientin content, and antioxidant activities of tigernut with YSD J2 were tested. YSD J2 elevated the levels of IAA and orientin by 65.74 % (Fig. 1A) and 212.53 % (Fig. 1B) compared to CK, respectively. Additionally, YSD J2 exhibits a significant reduction in MDA levels (Fig. 2C), alongside a marked rise in POD (Fig. 2B) and SOD activities (Fig. 2A) compared to CK. These three enzyme families exhibited patterns akin to those of IAA and orientin, indicating that the elevation in orientin mediated by YSD J2 may be engaged in enhancing the antioxidant activity of tigernut.Fig. 1 Phenotype, IAA content, and orientin content of tigernut in response to Pantoea sp. YSD J2 inoculation (YSD J2). Values are means ± SD (n = 3). CK is non bacterial inoculated plants. * or ** indicates a significant difference at the 0.05 or 0.01 level, respectively.

Fig. 1

Fig. 2 Antioxidant activities of tigernut in response to Pantoea sp. YSD J2 inoculation (YSD J2). Values are means ± SD (n = 3). CK is non bacterial inoculated plants. * or ** indicates a significant difference at the 0.05 or 0.01 level, respectively.

Fig. 2

3.2 Metabolomic study of tigernut inoculated with Pantoea sp. YSD J2

It has been reported that orientin is the main flavonoid of tigernut [20]. To futher explore the effect of YSD J2 on flavonoid synthesis and possible mechanisms of tigernut, we carried out a widely targeted flavonoids metabolic analysis. Totally 301 flavonoids metabolites were detected by UPLC-MS/MS in tigernut (Table S3). In accordance with the hierarchical cluster analysis (Fig. 3), YSD J2 was clustered into diverse branches compared with CK, and flavonoid distribution and levels in YSD J2 vs. CK groups was also significantly different. Besides, YSD J2 contained significantly accumulated flavonoids (such as naringenin chalcone, naringenin, homoeriodictyol, and orientin, etc.) (Table S3). And the detected DEMs mainly contained flavones and flavonols (Table S4).Fig. 3 The total flavonoids/metabolite heat map derived from UPLC-MS/MS profiling. In the above heat map, the red color indicates significant flavonoids accumulation, and the green color indicates significant flavonoids reduction.

Fig. 3

Most DEMs displayed significant accumulation in the tigernut incobulated with YSD J2, 55 DEMs were upregulated, and 14 DEMs were notably downregulated in the YSD J2 vs. CK groups (Table S3). Furthermore, cyanidin-3-O-(2″-O-glucosyl) glucoside was the metabolite with the maximum upregulated level, with log2FC value reaching 12.27, and the downregulated expression metabolite was 6-prenylnaringenin, reached −2.47 (Fig. 4A). Additionally, the metabolites in the YSD J2 vs. CK groups was enriched by KEGG, as seen in Fig. 4B, the changes in metabolites treated by YSD J2 mainly concerned these pathways: secondary metabolites biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis. According to the above findings, the unique distribution and accumulation patterns of the above DEMs were the primary factors leading to different flavonoids of tigernut in response to YSD J2 inoculation.Fig. 4 Metabolomics analysis of tigernut in response to Pantoea sp. YSD J2 inoculation. (A) The top 20 metabolites of YSD J2 vs. CK group difference multiple, and in the above difference multiple histogram, red represents up-regulated differential metabolite, green represents down-regulated differential metabolite. (B) KEGG pathway enrichment of DEMs in YSD J2 vs. CK group.

Fig. 4

3.3 Correlation analysis in IAA, DEMs and antioxidant activity

The correlations in the IAA content, antioxidant activity indicators (SOD, POD and MDA) of tigernut stems and DEMs in YSD J2 vs. CK groups were explored. The findings demonstrated that the 19 DEMs in tigernut were significantly correlated with IAA and enzymes families (p < 0.05). Moreover, those 19 most significant flavonoids were further analyzed, which indicated that uralenol, petunidin-3-O-glucoside-5-O-arabinoside, luteolin-7-O-glucuronide-(2 → 1)-glucuronide, kaempferol-3-O-neohesperidoside, cyanidin-3-O-(2″-O-glucosyl)glucoside, kaempferol-3-O-glucuronide-7-O-glucoside, quercetin-3-O-glucoside, luteolin-7-O-glucuronide-(2 → 1)-(2″-sinapoyl)glucuronide, and quercetin-4′-O-glucoside were positively related to the decrease of MDA content, increase of POD and SOD activities (Fig. 5, p < 0.01). Accumulation of nine flavonoids (five flavonols, two flavones, and two anthocyanidins) with YSD J2 inoculation is a crucial factor in enhancing the antioxidant ability of tigernut.Fig. 5 Correlation analysis of differential expressed metabolites (DEMs), IAA content, and antioxidant activity parameters (SOD, POD and MDA). * or ** indicates a significant difference at the 0.05 or 0.01 level, respectively.

Fig. 5

3.4 Transcriptome sequencing analysis by RNA-Seq

For further exploring the flavonoid biosynthesis mechanism in tigernut treated with YSD J2, transcriptome sequencing was performed, and 50.67 Gb clean data were acquired by QC sequencing, with 7.44 Gb clean data per sample. The GC level was 46.3–48.86 %, with Q30 based percentage of ＞90.76 % (Table S5). These data revealed that RNA-Seq profiling data were reliable. Altogether, 51344 unigenes more than 1 kb in length were acquired. The obtained N50 and N90 lengths were around 2004 bp and 643 bp, separately (Table S6). Next, the above unigenes were exposed to functional annotation on the basis of the KEGG, NR, SwissProt, GO, COG/KOG, PFAM, and TrEMBL databases, and 98370 unigenes annotations were acquired (Table S7). Clean sample data were found to be assembled and serialized based on the single gene pool.

