14-3-3 Lambda Protein Affects Anthocyanin Production in Arabidopsis thaliana during Drought Stress

Plants evolve to adapt to environmental stresses, including changes at the genetic and molecular levels. For bioengineers to utilize genetic manipulation to build tolerance into crops, a better understanding of the mechanism is needed. Published studies have demonstrated that 14-3-3 lambda (14-3-3λ) protein affect the phenylpropanoid (Pp) biosynthetic pathway and alters production of flavonoids and downstream compounds of importance for stress tolerance. The 14-3-3 family of proteins binds to many different client proteins and serves as signaling scaffolds. In this study 14-3-3λ knockout mutants were used to investigate changes in metabolite accumulation in the downstream Pp pathway. Amongst them are anthocyanins which are important antioxidants involved in a variety of plant functions including stress response. Investigating how drought stress influenced anthocyanin production identified nodes in the Pp pathway affected by 14-3-3λ. A metabolomics analysis employing high resolution mass spectrometry (HRMS) and metabolomics software was used to identify metabolites in 14-3-3 knockout which changed relative to wild-type A. thaliana (Columbia-0) during drought stress. The metabolites Cy-3-p-coumaurolysinapoylsophoroside-5-diglucoside, 3-caffeoylferuloylsophoroside5-succinoylglucoside, 3-caffeoylferuloylsophoroside-5-malonyldiglucoside, 3-feruloylsophoroside-5-succinoyl glucoside, petunidin-3,5-O-diglucoside and malvidin-3-O-p-coumarylmonoglucoside show significant differences in their profiles ranging from 18to > 500-fold between the Col-0 and 14-3-3λ knockout in wet and dry groups. The findings suggest that 14-3-3λ interacts along the CHS, and CHI nodes, which in turn regulate the downstream production of specific anthocyanins. The interaction of 14-3-3λ with CHS was confirmed using co-immunoprecipitation and co-localization studies. This study supports the hypothesis that manipulation of gene expression of 14-3-3λ can lead to development of drought tolerance in plants.

From previously published studies, it was suggested that 14-3-3 proteins affect production of anthocyanins which are a family of molecules involved in a variety of functions including defense (Dixon & Steele, 1999;He et al., 2010;Holton & Cornish, 1995).As one example anthocyanin loss of function lines of A. thaliana grown under high light conditions, showed no significant growth difference compared to controls, suggesting that anthocyanins major role in the Pp pathway may be focused on other functions such as defense and stress response (Misyura, Colasanti, & Rothstein, 2012).Numerous studies have been conducted on the anthocyanin biosynthesis in A. thaliana, but the role of 14-3-3 proteins on anthocyanin production remains unresolved and specific nodes of interaction in the anthocyanin pathway by 14-3-3 proteins has not been fully elucidated.
The focus of this research was to study effect of 14-3-3λ knockout on anthocyanin production through the Pp pathway in Arabidopsis thaliana, under drought stress (dry) conditions.Published research has identified the 14-3-3λ mutant as having greater sensitivity to dry conditions (Peethambaran, Chi Li, Dzugan, Xiang, & Balsamo, 2012), and also revealed that 14-3-3λ affects production of synapoyl maleate and lignin biosynthesis under dry conditions (Lindberg et al., 2014).These findings indicate that 14-3-3 proteins are interacting along with flavonoid synthesis in the Pp pathway.This study proves that specific nodes are affected by 14-3-3λ proteins in production of anthocyanins.A reverse genetics approach was applied to screen anthocyanin metabolites under dry and well hydrated (wet) conditions using A. thaliana 14-3-3λ homozygous TDNA knockout mutant and WT.The 14-3-3λ homozygous TDNA knockout mutant (SALK_075219) had significantly different amounts of total flavonoid, phenolics and antioxidants compared to Columbia-0 (WT) under dry conditions.This provided the rationale for investigating differentially regulated anthocyanins in 14-3-3λ knockout mutant.Comparing dry and wet conditions to 14-3-3λ knockouts and WT A. thaliana we observed significant increases in the metabolites listed in Table 1, for WT, with smaller changes in the 14-3-3λ knockout between the conditions.These data support the hypothesis that 14-3-3λ protein has a significant effect on the Cyanidin and Delphinidin nodes of the Kegg anthocyanin biosynthetic pathway.Moving further upstream of anthocyanins, in the Pp pathway these are metabolites produced by the action of Chalcone Synthase (CHS) and Chalcone Isomerase (CHI) nodes.The CHS gene has been shown to have an elevated transcription rate under environmental and pathogenic stress conditions resulting in accumulation of various flavonoids and anthocyanins (Feinbaum & Ausubel, 1988;Li & Strid, 2005).CHI mutants have been shown to have lower levels of flavonoids and high sensitivity to UV-B damage.When the CHS and CHI knockouts were drought stressed, lower expression of 14-3-3 was observed, suggesting that CHS and CHI interact with 14-3-3 during dry conditions.

Plant Growth Conditions, Drought Stressing and Sampling
Wild-type (Columbia-0), 14-3-3 T-DNA mutant Lambda (SALK_075219), CHS T-DNA mutant (SALK_077592) and CHI T-DNA mutant (SALK_ 034145) were purchased from Arabidopsis Biological Resource Center (ABRC, Ohio State University).Seeds were grown in a Sunshine Mix soil (Sun Gro Horticulture, Quincy, MI) and hydrated with Scotts peters professional water soluble fertilizer 20-20-20 (Scotts, Marysville, OH) as previously described (Lindberg et al., 2014).Genotypting of these knockout lines were conducted using polymerase chain reaction and western blot for the 14-3-3 T-DNA mutant Lambda (SALK_075219).Drought treatment was carried out on 5 week old plants, half of the plants were watered normally and the other half was not watered, soil moisture was monitored daily and leaf mass harvested from all plants when the drought treated soil moisture level reached ~30%, at which point wilting of the leaves was observed.Harvested leaves were immediately frozen in liquid nitrogen and freeze dried (LabConco Corporation, Kansas City, MO).The dried leaf samples were stored at -80 ºC until extraction.

