AaMYB108 is the core factor integrating light and jasmonic acid signaling to regulate artemisinin biosynthesis in Artemisia annua.

Artemisinin, a sesquiterpene compound synthesized and stored in the glandular trichome of Artemisia annua leaves, has been used to treat malaria. Previous studies have shown that both light and jasmonic acid (JA) can promote the biosynthesis of artemisinin, and the promotion of artemisinin by JA is dependent on light. However, the specific molecular mechanism remains unclear. Here, we report a MYB transcription factor, AaMYB108, identified from transcriptome analysis of light and JA treatment, as a positive regulator of artemisinin biosynthesis in A. annua. AaMYB108 promotes artemisinin biosynthesis by interacting with a previously characterized positive regulator of artemisinin, AaGSW1. Then, we found that AaMYB108 interacted with AaCOP1 and AaJAZ8, respectively. The function of AaMYB108 was influenced by AaCOP1 and AaJAZ8. Through the treatment of AaMYB108 transgenic plants with light and JA, it was found that the promotion of artemisinin by light and JA depends on the presence of AaMYB108. Taken together, our results reveal the molecular mechanism of JA regulating artemisinin biosynthesis depending on light in A. annua. This study provides new insights into the integration of light and phytohormone signaling to regulate terpene biosynthesis in plants.

Identification, characterization and functional analysis of grape (Vitis vinifera L.) mitochondrial transcription termination factor (mTERF) genes in responding to biotic stress and exogenous phytohormone.

BACKGROUND: Mitochondrial transcription termination factor (mTERF) is a large gene family which plays a significant role during plant growth under various environmental stresses. However, knowledge of mTERF genes in grapevine (Vitis L.) is limited. RESULTS: In this research, a comprehensive analysis of grape mTERF (VvmTERF) genes, including chromosome locations, phylogeny, protein motifs, gene structures, gene duplications, synteny analysis and expression profiles, was conducted. As a result, a total of 25 mTERF genes were identified from the grape genome, which are distributed on 13 chromosomes with diverse densities and segmental duplication events. The grape mTERF gene family is classified into nine clades based on phylogenetic analysis and structural characteristics. These VvmTERF genes showed differential expression patterns in response to multiple phytohormone treatments and biotic stresses, including treatments with abscisic acid and methyl jasmonate, and inoculation of Plasmopara viticola and Erysiphe necator. CONCLUSIONS: These research findings, as the first of its kind in grapevine, will provide useful information for future development of new stress tolerant grape cultivars through genetic manipulation of VvmTERF genes.

Jasmonate- and abscisic acid-activated AaGSW1-AaTCP15/AaORA transcriptional cascade promotes artemisinin biosynthesis in Artemisia annua.

Artemisinin, a sesquiterpene lactone widely used in malaria treatment, was discovered in the medicinal plant Artemisia annua. The biosynthesis of artemisinin is efficiently regulated by jasmonate (JA) and abscisic acid (ABA) via regulatory factors. However, the mechanisms linking JA and ABA signalling with artemisinin biosynthesis through an associated regulatory network of downstream transcription factors (TFs) remain enigmatic. Here we report AaTCP15, a JA and ABA dual-responsive teosinte branched1/cycloidea/proliferating (TCP) TF, which is essential for JA and ABA-induced artemisinin biosynthesis by directly binding to and activating the promoters of DBR2 and ALDH1, two genes encoding enzymes for artemisinin biosynthesis. Furthermore, AaORA, another positive regulator of artemisinin biosynthesis responds to JA and ABA, interacts with and enhances the transactivation activity of AaTCP15 and simultaneously activates AaTCP15 transcripts. Hence, they form an AaORA-AaTCP15 module to synergistically activate DBR2, a crucial gene for artemisinin biosynthesis. More importantly, AaTCP15 expression is activated by the multiple reported JA and ABA-responsive TFs that promote artemisinin biosynthesis. Among them, AaGSW1 acts at the nexus of JA and ABA signalling to activate the artemisinin biosynthetic pathway and directly binds to and activates the AaTCP15 promoter apart from the AaORA promoter, which further facilitates formation of the AaGSW1-AaTCP15/AaORA regulatory module to integrate JA and ABA-mediated artemisinin biosynthesis. Our results establish a multilayer regulatory network of the AaGSW1-AaTCP15/AaORA module to regulate artemisinin biosynthesis through JA and ABA signalling, and provide an interesting avenue for future research exploring the special transcriptional regulation module of TCP genes associated with specialized metabolites in plants.