Additionally, we detected 14391 DEGs, of which 5146 DEGs presented upregulation and 9245 DEGs showed downregulation. The findings are displayed in the volcano map and heatmap (Fig. S1). Meanwhile, we carried out enrichment analysis of DEGs to determine candidate functions and associated biological pathways of possible transcripts. The findings of KOG enrichment analysis suggested that DEGs were mostly enriched in “general function prediction only” (1318), “signal transduction mechanisms” (859), “posttranslational modification, protein turnover, chaperones” (855), “carbohydrate transport and metabolism” (576), “translation, ribosomal structure and biogenesis” (561), and “energy production and conversion” (549; Fig. 6A). Further, the DEGs between the YSD J2 vs. CK groups enriched 148 KEGG metabolic pathways, and 16 remarkably associated pathways were screened (p < 0.05) (Table S8), such as “secondary metabolite biosynthesis,” “ribosome,” “starch and sucrose metabolism,” “peroxisome”, “phenylpropanoid biosynthesis”, and “fatty acid metabolism” (Fig. 6B). According to GO results, DEGs were mainly associated with one subcategory of “cellular component”, i.e., “cellular anatanical entity”. In addition, numerous DEGs were associated with two subcategories of “biological process,” i.e., “cellular process” and “metabolic process”. Numberous DEGs were also associated with two subcategories of “molecular function,” i.e., “binding” and “catalytic activity” (Fig. 6C).Fig. 6 Transcriptomic analysis of tigernut in response to Pantoea sp. YSD J2 inoculation. (A) Bar graph of KOG classification. The horizontal coordinate indicates the functional classification (code) of KOG ID, and the vertical coordinate indicates the number of DEGs included, different classifications are indicated by different colors. The legend shows the code plus its functional description information. (B) KEGG pathway enrichment of DEGs in YSD J2 vs. CK. (C) Clusters of Gene Ontology (GO). The orange and blue colors represent the amount of up-regulated DEGs, and the amount of down-regulated DEGs, respectively. BP: Biological Process; CC: Cellular Component; MF: Molecular Function.

Fig. 6

3.5 Transcriptome and metabolome analyses of tigernut

KEGG enrichment analysis indicated that DEGs showed consistent expression profiles with DEMs associated with 6 pathways, including “secondary metabolites biosynthesis,” “metabolic pathways,” “flavonoid biosynthesis,” “isoflavonoid biosynthesis,” “flavone and flavonol biosynthesis,” and “anthocyanin biosynthesis” (Table S9). Our results revealed consistency and reliability of our metabolome and transcriptome results (Fig. 4, Fig. 6). In addition, 25 critical genes related to isoflavonoid biosynthesis, phenylpropanoid (flavonoid) biosynthesis, anthocyanin biosynthesis, and flavone and flavonol biosynthesis pathways showed remarkably diverse levels within YSD J2 treatment of tigernut (Table S10). Therefore, further studies were conducted to analyze the genes and corresponding metabolites associated with these pathways.

In the YSD J2 vs. CK groups, five of these genes (trans cinnamate 4-monooxygenase (CYP73A), flavonol synthase (FLS), anthocyanin 5-O-glucoside-6‴-O-malonyltransferase (5MaT1), isoflavone 7-O-glucoside-6″-O-malonyltransferase (IF7MAT), and flavonol-3-O-L-rhamnoside-7-O-glucosyltransferase (UGT73C6)) were significantly upregulated, while the other eight genes (chalcone synthase (CHS), chalcone reductase (CHR), caffeoyl-CoA O-methyltransferase (CCoAOMT), flavone synthase II (CYP93G1), naringenin 3-dioxygenase (F3H), leucoanthocyanidin reductase (LAR), anthocyanidin 3-O-glucosyltransferase (BZ1), and flavonoid 6-hydroxylase (CYP71D9)) were significantly downregulated (Fig. 8). In addition, seven chalcone isomerase (CHI) coding genes, four anthocyanidin synthase (ANS) coding genes, three shikimate O-hydroxycinnamoyltransferase (HCT) coding genes, two flavonoids 3′,5′-hydroxylase (CYP75A) genes, one anthocyanidin reductase (ANR) coding gene, and one bifunctional dihydro flavonol 4-reductase/flavanone 4-reductase (DFR) exhibited significant upregulation. These genes were associated with flavonoid synthesis in tigernut.

Phenylalanine ammonia-lyase (PAL) transforms phenylalanine into cinnamoyl-CoA, and the latter is subsequently transformed into p-coumaroyl-CoA under catalysis by CYP73A. The flavonoid were finally synthesized under the catalysis of certain enzymes (such as CHS, CHI, DFR, ANR, and HCT) (Fig. 8). Flavonoids showing differential accumulation were in consistence with the levels of the related genes (Fig. 9A). The 15 DEMs were closely related to the flavonoid synthesis pathway and were upregulated under YSD J2 treatment in tigernut compared to CK. Consequently, these genes were applied to network co-expression and characterization of genes regulating (flavonoids). Such genes such as 2-hydroxyisoflavanone dehydratase (HIDH), anthocyanidin 5,3-O-glucosyltransferase (GT1), CHI, CYP75B1, DFR, IF7MAT, pterocarpan synthas (PTS), CHS, CYP75A, ANR, HCT, FLS, isoflavone/4′-methoxyisoflavone 2′-hydroxylase (CYP81E), anthocyanin 5-O-glucoside-6‴-O-malonyltransferase (5MaT1), CYP93G1, and LAR were most closely connected with cyanidin-3-O-(2″-O-glucosyl) glucoside, apigenin-6-C-glucoside (isovitexin), 3-O-methylquercetin, apigenin-8-C-glucoside (vitexin), quercetin-3-O-rutinoside (rutin), butein, 3,9-dihydroxypterocarpan, isoliquiritigenin, and luteolin-7-O-glucuronide biosynthesis (Fig. 9B). Three hub metabolites (cyanidin-3-O-(2″-O-glucosyl) glucoside, apigenin-6-C-glucoside (isovitexin), and quercetin-3-O-glucoside (isoquercitrin)) were closely related to critical enzyme genes related to flavonoid synthesis. Moreover, HCT, CYP73A, CHI, DFR, ANS, FLS, flavanone 7-O-glucoside 2″-O-beta-L-rhamnosyltransferase (C12RT1), and ANR gene levels were associated with flavonols (4), flavonoid carbonoside (3), chalcones (3), flavanones (2), isoflavones (1), flavones (1), flavanonols (1), and anthocyanidins (1) synthesis in tigernut. Based on the above findings, such structural genes were identified as essential regulators of flavonoid biosynthesis in tigernut.

