Identification of Microbial Metabolites Elevating Vitamin Contents in

Article pubs.acs.org/JAFC

Identification of Microbial Metabolites Elevating Vitamin Contents in Barley Seeds Anam Yousaf,*,† Abdul Qadir,† Tehmina Anjum,‡ and Aqeel Ahmad‡ †

College of Earth and Environmental Sciences and ‡Institute of Agricultural Sciences, University of the Punjab, Lahore-54590, Pakistan ABSTRACT: The current investigation analyzes metabolites of Acetobacter aceti to explore chemical compounds responsible for the induction of vitamins in barley seeds. A bioactivity guided assay of bacterial extracts and chromatographic analyses of barley produce revealed 13 chemical compounds, which were subjected to principal component analysis (PCA). PCA determined four chemical compounds (i.e., quinolinic acid, pyridoxic acid, p-aminobenzoate, and α-oxobutanoic acid) highly associated with increased quantities of vitamins. Further experimentations confirmed that quinolinic acid and p-aminobenzoate were the most efficient vitamin inducers. The results indicated chloroform/ethanol (4:1) as the best solvent system for the extraction of active compounds from crude metabolites of A. aceti. Significant quantities of mevalonic acid were detected in the extracted fraction, indicating the possible induction of the isoprenoid pathway. Altogether, the current investigation broadens the frontiers in plant− microbe interaction. KEYWORDS: bioactivity guided assay, chromatographic analysis, fractionation, precursor, transition state

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INTRODUCTION

Bacterial Species and Barley Plants. A. aceti (FCBP-537) was procured from Fungal Culture Bank of Pakistan, University of the Punjab, Lahore, Pakistan. Cultures were maintained on Petri plates containing LB agar cultures medium (EZMix, Lahore, Pakistan). Barley seeds were obtained from Fungal Biotechnology Lab and grown in plastic pots under greenhouse conditions (22 ± 2 °C). These plants were used to perform the biological assay at the age of 2 weeks. Bacterial species was grown in 4 L of Czapek Dox broth medium (Sigma-Aldrich, Lahore, Pakistan) based upon sucrose (3%). Bacterial cells were removed by centrifugation leaving behind the cell free culture filtrate (CFCF), which was subjected to organic solvent extraction. Organic Solvent Extraction. CFCF was evaporated using a rotary evaporator at a temperature of 40 °C up to 1 L of remaining volume. Then, it was successively extracted with n-hexane, chloroform, ethyl acetate, and n-butanol. The former fraction of each organic solvent was separated before starting the extraction process with the next solvent. Thus, organic fractions of all solvents were prepared and evaporated yielding dry extracts. These dried fractions were checked for their ability to induce vitamin quantities in barley, and the best performing fraction was screened out. For this purpose, vitamin contents were analyzed in barley plants treated with different organic fractions. The comparison of organic treatments was made with control treatment (on which no fraction was applied). Then, the fraction inducing maximum vitamin quantities was selected for further downstream purification steps. This purification was carried out through chromatographic techniques, while checking the bioactivity of all subfractions at every downstream level. Hence, the process is termed as bioactivity guided isolation of active compounds. Bioactivity Guided Isolation of Active Compounds. The crude fraction exhibiting the maximum bioactivity was processed through column chromatography generating five subfractions, i.e.,

Modulation of plant metabolic processes under the influence of microbial treatments is a well-known phenomenon in agricultural sciences. Researchers have successfully utilized this technique to achieve numerous goals related to plant protection.1,2 Researchers have concluded that biological treatments bring large scale changes in plant metabolism.3 They may alter the rate of plant metabolic pathways, which modulate the quantities of plant biochemicals.4−6 Thus, a number of studies have reported changes in quantities of intrinsically very diverse biochemicals in plants.7,8 The primary objective of these studies was to explore the potential of microbes to enhance the quantities of vitamin contents in plant produce (barley seeds). Langston et al.9 and Rice et al.10 studied the roles of photo induction and sodium hydroxide inductions in elevated quantities of vitamins. In a parallel study, Lichtenthaler et al.11 also looked at light induced vitamin K1. All these studies provide a strong base to investigate microbes to achieve higher vitamin levels in plant produce. Quantities of water-soluble vitamins were primarily focused on in this study, which exhibit prime importance in defining food quality. The study screened out Acetobacter aceti, which altered the plant metabolic processes and enhanced the vitamin contents in barley seeds. However, the bacterial species has been rarely reported as a potential pathogen of pink disease of pineapple.12 Thus, it cannot be applied carelessly in agriculture fields. Therefore, it is imperative to identify the active compound in the bacterial metabolite mixture, which could be used to get an improved quality agriculture yield. Thus, the current investigation mainly focuses upon screening of bacterial metabolites to identify the specific biochemical substances that would help to improve the quality of agricultural produce. © 2015 American Chemical Society