An HD-ZIP-MYB complex regulates glandular secretory trichome initiation in Artemisia annua.

Plant glandular secretory trichomes (GSTs) produce various specialized metabolites. Increasing GST density represents a strategy to enhance the yield of these chemicals; however, the gene regulatory network that controls GST initiation remains unclear. In a previous study of Artemisia annua L., we found that a HD-ZIP IV transcription factor, AaHD1, promotes GST initiation by directly regulating AaGSW2. Here, we identified two AaHD1-interacting transcription factors, namely AaMIXTA-like 2 (AaMYB16) and AaMYB5. Through the generation and characterization of transgenic plants, we found that AaMYB16 is a positive regulator of GST initiation, whereas AaMYB5 has the opposite effect. Notably, neither of them regulates GST formation independently. Rather, they act competitively, by interacting and modulating AaHD1 promoter binding activity. Additionally, the phytohormone jasmonic acid (JA) was shown to be associated with the AaHD1-AaMYB16/AaMYB5 regulatory network through transcriptional regulation via a JASMONATE-ZIM DOMAIN (JAZ) protein repressor. These results bring new insights into the mechanism of GST initiation through regulatory complexes, which appear to have similar functions in a range of vascular plant taxa.

AaWRKY9 contributes to light- and jasmonate-mediated to regulate the biosynthesis of artemisinin in Artemisia annua.

Artemisinin, isolated from Artemisia annua, is recommended as the preferred drug to fight malaria. Previous research showed that jasmonate (JA)-mediated promotion of artemisinin accumulation depended on light. However, the mechanism underlying the interaction of light and JA in regulating artemisinin accumulation is still unknown. We identified a WRKY transcription factor, AaWRKY9, using transcriptome analysis. The glandular trichome-specific AaWRKY9 positively regulates artemisinin biosynthesis by directly binding to the promoters of AaDBR2 and AaGSW1. The key regulator in the light pathway AaHY5 activates the expression of AaWRKY9 by binding to its promoter. In addition, AaWRKY9 interacts with AaJAZ9, a repressor in the JA signalling pathway. AaJAZ9 represses the transcriptional activation activity of AaWRKY9 in the absence of methyl jasmonate. Notably, in the presence of methyl jasmonate, the transcriptional activation activity of AaWRKY9 is increased. Taken together, our results reveal a novel molecular mechanism underlying AaWRKY9 contributes to light-mediated and jasmonate-mediated to regulate the biosynthesis of artemisinin in A. annua. Our study provides new insights into integrating the two signalling pathways to regulate terpene biosynthesis in plants.

The WRKY transcription factor AaGSW2 promotes glandular trichome initiation in Artemisia annua.

Glandular secreting trichomes (GSTs) synthesize and secrete large quantities of secondary metabolites, some of which have well-established commercial value. An example is the anti-malarial compound artemisinin, which is synthesized in the GSTs of Artemisia annua. Accordingly, there is considerable interest in understanding the processes that regulate GST density as a strategy to increase artemisinin production. In this study, we identified a GST-specific WRKY transcription factor from A. annua, AaGSW2, which is positively regulated by the direct binding of the homeodomain proteins AaHD1 and AaHD8 to the L1-box of the AaGSW2 promoter. Overexpression of AaGSW2 in A. annua significantly increased GST density, while AaGSW2 knockdown lines showed impaired GST initiation. Ectopic expression of AaGSW2 homologs from two mint cultivars, Mentha spicata and Mentha haplocalyx, in A. annua also induced GST formation. These results reveal a molecular mechanism involving homeodomain and WRKY proteins that controls glandular trichome initiation, at least part of which is shared by A. annua and mint.

Morphophysiological and molecular evidence supporting the augmentative role of Piriformospora indica in mitigation of salinity in Cucumis melo L.