Orientin, or luteolin-8-C-glucoside, whose structure is 3′, 4′, 5, 7-tetrahydroxyyellow keto-8-D-glucopyranoside, is a flavonoid carbon glycoside monomer compound. Previous research has shown that orientin possesses several pharmacological effects, including anti-inflammatory, antioxidant, anticancer, cardioprotective, neuroprotective, and analgesic properties [[39], [40], [41], [42], [43]]. Up to now, two flavonoid C-glycoside biosynthetic pathways have been identified [44]. This study found that tigernut with YSD J2 treatment significantly accumulated orientin compared to CK (Fig. 1B). Based on transcriptomic and metabonomic analyses, we generated a proposed biosynthetic pathway for orientin in tigernut (Fig. 7).Fig. 7 Speculative pathways of flavonoid synthesis of the tigernut. The pathway was constructed based on the KEGG pathway and the scientific literature.

Fig. 7

Fig. 8 Heat map of gene expression levels (FPKM values) of key enzyme sites of the flavonoid synthesis pathway. Enzymes encoded by the differentially expressed genes were marked in red and the heatmaps showed log2 (FPKM+1) values of each DEGs. Each row of the heatmap represented one gene and each column represented one a group (from right to left, CK, YSD J2-treated). In the above heat map, the red color indicates the expression of genes significantly up-regulated, and the green color indicates the expression of genes significantly down-regulated.

Fig. 8

Fig. 9 (A) The heat map of significantly accumulated flavonoids that are enriched in flavonoid biosynthesis pathway. (B) Transcriptome and metabolome combined analysis network in tigernut in response to Pantoea sp. YSD J2 inoculation. In the above heat map, the red color indicates the expression of genes significantly up-regulated, and the green color indicates the expression of genes significantly down-regulated. In the network, the tiffany blue circles represent the DEGs and the purple triangle represent the corresponding metabolites.

Fig. 9

3.6 Quantitative real-time PCR verification

In order to ensure that our transcriptome data at a gene level were valid, a qRT-PCR experiment was carried out on eight essential genes (Fig. 10). These genes included CYP73A (Cluster-49537.1), CHI (Cluster-37365.0 and Cluster-50548.7), ANS (Cluster-49536.4 and Cluster-43695.0), FLS (Cluster-25595.0), HCT (Cluster-44099.0), and UGT73C6 (Cluster-51835.5). As a result, these genes under YSD J2 treatment were significantly upregulated by 2.81, 9.64, 1.73, 1.82, 12.90, 2.19, 2.70, and 18.10 folds, compared to CK, separately. These findings conformed to RNA-Seq data, suggesting that these data can be adopted for evaluating the upregulation and downregulation of the gene expression. Finally, YSD J2 treatment could increase the expression of flavonoid synthesis-associated genes to different degrees. Such critical genes in tigernut participated in flavonoid biosynthesis and were potential genes used in further cloning and gene function verification.Fig. 10 Expression levels of flavonoids biosynthesis genes of tigernut in response to Pantoea sp. YSD J2 inoculation. Cluster-xxxxx.x represents the ID of gene. The y-axis indicates the relative expression level (A: by qRT-PCR; B: FPKM values) of the genes. Each value is the mean of three replicates, and error bars indicate standard deviations. Statistical analysis of the data was performed by independent samples t-test using the SPSS 22.0 software (IBM, Chicago, IL, USA). *** above the columns are significantly different at p ≤ 0.001.

Fig. 10

3.7 Transcription factors (TFs) related to flavonoid biosynthesis

TFs can influence plant flavonoid biosynthesis. YSD J2 inoculation can activate TFs related to flavonoid biosynthesis in tigernut. The TFs associated with flavonoid biosynthesis were AP2/ERF-ERF, bHLH, GRAS, C2H2, NAC, MYB, bZIP, MYB-related, WRKY, AUX/IAA, and TRAF TFs (Fig. S2). The most abundant TFs were AP2/ERF-ERF and bHLH TFs. We obtained twenty-one AP2/ERF-ERF and five bHLH TFs (FPKM >10) (Fig. S3).

bHLH34 (Cluster-37505.3) was significantly upregulated under YSD J2 treatment and showed a positive correlation with tetahydroxyflavone-7-O-glucuronide, orientin, kaempferol-3-O-glucuronide and scutellarin, which indicates that the TF bHLH34 (Cluster-37505.3) may positively regulate flavonoid biosynthesis. Additionally, AP2/ERF-ERF (Cluster-10150.7 and Cluster-32230.0) were positively correlated with orientin. In contrast, the level of bHLH16 (Cluster-11980.2) showed significant negative correlation with those of tetahydroxyflavone-7-O-glucuronide, orientin, and kaempferol-3-O-glucuronide, and AP2/ERF-ERF (Cluster-22211.0) was significantly negatively related to that of tetahydroxyflavone-7-O-glucuronide, luteolin-7-O-glucuronide, and kaempferol-3-O-glucuronide, which indicated that AP2/ERF-ERF (Cluster-22211.0) may negatively regulate flavonoid accumulation in tigernut (Fig. S4).