MATERIALS AND METHODS

Received: Revised: Accepted: Published: 7304

April 30, 2015 July 1, 2015 July 14, 2015 July 14, 2015 DOI: 10.1021/acs.jafc.5b01817 J. Agric. Food Chem. 2015, 63, 7304−7310

Article

Journal of Agricultural and Food Chemistry

Figure 1. Quantities of vitamin contents in barley treated with organic fractions of Acetobacter aceti extracts. Control treatment refers to no application of bacterial extracts. Error bars show the standard error among each treatment, while different alphabetic letters show significant difference between treatments, calculated through Duncan’s multiple range test (DMRT) at p ≤ 0.05. Statistical analysis was performed through DSAASTAT (Onofri, Italy).

Figure 2. Quantities of water-soluble vitamins in barley obtained from treated plants. Plants had been treated with subfractions of chloroform extracts, prepared in, i.e., 20, 40, 60... 100% concentrations of chloroform in ethyl acetate (plotted on X-axis). The quantities of vitamins in g/kg have been presented on the Y-axis. Error bars have been plotted to represent the standard error among each data set, whereas changing lowercase letters represent significant difference in data. Data were statistically analyzed through Duncan’s multiple range test the (DMRT) at p ≤ 0.05 and using the MS-Excel add-in statistical package DSAASTAT (Onofri, Italy). SF01, SF02, SF03, SF04, and SF05. The biochemical profile of each subfraction was estimated through thin layer chromatography, while bioactivity was studied by applying subfractions on barley plants. Organic subfraction induced maximum quantities in barley were analyzed through chromatographic techniques to identify the specific compound(s) responsible for vitamin induction in the greenhouse assay. Barley plants were grown under 24 ± 2 °C in a greenhouse, and watering was carried out when needed. Vitamin contents of barley were quantified, and the best organic fraction was screened out. The organic fraction was further fractionated into 5 subfractions (i.e., FR01, FR02, FR03, FR04, and FR05) through column chromatography (CC). For this purpose, the concentrate of the previous fraction was mixed with a small quantity of silica gel of Uni-Chem (mesh 60−100) to make its slurry. A two third portion of the column (200 g) was filled with silica gel, and then a thin layer of prepared slurry was uniformly spread over the surface of the silica column. Moreover, the slurry was again overlaid by a uniform thin layer of silica gel. A stepwise elution of samples was performed with a solvent system of ethanol/chloroform, having a constant increment of 20% in concentration of chloroform.

All prepared subfractions were studied for vitamin elevation potential, through the bioactivity guided assay. Therefore, all these subfractions were applied to barley plants, and plants were allowed to grow up to seed harvesting stage. These seeds were crushed and tested for watersoluble vitamin quantities (i.e., ascorbic acid, niacin, panthothenic acid, pyridoxine thiamine, folic acid, and riboflavin). These quantities were quantified by measuring peak height and peak area of gas chromatograms of vitamins along with some other contaminants. Moreover, preparative thin layer chromatography (PTLC) was performed to remove the maximum possible contaminants from the fractions. PTLC was performed through glass plates (20 × 20 cm2) coated with silica. Bioactive compounds were isolated with the minimum possible number of contaminants before performing gas chromatography−mass spectrometery (GCMS). Gas Chromatography−Mass Spectrometery. GCMS was performed on a gas chromatography unit of Agilent Technologies (7890A), accompanied by a mass spectrometer unit of (5975C) and column HP-5MS. Operation was started from 70 °C temperature with continuous increments of 5 °C/min up to 200 °C temperature. 7305

DOI: 10.1021/acs.jafc.5b01817 J. Agric. Food Chem. 2015, 63, 7304−7310

Article


Figure 3. GCMS profiles of the two most active chloroform fractions, i.e., 80% (A) and 100% (B). Standard run of most active compounds were performed providing identical GCMS conditions on the same GCMS apparatus. Quinolinic acid (C), pyridoxic acid (D), p-aminobenzoate (E), and α-oxobutanoic acid (F). X-Axis has been marked with the time elapsed for chemical elution, and the Y-axis represents the height of peaks dependent upon chemical quantities.