Salinity is one of the major limiting factors in plant growth and productivity. Cucumis melo L. is a widely cultivated plant, but its productivity is significantly influenced by the level of salinity in soil. Symbiotic colonization of plants with Piriformospora indica has shown a promotion in plants growth and tolerance against biotic stress. In this study, physiological markers such as ion analysis, antioxidant determination, proline content, electrolyte leakage and chlorophyll measurement were assessed in melon cultivar under two concentrations (100 and 200 mM) of NaCl with and without P. indica inoculation. Results showed that the endophytic inoculation consistently upregulated the level of antioxidants, enhanced plants to antagonize salinity stress. The expression level of an RNA editing factor (SLO2) which is known to participate in mitochondria electron transport chain was analyzed, and its full mRNA sequence was obtained by rapid amplification of cDNA ends (RACE). Under salinity stress, the expression level of SLO2 was increased, enhancing the plant's capability to adapt to the stress. However, P. indica inoculation further elevated the expression level of SLO2. These findings suggested that the symbiotic association of fungi could help the plants to tolerate the salinity stress.

CmRAV1 shows differential expression in two melon (Cucumis melo L.) cultivars and enhances salt tolerance in transgenic Arabidopsis plants.

The growth and development of melon (Cucumis melo L.) are severely affected by soil salinization in many areas of the world, but the understanding of the molecular mechanisms underlying salt tolerance in melon remains limited. In this study, a new RAV (related to ABI3/VP1) gene, CmRAV1, was identified in melon. Protein structure homology analysis revealed that CmRAV1 contains an AP2 domain and a B3 domain, and subcellular localization assay revealed that CmRAV1 is localized in the nucleus. The transcript level of CmRAV1 was closely correlated with NaCl treatment, and the expression pattern of CmRAV1 differed between two cultivars (salt-tolerant and salt-sensitive cultivars) under NaCl treatment. In addition, yeasts transformed with CmRAV1 showed notably improved growth on medium containing 200 mM NaCl compared with wild-type ones. The overexpression of CmRAV1 in transgenic Arabidopsis thaliana resulted in enhanced salt tolerance at the seed germination and seedling growth stages. This study demonstrated that the expression of CmRAV1 was associated with saline stress and can potentially be utilized to improve plant salt tolerance.

Effect of glycine nitrogen on lettuce growth under soilless culture: a metabolomics approach to identify the main changes occurred in plant primary and secondary metabolism.

BACKGROUND: Lettuce is a significant source of antioxidants and bioactive compounds. Nitrate is a cardinal fertilizer in horticulture and influences vegetable yield and quality; however, the negative effects of nitrate on the biosynthesis of flavonoids require further study. It is expected that using fertilizers containing organic nitrogen (N) could promote the synthesis of health-promoting compounds. RESULTS: Lettuces were hydroponically cultured in media containing 9 mmol L-1 nitrate or 9 mmol L-1 glycine for 4 weeks. Primary and secondary metabolites were analyzed using gas chromatography/mass spectrometry (GC/MS) and ultra-performance liquid chromatography/ion mobility spectrometry/quadrupole time-of-flight mass spectrometry (UPLC/IMS/QTOF-MS). Data analysis revealed that 29 metabolites were significantly altered between nitrate and glycine treatments. Metabolites were tentatively identified by comparison with online databases, literature and standards and using collision cross-section values. Significant differences in flavonoid biosynthesis, phenolic biosynthesis and the tricarboxylic acid (TCA) cycle response were observed between N sources. Compared with nitrate, glycine promoted accumulation of glycosylated flavonoids (quercetin 3-glucoside, quercetin 3-(6″-malonyl-glucoside), luteolin 7-glucuronide, luteolin 7-glucoside), ascorbic acid and amino acids (l-valine, l-leucine, l-glutamine, asparagine, l-serine, l-ornithine, 4-aminobutanoic acid, l-phenylalanine) but reduced phenolic acids (dihydroxybenzoic acid hexose isomers 1 and 2, chicoric acid, chicoric acid isomer 1) and TCA intermediates (fumaric, malic, citric and succinic acids). CONCLUSION: The novel methodology applied in this study can be used to characterize metabolites in lettuce. Accumulation of glycosylated flavonoids, amino acids and ascorbic acid in response to glycine supply provides strong evidence supporting the idea that using amino acids as an N source alters the nutritional value of vegetable crops. © 2017 Society of Chemical Industry.

The endomembrane system: how does it contribute to plant secondary metabolism?