4 Discussion

Tigernut, known as chufas, is the second most abundant species within Cyperaceae family. Cyperus is the type genus and core taxon of the Cyperaceae family [45]. Recently, tigernut has been recognized for its high-quality oils, similar to olive oil, and its rich components like lactones, flavonoids, coumarins, steroids glycosides, cardiac glycosides, and triterpenoids [15,16]. These elements offer various pharmaceutical benefits, including antioxidation, antibacterial, and anticoagulation effects, and they help improve blood microcirculation and prevent ischemic stroke [17,18]. Given the numerous pharmacological effects of tigernut, it is crucial to study its secondary metabolites and their synthesis mechanisms.

Auxin critically affects plant growth through concentration changes that regulate multiple processes. IAA functions as the predominant natural auxin in plants [46]. Our results indicate that IAA levels in tigernut significantly increased under the YSD J2 treatment compared to the CK. This was in consistence with previous research results, IAA not only promotes growth, but also induces flavonoid accumulation and enhances the expression of flavonoid pathway enzyme genes [[47], [48], [49], [50], [51]]. Antioxidants belong to plant stress tolerance indicators [52,53]. POD and SOD represent key plant antioxidant enzymes families, and the alterations can show plant's stress resistance [54]. Under normal conditions, enzymes in plants are coordinated mutually, while enzyme activity within this defense system is the decisive factor in controlling damage, better reflecting the adaptability of plants to adversity. This study evaluated the activity of antioxidants in both uninoculated and inoculated plants and found that compared with CK, tigernut inoculated with YSD J2 showed higher SOD and POD activities, and lower MDA content. The results indicate that YSD J2 inoculation significantly promotes plant growth and development.

Plant flavonoids exhibit appropriate biochemical activity and additional bioactivities in different diseases [55,56]. Flavonoids are important components in the tigernut stem, including 7-hydroxyl-6-methoxy radical coumarin, isoorientin, and orientin. Orientin accounts for 65.4 % (mass percentage) of the total three monomers and is the main component of the monomer compounds [20]. The biosynthesis of active ingredients in medicinal crops results from the interaction between crop genotypes and specific environmental conditions, with certain genes playing a crucial role. Critical enzyme genes related to secondary metabolic pathways are fully expressed only under specific ecological conditions. However, most studies have focused on external environmental effects on biosynthesis [57], neglecting the internal environment of the plants.

The distribution and accumulation of PGPMs in medicinal plants are tightly associated with external environmental factors, such as humidity, temperature, and other climatic conditions, as well as the growth period of medicinal plants, which are critical internal factors that influence medicinal plant growth and metabolism. To systematically study the pathway, species, and differential genes of endophyte-mediated secondary metabolite accumulation in medicinal plants, metabolome (LC-MS/MS) analysis plus transcriptome analysis was conducted to explore the mechanism of flavonoid biosynthesis of tigernut with YSD J2. In the current study, 301 flavonoids were found in tigernut and 55 significantly upregulated DEMs w associated with flavonoid synthesis pathway were identified. Therefore, such unique DEMs distribution and accumulation modes accounted for the primary factor leading to different flavonoids of tigernut under YSD J2. PGPMs can directly produce various metabolites and plant hormones, such as GA, ABA, SA, and IAA, to promote plant development [58] and produce flavonoids as antioxidant regulators. In addition, horizontal gene exchange between plants and microorganisms may lead to changes in plant secondary metabolites [59]. Many studies have shown that some PGPMs "reprocesses" plant metabolites into other compounds, leading to degradation of certain toxic compounds or other plant functions [60]. PGPMs may interact differently in different plants species, which requires further in-depth research. The present study found that YSD J2 treatment significantly enriched nine compounds, namely uralenol, petunidin-3-O-glucoside-5-O-arabinoside, luteolin-7-O-glucuronide-(2 → 1)-glucuronide, kaempferol-3-O-neohesperidoside, cyanidin-3-O-(2″-O-glucosyl)glucoside, kaempferol-3-O-glucuronide-7-O-glucoside, quercetin-3-O-glucoside, luteolin-7-O-glucuronide-(2 → 1)-(2″-sinapoyl)glucuronide, and quercetin-4′-O-glucoside, which futher enhanced antioxidant activity. Our previous research has shown that YSD J2 exhibited multiple growth- and metabolism-related abilities [31]. It was speculated that the IAA produced by YSD J2 significantly activated the expression of CHS, CHR, and CHI, which are widely distributed in the phloem of vascular bundle cells of the cell wall, resulting in a significant accumulation of flavonoids. In the present study, levels of IAA in tigernut significantly increased in comparison to CK under YSD J2 treatment. The qRT-PCR results revealed a significant upregulation of CHI gene expression in the YSD J2 treatment groups, confirming the above speculation. At the same time, the YSD J2 treatment significantly reduced the content of vitexin and isovitexin, which led to the accumulation of apigenin, causing the reaction to enter flavone and flavonol biosynthesis or form flavanonols to accumulate orientin further.