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Operation was further continued up to 240 °C at a continuous increment of 10 °C/min. The flow rate of carrier gas (helium) was maintained 1 mL/min during the entire operation. Mass spectra of compounds were identified using TURBOMASS (Perkinelmer, Shelton, USA) equipped with a mass spectral NIST library. Data Analysis. Statistical analysis of data was carried out through analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) at p ≤ 0.05. Statistical operations were performed using the statistical package of MS-Excel based add-in DSAASTAT (Onofri, Italy). Principal component analysis (PCA) was performed to determine the most active bacterial metabolite, and the screened chemical was applied to barley plants to verify the results of the study. Activity Verification of Identified Compound. Purified chemicals were purchased from Sigma-Aldrich (Lahore) and applied separately to sets of barley plants of 2 weeks of age, treating the control with distilled sterilized water. Seeds of the plants were tested for vitamin contents, and data were compared with the standard GCMS run of purified compounds to test the efficacy of treatment. The whole experiment was independently repeated thrice to ensure the authenticity of the results.

RESULTS Chloroform fraction provided the maximum levels of most of the tested vitamins. Significantly higher quantities of ascorbic acid, pyridoxine, folic acid and riboflavin were recorded in barley. Niacin and pantothenic acid were recorded in equal quantities in both treatments of chloroform and ethyl acetate, whereas n-butanol exhibited nonsignificantly more amounts of thiamine contents than chloroform treatment. n-Hexane was recorded with nonsignificantly lower quantities of panthothenic acid and thiamine contents than the respective values of control treatment (Figure 1). Eighty percent chloroform treatment induced the highest quantities of vitamins in barley. It was most efficient for inducing ascorbic acid content in barley seeds. Moreover, the quantity of riboflavin was also elevated by 80% chloroform in the most efficient way. Pyrodixine was recorded to be significantly elevated after getting 80% chloroform treatment. Similarly, the quantity of niacin was found to be significantly 7306


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higher in 80% chloroform than in all other treatments. The second most efficient treatment in terms of vitamins induction was 100% chloroform. It induced ascorbic acid at significantly higher concentrations than 20, 40, and 60% chloroform treatments. Induction in the quantity of pyrodoxine was also significantly higher in 100% chloroform treatment than 20, 40, and 60%. A similar trend was found in the case of riboflavin, which was detected in significantly higher amounts in 100 chloroform treatments than 20%, 40%, and 60% treatments. Thiamine was the only vitamin, which did not show any significant variation in its quantities among all treatments; however, the quantity folic acid was recorded to be significantly greater in the 80% treatment in comparison to the respective quantities in 20, 40, and 60% treatment (Figure 2). Two fractions (i.e., 80% and 100%) possessed the most efficient vitamin induction activities and were thus analyzed by GCMS to figure out active compounds. Thirteen chemical compounds were detected in the most active (80%) chloroform fraction. These chemicals were identified as thymol, eugenol, globulol, mevalonic lactone, quinolinic acid, E-8-octadecacen-1-ol acetate (ODCA), pentadenoic acid, phthalic ester, octadecatrienoic acid (ODTA), pyridoxic acid, p-aminobenzoate, α-oxobutanoic acid, and glycine. The second most active (100%) chloroform fraction was missing in quinolinic acid and ODCA. However, the concentration of most of the compounds (i.e., quinolinic acid, ODCA, pentadenoic acid, phthalic ester, ODTA, p-aminobenzoate, and α-oxobutanoic acid) was lower in the 100% chloroform fraction than in the 80% fraction (Figure 3). Chemical properties of all detected compounds were recorded in order to evaluate the principal compound. All 13 compounds had peaks sufficiently apart with good resolution. Thymol was the first compound detected at 8.03 min of the chromatographic run with a peak area of 4.7E. Eugenol was eluted at 8.88 min and recorded a peak area of 4.9E. Mass spectra of globulol (peak area: 5.1E) and mevalonic lactone (peak area: 4.3E) were recorded after chromatographic runs of 9.17 and 9.29 min, respectively. Quinolinic acid with a peak area of 4.7E was detected at 12.33 min. Pyridoxic acid (19.67 min) exhibited the highest peak area 7.4E. The second highest peak area (6.5E) was recorded in the case of p-aminobenzoate, which had the retention time of 20.41 min. α-Oxobutanoic acid and glycine (peak area: 5.3E and 4.2E, respectively) were the last eluting compounds with retention times of 21.18 and 21.28 min, respectively (Table 1). Pyridoxic acid was a compound having maximum affinity with the concentration of chloroform in the extraction solvent. p-Aminobenzoate had a lower affinity with chloroform concentration than pyridoxic acid. However, it exhibited stronger association with vitamin induction than pyridoxic acid. Similarly, α-oxobutanoic acid had the third strongest association with chloroform concentration, but it recorded the strongest correlation with vitamins induction. Quinolinic acid recorded the third strongest relationship with vitamin induction, just after p-aminobenzoate; but it was fourth according to its interrelationship with chloroform concentration. All these four compounds (i.e., quinolinic acid, paminobenzoate, pyridoxic acid, and α-oxobutanoic acid) collectively construct a group (∞) and are closely placed over a PCA plot. Similarly, thymol, globulol, mevalonic lactone, and ODCA were members of a second group (β), and this group had more variable association with chloroform concentration than with vitamin induction. Mevalonic lactone