New organelle acquisition through neofunctionalization of the endomembrane system (ES) with respect to plant secondary metabolism is a key evolutionary strategy for plant adaptation, which is overlooked due to the complexity of angiosperms. Bryophytes produce a broad range of plant secondary metabolites (PSMs), and their simple cellular structures, including unique organelles, such as oil bodies (OBs), highlight them as suitable model to investigate the contribution of the ES to PSMs. In this opinion, we review latest findings on the contribution of the ES to PSM biosynthesis, with a specific focus on OBs, and propose that the ES provides organelles and trafficking routes for PSM biosynthesis, transportation, and storage. Therefore, future research on ES-derived organelles and trafficking routes will provide essential knowledge for synthetic applications.

Elevation of artemisinin content by co-transformation of artemisinin biosynthetic pathway genes and trichome-specific transcription factors in Artemisia annua.

Artemisinin, derived from Artemisia annua, is currently used as the first-line treatment for malaria. However, wild-type plants have a low artemisinin biosynthesis rate. Although yeast engineering and plant synthetic biology have shown promising results, plant genetic engineering is considered the most feasible strategy, but it is also constrained by the stability of progeny development. Here we constructed three independent unique overexpressing vectors harboring three mainstream artemisinin biosynthesis enzymes HMGR, FPS, and DBR2, as well as two trichomes-specific transcription factors AaHD1 and AaORA. The simultaneous co-transformation of these vectors by Agrobacterium resulted in the successful increase of the artemisinin content in T0 transgenic lines by up to 3.2-fold (2.72%) leaf dry weight compared to the control plants. We also investigated the stability of transformation in progeny T1 lines. The results indicated that the transgenic genes were successfully integrated, maintained, and overexpressed in some of the T1 progeny plants' genomes, potentially increasing the artemisinin content by up to 2.2-fold (2.51%) leaf dry weight. These results indicated that the co-overexpression of multiple enzymatic genes and transcription factors via the constructed vectors provided promising results, which could be used to achieve the ultimate goal of a steady supply of artemisinin at affordable prices around the world.

Vacuoles in Bryophytes: Properties, Biogenesis, and Evolution.

Vacuoles are the most conspicuous organelles in plants for their indispensable functions in cell expansion, solute storage, water balance, etc. Extensive studies on angiosperms have revealed that a set of conserved core molecular machineries orchestrate the formation of vacuoles from multiple pathways. Usually, vacuoles in seed plants are classified into protein storage vacuoles and lytic vacuoles for their distinctive morphology and physiology function. Bryophytes represent early diverged non-vascular land plants, and are of great value for a better understanding of plant science. However, knowledge about vacuole morphology and biogenesis is far less characterized in bryophytes. In this review, first we summarize known knowledge about the morphological and metabolic constitution properties of bryophytes' vacuoles. Then based on known genome information of representative bryophytes, we compared the conserved molecular machinery for vacuole biogenesis among different species including yeast, mammals, Arabidopsis and bryophytes and listed out significant changes in terms of the presence/absence of key machinery genes which participate in vacuole biogenesis. Finally, we propose the possible conserved and diverged mechanism for the biogenesis of vacuoles in bryophytes compared with seed plants.

Transcriptional regulation of flavonoid biosynthesis in Artemisia annua by AaYABBY5.

Artemisia annua is a medicinal plant rich in terpenes and flavonoids with useful biological activities such as antioxidant, anticancer, and antimalarial activities. The transcriptional regulation of flavonoid biosynthesis in A. annua has not been well-studied. In this study, we identified a YABBY family transcription factor, AaYABBY5, as a positive regulator of anthocyanin and total flavonoid contents in A. annua. AaYABBY5 was selected based on its similar expression pattern to the phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavonol synthase (FLS) genes. A transient dual-luciferase assay in Nicotiana bethamiana with the AaYABBY5 effector showed a significant increase in the activity of the downstream LUC gene, with reporters AaPAL, AaCHS, AaCHI, and AaUFGT. The yeast one-hybrid system further confirmed the direct activation of these promoters by AaYABBY5. Gene expression analysis of stably transformed AaYABBY5 overexpression, AaYABBY5 antisense, and control plants revealed a significant increase in the expression of AaPAL, AaCHS, AaCHI, AaFLS, AaFSII, AaLDOX, and AaUFGT in AaYABBY5 overexpression plants. Moreover, their total flavonoid content and anthocyanin content were also found to increase. AaYABBY5 antisense plants showed a significant decrease in the expression of flavonoid biosynthetic genes, as well as a decrease in anthocyanin and total flavonoid contents. In addition, phenotypic analysis revealed deep purple-pigmented stems, an increase in the leaf lamina size, and higher trichome densities in AaYABBY5 overexpression plants. Together, these data proved that AaYABBY5 is a positive regulator of flavonoid biosynthesis in A. annua. Our study provides candidate transcription factors for the improvement of flavonoid concentrations in A. annua and can be further extended to elucidate its mechanism of regulating trichome development.