In tigernut, flavonoid accumulation was markedly related to phenylpropanoid (flavonoid) biosynthesis, anthocyanin biosynthesis, isoflavonoid biosynthesis, and flavone and flavonol biosynthesis pathways, which specifically target hundreds of genes related to flavonoid biosynthesis. Flavonoid accumulation modes of tigernut were significantly different under YSD J2 treatments, associated genes closely related to this pathway also exhibited differential expression. Consequently, this study analyzed different flavonoid biosynthesis-related transcriptional reprogramming cascades and metabolite synthesis flow in tigernut. According to our results, PAL is responsible for catalyzing phenylalanine transformation into cinnamoyl-CoA. Subsequently, CYP73A catalyzes cinnamoyl-CoA isomerization into p-coumaroyl-CoA. CYP73A-encoding genes of tigernut were significantly upregulated under YSD J2 treatments. Moreover, we found that the above gene levels and upstream metabolic compounds accumulation facilitated downstream flavonoid biosynthesis within tigernut. Moreover, eleven CHI coding genes (Cluster-25300.1, Cluster-25300.7, Cluster-25300.8, Cluster-37365.0, Cluster-37365.3, Cluster-37365.4, Cluster-50548.1, Cluster-50548.10, Cluster-50548.5, Cluster-50548.6, Cluster-50548.7) were significantly upregulated by YSD J2 treatment. CHI is responsible for the isomerization of naringenin chalcone into naringenin, the frequently adopted precursor for synthesizing diverse plant flavonoids, such as chalcones (isoliquiritigenin, butein), flavanones (homoeriodictyol), flavanols (afzelechin, epigallocatechin, luteoforol), flavonols (quercetin, isoquercitrin, 3-O-methyl quercetin), flavanonols (dihydro kaempferol), anthocyanidins (cyanidin-3-O-rutinoside, cyanidin-3-O-sophoroside), flavones (luteolin-7-O-glucuronide), isoflavones (3,9-dihydroxypterocarpan, 6″-O-malonyl genistin) detected in this study. These DEMs possess potent anti-inflammatory and antioxidative effects, which can regulate some signal transducers to alleviate relevant disorders [61,62].

5 Conclusions

The physiological, transcriptomic and flavonoid metabolomic analyses of tigernut were compared under Pantoea sp. YSD J2 inoculation. Pantoea sp. YSD J2 considerably improved IAA and orientin content. This work indicated flavonoid metabolites related to enhancing tigernut antioxidant capacity upon Pantoea sp. YSD J2 inoculation, and several candidate genes related to DEMs were detected. In conclusion, the findings provide a foundation for investigating the regulatory effect of Pantoea sp. YSD J2 on tigernut development and flavonoid biosynthesis.

Data availability statements

All relevant data are within the manuscript and its Additional files.

Funding

This work was supported by the Shanghai Jiaotong University Talent Foundation (No. WH221615001), Program for Shanghai Leading Talent, and the Shanghai Agriculture Applied Technology Development Program (Grant No. 2020-2-1).

CRediT authorship contribution statement

Saisai Wang: Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation, Conceptualization. Yanna Huang: Writing – review & editing, Formal analysis, Data curation, Conceptualization. Yu Sun: Writing – review & editing, Formal analysis, Data curation, Conceptualization. Jinbin Wang: Writing – review & editing, Formal analysis, Data curation, Conceptualization. Xueming Tang: Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

All authors declares that they have no conflict of interest.

Appendix A Supplementary data

The following is the Supplementary data to this article.Multimedia component 1

Multimedia component 1

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e35966.
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References