Table 1. Chemical Compounds in Chloroform Fractions (with Concentrations of 80% and 100%) Were Identified through GCMS Analysisa serial no. 1 2 3 4 5 6 7 8 9 10 11 12 13

chemical thymol eugenol globulol mevalonic lactone quinolinic acid E-8-octadecacen-1-ol acetate (ODCA) pentadenoic acid phthalic ester (methyl octyl ester) octadecatrienoic acid (ODTA) pyridoxic acid p-aminobenzoate α-oxobutanoic acid glycine

retention time (min)

total peak area (E)

8.03 8.88 9.17 9.29 12.33 13.68

4.7 4.9 5.1 4.3 4.7 5.6

13.85 14.08

5.8 5.9

15.75 19.67 20.41 21.18 21.28

6.1 7.4 6.5 5.3 4.2

a

MZmine (Pluskal, Okinawa, Japan) was used to analyze chromatograms and mass spectra. Properties of each compound were recorded and mentioned against it. Retention time is the time taken by chemicals for their individual elution and has been calculated in minutes (min), whereas total peak area has been calculated through MZmine unit of relativity (E).

Figure 4. Principal component analysis (PCA) of all detected compounds through GCMS analysis of chloroform subfractions of A. aceti extracts. Thirteen chemical componds detected in GCMS analysis were 1, thymol; 2, eugenol; 3, globulol; 4, mevalonic lactone; 5, quinolinic acid; 6, E-8-octadecacen-1-ol acetate (ODCA); 7, pentadenoic acid; 8, phthalic ester; 9, octadecatrienoic acid (ODTA); 10, pyridoxic acid; 11, p-aminobenzoate; 12, α-oxobutanoic acid; and 13, glycine. Association of all compounds was calculated with the concentration of chloroform in extraction solvent and induction of vitamins. The statistical package DSAASTAT (Onofri, Italy) was used to perform PCA analysis.

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Figure 5. Standard mass spectra of active compounds taken from the NIST library, i.e., quinolinic acid (A), pyridoxic acid (B), p-aminobenzoate (C), α-oxobutanoic acid (D). Mass spectrum recorded during an experimental run of A. aceti fractions has been tagged with the English alphabet followed by symbol -’-. Compoounds were identified using TURBOMASS (Perkinelmer, Shelton, USA) equipped with the NIST library.

had the strongest association with chloroform concentration as well as vitamin induction among all the members of group β. Thymol was the least associated member of group β with vitamin induction. Eugenol, pentadenoic acid, phthalic ester, ODTA, and glycine were found in inverse relationship with vitamins induction; however, they were positively associated with concentration of chloroform in extraction solvent (Figure 4). All compounds which occupied distinct positions in the correlation matrix plot were purified up to >99% to test their effectiveness for the induction of vitamin contents. Mass spectra of bioactive compounds were recorded through TURBOMASS (Perkinelmer, Shelton, USA) and their

respective compounds in the NIST library compared. More than 800 similarity scores were calculated in the case of all identified compounds (Figure 5). Activity Verification of Identified Compounds. Maximum quantities of vitamins were induced after the application of p-aminobenzoate. It recorded significantly higher quantities of six vitamins, i.e., ascorbic acid, niacin, pathothenic acid, pyridoxine, and thiamine and folic acid (Figure 6). However, the highest quantities of riboflavin were recorded in the case of quinolinic acid and p-aminobenzoate. Quinolinic acid was the second best inducer of vitamins in barley seeds. However, pyridoxic acid and α-oxobutanoic acid had a variable behavior 7308