An R2R3-MYB Transcription Factor Positively Regulates the Glandular Secretory Trichome Initiation in Artemisia annua L.

Artemisia annua L. is known for its specific product "artemisinin" which is an active ingredient for curing malaria. Artemisinin is secreted and accumulated in the glandular secretory trichomes (GSTs) on A. annua leaves. Earlier studies have shown that increasing GST density is effective in increasing artemisinin content. However, the mechanism of GST initiation is not fully understood. To this end, we isolated and characterized an R2R3-MYB gene, AaMYB17, which is expressed specifically in the GSTs of shoot tips. Overexpression of AaMYB17 in A. annua increased GST density and enhanced the artemisinin content, whereas RNA interference of AaMYB17 resulted in the reduction of GST density and artemisinin content. Additionally, neither overexpression lines nor RNAi lines showed an abnormal phenotype in plant growth and the morphology of GSTs. Our study demonstrates that AaMYB17 is a positive regulator of GSTs' initiation, without influencing the trichome morphology.

Transcriptomic analysis reveals the parallel transcriptional regulation of UV-B-induced artemisinin and flavonoid accumulation in Artemisia annua L.

UV-B radiation is a pivotal photomorphogenic signal and positively regulates plant growth and metabolite biosynthesis. In order to elucidate the transcriptional regulation mechanism underlying UV-B-induced artemisinin and flavonoid biosynthesis in Artemisia annua, the transcriptional responses of A. annua L. leaves to UV-B radiation were analyzed using the Illumina transcriptome sequencing. A total of 10705 differentially expressed genes (DEGs) including 533 transcription factors (TFs), were identified. Based on the expression trends of the differentially expressed TFs as well as artemisinin and flavonoid biosynthesis genes, we speculated that TFs belonging to 6 clusters were most likely to be involved in the regulation of artemisinin and/or flavonoid biosynthesis. The regulatory relationship between TFs and artemisinin/flavonoid biosynthetic genes was further studied. Dual-LUC assays results showed that AaMYB6 is a positive regulator of AaLDOX which belongs to flavonoid biosynthesis pathway. In addition, we identified an R2R3 MYB TF, AaMYB4 which potentially mediated both artemisinin and flavonoid biosynthesis pathways by activating the expression of AaADS and AaDBR2 in artemisinin biosynthesis pathway and AaUFGT in flavonoid biosynthesis pathway. Overall, our findings would provide an insight into the elucidation of the parallel transcriptional regulation of artemisinin and flavonoid biosynthesis in A. annua L. under UV-B radiation.

Parallel Transcriptional Regulation of Artemisinin and Flavonoid Biosynthesis.

Plants regulate the synthesis of specialized compounds through the actions of individual transcription factors (TFs) or sets of TFs. One such compound, artemisinin from Artemisia annua, is widely used as a pharmacological product in the first-line treatment of malaria. However, the emergence of resistance to artemisinin in Plasmodium species, as well as its low production rates, have required innovative treatments such as exploiting the synergistic effects of flavonoids with artemisinin. We overview current knowledge about flavonoid and artemisinin transcriptional regulation in A. annua, and review the dual action of TFs and structural genes that can regulate both pathways simultaneously. Understanding the concerted action of these TFs and their associated structural genes can guide the development of strategies to further improve flavonoid and artemisinin production.

AaABCG40 Enhances Artemisinin Content and Modulates Drought Tolerance in Artemisia annua.