1 Liu W. Feng Y. Yu S. Fan Z. Li X. Li J. Yin H. The flavonoid biosynthesis network in plants Int. J. Mol. Sci. 22 2021 12824 10.3390/ijms222312824
2 Sasaki N. Nakayama T. Achievements and perspectives in biochemistry concerning anthocyanin modification for blue flower coloration Plant Cell Physiol. 56 2015 28 40 10.1093/pcp/pcu097 25015943
3 Nahed L. Nasra Z. Hanaa A.E. El-Rahman S.M.A. The utilization of yellow and red onion peels and their extracts as antioxidant and antimicrobial in preservation of beef burger during storage Adv. J. Food Sci. Technol. 10 1 2022 1 9
4 Farhood B. Mortezaee K. Goradel N.H. Khanlarkhani N. Salehi E. Nashtaei M.S. Najafi M. Sahebkar A. Curcumin as an anti-inflammatory agent: implications to radiotherapy and chemotherapy J. Cell. Physiol. 234 5 2019 5728 5740 10.1002/jcp.27442 30317564
5 Khandelwal N. Chander Y. Kumar R. Riyesh T. Dedar R.K. Kumar M. Gulati B.R. Sharma S. Tripathi B.N. Barua S. Kumar N. Antiviral activity of Apigenin against buffalopox: novel mechanistic insights and drug-resistance considerations Antivir. Res. 181 2020 104870 10.1016/j.antiviral.2020.104870
6 Imran M. Aslam Gondal T. Atif M. Shahbaz M. Batool Qaisarani T. Hanif Mughal M. Salehi B. Martorell M. Sharifi-Rad J. Apigenin as an anticancer agent Phytother Res. 34 8 2020 1812 1828 10.1002/ptr.6647 32059077
7 Liu X. Zhang Y. Liu L. Pan Y. Hu Y. Yang P. Liao M. Protective and therapeutic effects of nanoliposomal quercetin on acute liver injury in rats BMC Pharmacol. Toxicol. 21 1 2020 11 10.1186/s40360-020-0388-5 32059743
8 Mahabady M.K. Shamsi M.M. Ranjbar R. Tabandeh M.R. Khazaeel K. Quercetin improved histological structure and upregulated adiponectin and adiponectin receptors in the placenta of rats with gestational diabetes mellitus Placenta 106 2021 49 57 10.1016/j.placenta.2021.02.008 33640737
9 Orzelska-Górka J. Szewczyk K. Gawrońska-Grzywacz M. Kędzierska E. Głowacka E. Herbet M. Dudka J. Biała G. Monoaminergic system is implicated in the antidepressant-like effect of hyperoside and protocatechuic acid isolated from Impatiens glandulifera Royle in mice Neurochem. Int. 128 2019 206 214 31077758
10 Carvalho T.T. Mizokami S.S. Ferraz C.R. Manchope M.F. Borghi S.M. Fattori V. Calixto-Campos C. Camilios-Neto D. Casagrande R. Verri W.A. Jr. The granulopoietic cytokine granulocyte colony-stimulating factor (G-CSF) induces pain: analgesia by rutin Inflammopharmacology 27 6 2019 1285 1296 10.1007/s10787-019-00591-8 30945072
11 Qi C. Shao Y. Liu X. Wang D. Li X. The cardioprotective effects of icariin on the isoprenaline-induced takotsubo-like rat model: involvement of reactive oxygen species and the TLR4/NF-kappa B signaling pathway Immunopharmacol vol. 74 2019 105733 10.1016/j.intimp.2019.105733
12 Zhang M. Lu X. Ren T. Marowa P. Meng C. Wang J. Yang H. Li C. Zhang L. Xu Z. Heterologous overexpression of Apocynum venetum flavonoids synthetase genes improves Arabidopsis thaliana salt tolerance by activating the IAA and JA biosynthesis pathways Front. Plant Sci. 14 2023 1123856 10.3389/fpls.2023.1123856
13 Ayeh-Kumi P.F. Tetteh-Quarcoo P.B. Duedu K.O. Obeng A.S. Addo-Osafo K. Mortu S. Asmah R.H. A survey of pathogens associated with Cyperus esculentus L (tiger nuts) tubers sold in a Ghanaian city BMC Res. Notes 7 2014 343 10.1186/1756-0500-7-343 24906387
14 Aduwamai U.H. Umaru Umaru I.J. Aminu A. Umaru K.I. Influence of different processing methods on proximate and anti-nutritional value of tigernuts (Cyperus esculentus L.) GSC Biol. Pharm. Sci. 3 2018 29 34 10.30574/gscbps.2018.3.3.0039
15 Bado S. Bazongo P. Son G. Kyaw M.T. Forster B.P. Nielen S. Lykke A.M. Ouedraogo A. Bassole I.H. Physicochemical characteristics and composition of three morphotypes of Cyperus esculentus tubers and tuber oils J. Anal. Methods. Chem. 673547 2015 10.1155/2015/673547
16 Nyarko H.D. Tagoe D.N.A. Aniweh Y. Assessment of microbiological safety of tiger nuts (Cyperus esculentus L.) in the Cape Coast Metropolis of Ghana Arch. Appl. Sci. Res. 3 2011 362 331
17 Jing S.Q. Wang S.S. Li Q. Zheng L. Yue L. Fan S.L. Tao G.J. Dynamic high pressure microfluidization-assisted extraction and bioactivities of Cyperus esculentus (C. esculentus L.) leaves flavonoids Food Chem. 192 2016 319 327 10.1016/j.foodchem.2015.06.097 26304354
18 Jing S.Q. Wang S.S. Zhong R.M. Zhang J.Y. Wu J.Z. Tu Y.X. Pu Y. Yan L.J. Neuroprotection of Cyperus esculentus L. orientin against cerebral ischemia/reperfusion induced brain injury Neural Regen. Res. 15 3 2020 548 556 10.4103/1673-5374.266063 31571667
19 Ma J.N. Shan C.B. Feng X. Ma Y. Wang L.W. Zhang X.Q. Zhao X.Q. Lu Z.Y. Zhang D.J. Ma C.M. Investigation on the relationship between stem and leaf metabolites and tuber morphology of Cyperus esculentus Chin. J. Oil Crop Sci. 2022 1 10 10.19802/j.issn.1007-9084.2022208
20 Wang S.S. Zhang M.F. Qi R. Jing S.Q. Tu Y.X. Wang W.L. Component isolation and identification and antioxidant activity analysis of Cyperus esculentus alcohol extraction Food Ind. 6 2017 185 189