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Figure 6. Vitamin contents in barley seeds treated with different biochemicals (i.e., quinolinic acid, pyridoxic acid, p-aminobenzoate, and αoxobutanoic acid). Control treatment refers to no chemical application. Data were statistically analyzed through analysis of variance (ANOVA) using DSAASTAT (Onofri, Italy), and error bars show standard error of the data, and each change in lowercase letters represents the significant difference among treatments calculated through Duncan’s multiple range test (DMRT) at p ≤ 0.05.

extracts have more amounts of active compounds and are more specific in action. GCMS analysis of biologically active fractions identified the compounds present in them; however, PCA analysis determined the activity of each identified biochemical and arranged them in a hierarchal manner on the basis of their ability to induce vitamins. Four compounds were found to be strongly associated with the induction of vitamins; however, the interactions among these chemicals have not been studied. It might be possible that they have a cumulative effect on vitamin biosynthesis or that they have synergistic effects on vitamin metabolism.18,19 All the biochemicals identified in this research are either precursors of vitamin biosynthesis pathways (e.g., quinolinic acid and pyridoxic acid)20,21or they are facilitators of these pathways (e.g., p-aminobenzoate and α-oxobutanoic).22,23 Thus, the increased production of vitamins in barley can be supported with the logic that “providing precursors of vitamins would facilitate plants in their production process.”24,25 PCA analysis categorized the compounds on the basis of their affinity with induced vitamin quantities and concentration of chloroform in extraction solvent. This revealed mevalonic acid as the most active compound among group β of the identified compounds. Mevalonic acid was the compound with the highest concentration in extracted fraction, as compared to the other compounds of group β. Moreover, this compound plays a key role in the isoprenoid pathway for the biosynthesis of squalene,26 which is responsible for the ultimate production of sterols.27 GCMS analysis also revealed the presence of glycine in tested fractions. Glycine takes part in the process of folate biosynthesis for the production of glutamic acid. It is a three step former biochemical reaction just equivalent to the production of 5,10methenyltetrahydrofolate polyglutamate. Logically, glycine would be in positive relationship with vitamin induction; however, it was inversely related with vitamin quantities. These findings indicate some complexities in folate metabolism, which still need to be resolved. Pyridoxic acid treatment on plants results in the decreased sensitivity of plant photosystem II and protects plants against photooxidative stress.28 However, the decreased sensitivity of the photosystem II reaction center

with respect to each individual vitamin. They could not induce significant quantities of most of the vitamins with respect to the control treatment. However, pyridoxic acid inhibited the production of ascorbic acid in barley seeds (Figure 6).

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DISCUSSION The current investigation describes an array of microbial metabolites which affect plant biochemicals. This study has highlighted the metabolites which more specifically enhanced nutritional quality related biochemicals in barley seeds. Barley is a staple food crop which can be routinely consumed by humans and animals. Therefore, the current investigation is an advancement in the improvement of food quality of large populations. It recommends the microbial application for getting augmented agricultural yields in terms of nutritional contents. The solubility of different compounds is extremely variable in a single organic solvent.13 Similarly, different organic solvents dissolve highly variable amounts of a single compound.14 Thus, the optimization of solvent is necessarily required to isolate a desired compound with minimum number of contaminants; and this optimization is imperative when an active compound is needed to be isolated from a mixture containing thousands of bacterial metabolites.15 This study suggests a solvent system of 80% chloroform and 20% ethanol to isolate active compounds with a minimum number of impurities from bacterial metabolites. The individual purity index of these metabolites is >99.9%. However, their concentrations in subfractions were recorded as quinolinic acid (1.09%), pyridoxic acid (50.54%), p-aminobenzoate (36.26%), and α-oxobutanoic acid (12.11%). Plants respond to external applications of chemicals, while metabolites of A. aceti contain a number of chemicals which had unknown effects on plants. Hence, the purification of active compounds becomes very important in order to reduce the number of undesired compounds. Furthermore, the composition solvent system affects the quantity and quality of compounds being extracted.16,17 The current study approves the two step extraction of A. aceti metabolites: first, the extraction with 100% chloroform and subsequent extraction with an 80% chloroform solvent system in ethanol (chloroform/ethanol (4:1) solvent system). Thus, the resulting 7309


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Journal of Agricultural and Food Chemistry hampers light acclimation and reduced ascorbate content.29 Moreover, the quenching of O2 starts proteolytic degradation of ascorbate and lowers its contents in plant tissues.30 All these studies support the decreased ascorbic acid contents recorded after treating barley plants with pyridoxic acid. The study concludes that two biochemicals, i.e., p-aminobenzoate and quinolinic acid can induce vitamin contents in barley seeds. However, p-aminobezoate is recommended as the best vitamin inducer. All of these two biochemicals can be isolated from the metabolites of A. aceti using an organic solvent system (chloroform/ethanol 4:1). This study would be a step toward the cultivation of food crops with improved nutritional values.