The phytohormone Abscisic acid (ABA) regulates plant growth, development, and responses to abiotic stresses, including senescence, seed germination, cold stress and drought. Several kinds of researches indicate that exogenous ABA can enhance artemisinin content in A. annua. Some transcription factors related to ABA signaling are identified to increase artemisinin accumulation through activating the artemisinin synthase genes. However, no prior study on ABA transporter has been performed in A. annua. Here, we identified a pleiotropic drug resistance (PDR) transporter gene AaPDR4/AaABCG40 from A. annua. AaABCG40 was expressed mainly in roots, leaves, buds, and trichomes. GUS activity is primarily observed in roots and the vascular tissues of young leaves in proAaABCG40: GUS transgenic A. annua plants. When AaABCG40 was transferred into yeast AD12345678, yeasts expressing AaABCG40 accumulated more ABA than the control. The AaABCG40 overexpressing plants showed higher artemisinin content and stronger drought tolerance. Besides, the expression of CYP71AV1 in OE-AaABCG40 plants showed more sensitivity to exogenous ABA than that in both wild-type and iAaABCG40 plants. According to these results, they strongly suggest that AaABCG40 is involved in ABA transport in A. annua.

High-Level Patchoulol Biosynthesis in Artemisia annua L.

Terpenes constitute the largest class of secondary metabolites in plants. Some terpenes are essential for plant growth and development, membrane components, and photosynthesis. Terpenes are also economically useful for industry, agriculture, and pharmaceuticals. However, there is very low content of most terpenes in microbes and plants. Chemical or microbial synthesis of terpenes are often costly. Plants have the elaborate and economic biosynthetic way of producing high-value terpenes through photosynthesis. Here we engineered the heterogenous sesquiterpenoid patchoulol production in A. annua. When using a strong promoter such as 35S to over express the avian farnesyl diphosphate synthase gene and patchoulol synthase gene, the highest content of patchoulol was 52.58 µg/g DW in transgenic plants. When altering the subcellular location of the introduced sesquiterpene synthetase via a signal peptide, the accumulation of patchoulol was observably increased to 273 µg/g DW. This case demonstrates that A. annua plant with glandular trichomes is a useful platform for synthetic biology studies.

Influence of bio-fertilizer containing beneficial fungi and rhizospheric bacteria on health promoting compounds and antioxidant activity of Spinacia oleracea L.

BACKGROUND: This study evaluates the influences of bio fertilizers containing mycorrhizal fungi (Glomus fasciculatum, Glomus mosseae) individually or in combination with N-fixer (Azotobacter chroococcum), K solubilizer (Bacillus mucilaginous) and P solubilizer (Bacillus megaterium) on soil fertility and phytochemical levels of spinach. RESULTS: Root colonization by mycorrhizal fungi was increased in the presence of bacterial inoculation in comparison to individual inoculation treatments. Inoculation of bio fertilizer containing mycorrhizal fungi and bacterial species considerably augmented the concentration of total phenolic compounds, flavonoids and phenolic acid contents. The 1, 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging capacity of spinach was found to be positively coincided with flavonoid contents, while partially correlated with total phenolic compounds and phenolic acids. Further, the HPLC analysis showed that significantly higher antioxidant activity of spinach was correlated with quercetin contents and chlorogenic acid. Chlorophyll contents were also increased following the bio fertilization treatments. CONCLUSION: Results revealed that these microbes are useful tool for improving health promoting compounds in spinach.

Elevation of secondary metabolites synthesis in Brassica campestris ssp. chinensis L. via exogenous inoculation of Piriformospora indica with appropriate fertilizer.

This work evaluated the impact of exogenous soil inoculation of beneficial fungal strain Piriformospora indica on phytochemical changes and the related genes expression of Chinese cabbage (Brassica campestris ssp. chinensis L.) by greenhouse pot experiments. High performance liquid chromatography (HPLC) affirmed that among the different combinations of fungal and organic fertilizer treatments, the phenolic acids and flavonoids were considerably enriched in organic fertilizer and fungi (OP) followed by organic fertilizer, biochar, fungi (OBP) treated plants. The antiradical activity was higher in OP (61.29%) followed by P (60%) and organic fertilizer (OF) (53.84%) inoculated plants which positively correlated with chlorophyll, carotenoids and flavonoids level (P<0.05). Furthermore, results showed that the exogenous application of P. indica significantly (P<0.05) enhanced plant growth, as well as stimulating the activation of chlorophyll, carotenoids and other antioxidant related pathways. The RT-qPCR analysis indicated that key FLS gene triggering the synthesis of kaemferol was up-regulated by the inoculation of P. indica. In conclusion, the results revealed that organic fertilizer and P. indica (OP) is the most appropriate combination for improving phytochemical and antiradical properties in Pakchoi.