21 Ro D.K. Paradise E.M. Ouellet M. Fisher K.J. Newman K.L. Ndungu J.M. Ho K.A. Eachus R.A. Ham T.S. Kirby J. Chang M.C. Withers S.T. Shiba Y. Sarpong R. Keasling J.D. Production of the antimalarial drug precursor artemisinic acid in engineered yeast Nature 440 7086 2006 940 943 16612385
22 Leonard E. Lim K.H. Saw P.N. Koffas M.A. Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli Appl. Environ. Microbiol. 73 12 2007 3877 3886 10.1128/AEM.00200-07 17468269
23 Zhai X. Jia M. Chen L. Zheng C.j. Rahman K. Han T. The regulatory mechanism of fungal elicitor-induced secondary metabolite biosynthesis in medical plants Crit. Rev. Microbiol. 43 2 2017 238 261 10.1080/1040841X.2016.1201041 27936989
24 Gomez O.C. Hortolan Luiz J.H. Endophytic fungi isolated from medicinal plants: future prospects of bioactive natural products from Tabebuia/Handroanthus endophytes Appl. Microbiol. Biotechnol. 102 21 2018 9105 9119 10.1007/s00253-018-9344-3 30203146
25 Chen L. Shi H. Heng J. Wang D. Bian K. Antimicrobial, plant growth-promoting and genomic properties of the peanut endophyte Bacillus velezensis LDO2 Microbiol. Res. 218 2019 41 48 10.1016/j.micres.2018.10.002 30454657
26 Cui J.L. Wang C.L. Guo S.X. Xiao P.G. Wang M.L. Stimulation of dragon's blood accumulation in Dracaena cambodiana via fungal inoculation Fitoterapia 87 2013 31 36 10.1016/j.fitote.2013.03.012 23518260
27 Bidabadi S.S. Mehralian M. Seed bio-priming to improve germination, seedling growth and essential oil yield of Dracocephalum kotschyi Boiss, an endangered medicinal plant in Iran Gesunde Pflanz. 72 2020 17 27 10.1007/s10343-019-00478-2
28 Delshadi S. Ebrahimi M. Shirmohammadi E. Influence of plant-growth-promoting bacteria on germination, growth and nutrient uptake of Onobrychis sativa L. under drought stress J. Plant Interact. 12 1 2017 200 208
29 Zhao Y. Sun C. Wang S. Zhang M. Li Y. Xue Q. Guo Q. Lai H. Widely targeted metabolomic, transcriptomic, and metagenomic profiling reveal microbe-plant-metabolic reprogramming patterns mediated by Streptomyces pactum Act12 enhance the fruit quality of Capsicum annuum L Food Res. Int. 166 2023 112587 10.1016/j.foodres.2023.112587
30 Zapata-Sifuentes G. Hernandez-Montiel L.G. Saenz-Mata J. Fortis-Hernandez M. Blanco-Contreras E. Chiquito-Contreras R.G. Preciado-Rangel P. Plant growth-promoting rhizobacteria improve growth and fruit quality of cucumber under greenhouse conditions Plants 11 12 2022 10.3390/plants11121612
31 Wang S.S. Wang J.B. Zhou Y.F. Huang Y.N. Tang X.M. Isolation, classification, and growth-promoting effects of pantoea sp. YSD J2 from the aboveground leaves of Cyperus esculentus L. var. sativus Curr. Microbiol. 79 2 2022 66 10.1007/s00284-021-02755-8 35059843
32 Ham S.H. Yoon A.R. Oh H.E. Park Y.G. Plant growth-promoting microorganism Pseudarthrobacter sp. NIBRBAC000502770 enhances the growth and flavonoid content of geum aleppicum Microorganisms 10 6 2022 1241 10.3390/microorganisms10061241 35744759
33 Dimitrijević S. Pavlović M. Maksimović S. Ristić M. Filipović V. Antonović D. Dimitrijević-Branković S. Plant growth-promoting bacteria elevate the nutritional and functional properties of black cumin and flaxseed fixed oil J. Sci. Food Agric. 98 4 2018 1584 1590 10.1002/jsfa.8631 28833158
34 Zhao Y. Sun C. Wang S. Zhang M. Li Y. Xue Q. Guo Q. Lai H. Widely targeted metabolomic, transcriptomic, and metagenomic profiling reveal microbe-plant-metabolic reprogramming patterns mediated by Streptomyces pactum Act12 enhance the fruit quality of Capsicum annuum L Food Res. Int. 166 2023 112587 10.1016/j.foodres.2023.112587
35 Wang S.S. Yang F.F. Nie Z.X. Qian L. Ma X.F. Jing S.Q. Optimization of extraction technology of flavonoids from Cyperus esculentus L. by ultrasonic treatment followed by microwave treatment Food Ind 11 2014 189 193
36 Ullah I. Waqas M. Khan M.A. Exogenous ascorbic acid mitigates flood stress damages of Vigna angularis Appl. Biol. Chem. 60 2017 603 614
37 Zhou Y. Xu X. Chen Y. Gao J. Shi Q. Tian L. Cao L. Combined metabolome and transcriptome analyses reveal the flavonoids changes and biosynthesis mechanisms in different organs of Hibiseu manihot L Front. Plant Sci. 13 2022 10.3389/fpls.2022.817378
38 Chen S. Zhou Y. Chen Y. Gu J. Fastp: an ultra-fast all-in-one FASTQ preprocessor Bioinformatics 34 17 2018 i884 i890 10.1093/bioinformatics/bty560 30423086
39 Gou K.J. Zeng R. Ren X.D. Dou Q.L. Yang Q.B. Dong Y. Qu Y. Anti-rheumatoid arthritis effects in adjuvant-induced arthritis in rats and moleular docking studies of Polygonum orientale L. extracts Immunol. Lett. 201 2018 59 69 10.1016/j.imlet.2018.11.009 30471320
40 Nagai S. Matsumoto C. Shibano M. Fujimori K. Suppression of fatty acid and triglyceride synthesis by the flavonoid orientin through decrease of C/EBPdelta expression and inhibition of PI3K/Akt-FOXO1 signaling in adipocytes Nutrients 10 2 2018 10.3390/nu10020130
41 Qi S. Feng Z. Li Q. Qi Z. Zhang Y. Inhibition of ROS-mediated activation Src-MAPK/AKT signaling by orientin alleviates H2O2-induced apoptosis in PC12 cells Drug Des. Dev. Ther. 12 2018 3973 3984 10.2147/DDDT.S178217