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(16) Kaufmann, B.; Christen, P. Recent extraction techniques for natural products: microwave-assisted extraction and pressurised solvent extraction. Phytochem. Anal. 2002, 13, 105−113. (17) Sultana, B.; Anwar, F.; Ashraf, M. Effect of extraction solvent/ technique on the antioxidant activity of selected medicinal plant extracts. Molecules 2009, 14, 2167−2180. (18) Baider, A.; Cohen, Y. Synergistic interaction between BABA and mancozeb in controlling Phytophthora infestans in potato and tomato and Pseudoperonospora cubensis in cucumber. Phytoparasitica 2003, 31 (4), 399−409. (19) Brent, K. J.; Hollomon, D. W. Fungicide Resistance in Crop Pathogens: How Can It Be Managed? Fungicide Resistance Action Committee; Crop life International: Belgium, 2007. (20) Sauve, A. A. NAD+ and vitamin B3: from metabolism to therapies. J. Pharmacol. Exp. Ther. 2008, 324 (3), 883−893. (21) Gropper, S.; Smith, J. Water Soluble Vitamins. Advanced Nutrition and Human Metabolism. Section III: The Regulatory Nutrition; Wadsworth: Belmont, CA, 2012; pp 307−370, Chapter 9. (22) Robins, D. J.; Yee, R. B.; Bentley, R. Biosynthetic precursors of vitamin K as growth promoters for Bacteroides melaninogenicus. J. Bacteriol. 1973, 116 (2), 965−971. (23) Velíšek, J.; Cejpek, K. Biosynthesis of food constituents: Vitamins. 2. Water-Soluble Vitamins: Part 1 − a Review. Czech Journal of Food Science 2007, 25 (2), 49−64. (24) Pourcel, L.; Moulin, M.; Fitzpatrick, T. B. Examining strategies to facilitate vitamin B1 biofortification of plants by genetic engineering. Front. Plant Sci. 2013, 4, 160. (25) Mohammed, Y.; Lee, B.; Kang, Z.; Du, G. Development of a two-step cultivation strategy for the production of vitamin B12 by Bacillus megaterium. Microb. Cell Fact. 2014, 13, 102. (26) Closa, M.; Vranová, E.; Bortolotti, C.; Bigler, L.; Arró, M.; Ferrer, A.; Gruissem, W. The Arabidopsis thaliana FPP synthase isozymes have overlapping and specific functions in isoprenoid biosynthesis, and complete loss of FPP synthase activity causes early developmental arrest. Plant J. 2010, 63, 512−525. (27) Masferrer, A.; ArroÂ , M.; Manzano, D.; Schaller, H.; FernaÂ ndez-Busquets, X.; MoncaleaÂ n, P.; FernaÂ ndez, B.; Cunillera, N. R.; Boronat, A.; Ferrer, A. Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cytokinin levels. Plant J. 2002, 30 (2), 123−132. (28) Titiz, O.; Tambasco-Studart, M.; Warzych, E.; Apel, K.; Amrhein, N.; Laloi, C.; Fitzpatrick, T. B. PDX1 is essential for vitamin B6 biosynthesis, development and stress tolerance in Arabidopsis. Plant J. 2006, 48, 933−946. (29) Havaux, M.; Ksas, B.; Szewczyk, A.; Rumeau, D.; Fabrice Franck, F.; Caffarri, S.; Christian Triantaphylidès, C. Vitamin B6 deficient plants display increased sensitivity to high light and photo-oxidative stress. BMC Plant Biol. 2009, 9, 130. (30) Ehrenshaft, M.; Bilski, P.; Li, M. Y.; Chignell, C. F.; Daub, M. E. A highly conserved sequence is a novel gene involved in de novo vitamin B6 biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 9374− 9378.

AUTHOR INFORMATION

Corresponding Author

*Tel: +92 322 414 7507. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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Identification of Microbial Metabolites Elevating Vitamin Contents in

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