42 Yuan L. Wang J. Xiao H. Wu W. Wang Y. Liu X. MAPK signaling pathways regulate mitochondrial-mediated apoptosis induced by isoorientin in human hepatoblastoma cancer cells Food Chem. Toxicol. 53 2013 62 68 10.1016/j.fct.2012.11.048 23220614
43 Yuan L. Wei S. Wang J. Liu X. Isoorientin induces apoptosis and autophagy simultaneously by reactive oxygen species (ROS)-related p53, PI3K/Akt, JNK, and p38 signaling pathways in HepG2 cancer cells J. Agric. Food Chem. 62 23 2014 5390 5400 10.1021/jf500903g 24841907
44 Feng C.Y. Li S.S. Taguchi G. Wu Q. Yin D.D. Gu Z.Y. Wu J. Xu W.Z. Liu C. Wang L.S. Enzymatic basis for stepwise C-glycosylation in the formation of flavonoid di-C-glycosides in sacred lotus (Nelumbo nucifera Gaertn.) Plant J. 106 2 2021 351 365 10.1111/tpj.15168 33486798
45 Huygh W. Larridon I. Reynders M. Muasya A.M. Govaerts R. Goetghebeur S.P. Nomenclature and typification of names of genera and subdivisions of genera in cypereae (cyperaceae): 1. names of genera in the cyperus clade Taxon 59 6 2010 1883 1890 10.1002/tax.596021
46 Zhao Z. Andersen S.U. Ljung K. Dolezal K. Miotk A. Schultheiss S.J. Lohmann J.U. Hormonal control of the shoot stem-cell niche Nature 465 2010 1089 1092 10.1038/nature09126 20577215
47 Lee Y. Lee D.E. Lee H.S. Kim S.K. Lee W.S. Kim S.H. Kim M.W. Influence of auxins, cytokinins, and nitrogen on production of rutin from callus and adventitious roots of the white mulberry tree (Morus alba L.) Plant Cell Tiss Org 105 1 2011 9 19 10.1007/s11240-010-9832-3
48 Lewis D.R. Ramirez M.V. Miller N.D. Vallabhaneni P. Ray W.K. Helm R.F. Winkel B.S. Muday G.K. Auxin and ethylene induce flavonol accumulation through distinct transcriptional networks Plant. Physiol. 156 1 2011 144 164 10.1104/pp.111.172502 21427279
49 Jeong C.S. Murthy H.N. Hahn E.J. Lee H.L. Paek K.Y. Inoculum size and auxin concentration influence the growth of adventitious roots and accumulation of ginsenosides in suspension cultures of ginseng (Panax ginseng C.A. Meyer) Acta Physiol. Plant. 31 1 2009 219 10.1007/s11738-008-0206-y
50 Lister C.E. Lancaster J.E. Walker J.R.L. Developmental changes in enzymes of flavonoid biosynthesis in the skins of red and green apple cultivars J. Sci. Food Agric. 71 1996 313 320 10.1002/(sici)1097-0010(199607)71:3<313::aid-jsfa586>3.0.co;2-n
51 Max C.S. External control of anthocyanin formation in apple Sci. Hortic. 42 3 1990 181 218 10.1016/0304-4238(90)90082-P
52 Pan S.S. Rasul F. Li W. Tian H. Mo Z.W. Duan M.Y. Tang X.R. Roles of plant growth regulators on yield, grain qualities and antioxidant enzyme activities in super hybrid rice (Oryza sativa L.) Rice 6 2013 9 10.1186/1939-8433-6-9 24280625
53 Esfahani M.N. Mostajeran A. Rhizobial strain involvement in symbiosis efficiency of chickpea-rhizobia under drought stress: plant growth, nitrogen fixation and antioxidant enzyme activities Acta Physiol. Plant. 33 4 2011 1075 1083 10.1007/s11738-010-0635-2
54 He J. Qin J. Long L. Ma Y. Li H. Li K. Jiang X. Liu T. Polle A. Liang Z. Luo Z. Net cadmium flux and accumulation reveal tissue-specific oxidative stress and detoxification in Populus × canescens Physiol. Plantarum 143 1 2011 50 63 10.1111/j.1399-3054.2011.01487.x
55 Gentile D. Fornai M. Pellegrini C. Colucci R. Blandizzi C. Antonioli L. Dietary flavonoids as a potential intervention to improve redox balance in obesity and related co-morbidities: a review Nutr. Res. Rev. 31 2 2018 239 247 10.1017/S0954422418000082 29871706
56 Williamson G. Kay C.D. Crozier A. The bioavailability, transport, and bioactivity of dietary flavonoids: a review from a historical perspective Compr Rev Food Sci F 17 5 2018 1054 1112 10.1111/1541-4337.12351
57 Lillo C. Lea U.S. Ruoff P. Nutrient depletion as a key factor for manipulating gene expression and product formation in different branches of the flavonoid pathway Plant Cell Environ. 31 5 2008 587 601 10.1111/j.1365-3040.2007.01748.x 18031469
58 Shen L. Li L.Y. Zhang X.J. Li M. Song Y.C. A new indole derivative from endophyte myrothecium roridum IFB-E091 in artemisia annua Yao Xue Xue Bao 50 10 2015 1305 1308 26837178
59 Ambrose K.V. Koppenhöfer A.M. Belanger F.C. Horizontal gene transfer of a bacterial insect toxin gene into the Epichloë fungal symbionts of grasses Sci. Rep. 4 2014 5562 10.1038/srep05562 24990771
60 Sun K. Liu J. Gao Y. Jin L. Gu Y. Wang W. Isolation, plant colonization potential, and phenanthrene degradation performance of the endophytic bacterium Pseudomonas sp. Ph6-gfp Sci. Rep. 4 2014 5462 10.1038/srep05462 24964867
61 Zhang X. Zhang L. Zhang D. Su D. Li W. Wang X. Chen Q. Cai W. Xu L. Cao F. Zhang D. Yu X. Li Y. Comprehensive analysis of metabolome and transcriptome reveals the mechanism of color formation in different leave of Loropetalum Chinense var. Rubrum BMC Plant Biol. 23 1 2023 133 10.1186/s12870-023-04143-9 36882694
62 Pan S.S. Rasul F. Li W. Tian H. Mo Z.W. Duan M.Y. Tang X.R. Roles of plant growth regulators on yield, grain qualities and antioxidant enzyme activities in super hybrid rice (Oryza sativa L.) Rice 6 2013 9 10.1186/1939-8433-6-9 24280625