Metabolite Extraction and Isolation
Metabolites were extracted using a modification of the method described by (Lindberg et al., 2014).Briefly, frozen tissue was ground with a mortar and pestle, then suspended in extraction solvent (Methanol:Acetone; 1:1 v/v), followed by addition of 1 µg of apeginin (10 µg mL -1 ) as an internal standard.The supernatant was transferred to a BD Falcon tube (ThermoFisher Scientific, Waltham, MA).The pellet was extracted two more times with extraction solvent and dried using a Savant SpeedVac centrifugal evaporator (Savant Instruments Inc., Farmingdale, NY).Chlorophyll was precipitated by addition of acetone then water at a 70:40 v/v ratio and centrifugation at 13,000 × g.The supernatant was dried using the Savant SpeedVac, samples were reconstituted in a 10:90 ratio of methanol/0.1% formic acid in water and analyzed by LC-MS.

LC-MS Conditions
Samples were analyzed by Liquid Chromatography/Mass Spectrometry using an Accela UHPLC interfaced to an Exactive Plus ion trap mass spectrometer with a HESI source (ThermoFisher Scientific, San Jose, CA).Chromatographic separations were achieved employing a 2.1 × 150 mm, 5 m, Imtakt Flavonoid RP18 column (Portland, OR) with gradient elution at 0.6 mL/min.The column temperature was maintained at 65 C.Mobile phase A was water with 0.1% formic acid and mobile phase B was 98:2 acetonitrile:water with 0.1% formic acid.Mobile phase A was held at 100% for 0.5 min and then a three step linear gradient was formed from 0% to 20% mobile phase B over 5.5 min, to 60% mobile phase B in 2 min and then to 95% phase B in 4 min.The final composition was held for 1 min before returning to the initial conditions.Positive and negative electrospray ionization (ESI) data were acquired (separate injections) from m/z 200 to m/z 1200 with a mass accuracy within 5 ppm at 35 000 resolutions.A single 10 L injection was used for each ionization mode.Instrumental settings follow: maximum injection time 10 msec, capillary temperature 320 C; tube lens voltage 175 V; ESI spray voltage 4.3 kV for positive ion mode, 3.6 kV for negative ion mode; sheath gas 2 arbitrary units (arbs).

Total Flavonoid, Phenolic, Antioxidant and Free Radical Scavenging Analysis
Analysis was performed using modifications of the methods described by Kiranmai et al., and Mahboubi et al., (Kiranmai et al., 2011;Mahboubi, Kazempour, & Boland Nazar, 2013), the response from each assay was calculated using 4PL curve fitting and expressed as µg mL -1 .

Total Phenolic
Total phenolic contents in the sample extracts was determined using the Folin-Ciocalteu's reagent (Folin & Denis, 1912) and the method described by Mahboubi et al.We use 25 µL aliquot of each sample extract and diluted calibrator solution (Gallic acid 15.6 to 1000 µg mL -1 in methanol) was mixed with 0.125 mL of Folin-Ciocalteu's reagent (10%).After approximately 5 minute, 0.1 mL of 7.5% (w/v) sodium carbonate solution was added and mixed.That was followed by incubation for 1 hour and measure absorbance at 765 nm.

Total Flavonoid
For total flavonoid contents we use a 25 µL aliquot of each sample extract and diluted calibrator solution (Quercetin 15.6 to 1000 µg mL -1 in methanol) and mix with 75 µL of ethanol (95%), 0.5 µL of aluminum chloride (10%), 0.5 µL potassium acetate (1 M) and 140 µL deionized water.That was followed by incubation at RT for 30 minutes and measure absorbance at 415 nm.

Total Antioxidant
For total antioxidant activity was determined using the method described by Kiranmai et al., we use 100 µL of each sample and diluted calibrator solution (Ascorbic acid 78 to 5000 µg mL -1 in methanol) and mix separately with 1.0 mL of a mixture of (0.6 M Sulfuric Acid/28 mM Sodium phosphate/4 mM Ammonium Molybdate).That was followed by incubation at 95 °C for 90 minutes, measure absorbance of the reaction mixture at 695 nm.

Protein Extraction, Quantification and Western Blot
Protein extraction was carried out on freshly collected 0.1 gram plant leaf weight, frozen in Liquid Nitrogen and maintained frozen while samples were pulverized to a fine powder and mixed Laemmli buffer and boil at 95 °C for 10 min.Centrifugate at 1000 × g for 5 minutes at 4 °C.

Protein Quantitation
Protein was quantitated with a Thermo Scientific Pierce 660 nm Protein Assay kit (cat #22662), in a ready-to-use format (Thermo Fisher Scientific, Waltham, MA).A 10 µL aliquot of the kit pre-made Albumin standard solutions ranging from 0.125 to 2000 µg mL -1 and 10µL aliquots of the unknown protein samples extracts were added to wells in a clear flat bottom 96 well plate.An aliquot of 150 µL of the kit developing reagent mixture was placed in the well of the microtiter plate containing samples and the plate incubate for 1 min at room temperature, then read at 660 nm on a Spectramax 386 plus plate reader (Molecular Devices, Sunnyvale, CA).Protein concentration was calculated using a linear curve fit in Softmax Pro ver 5.0 (Molecular Devices, Sunnyvale, CA) and unknown sample concentration read of the calibration curve.

Western Blot
For the gel electrophoresis the Bio Rad Mini Protean TGX precast gel was used 4-15% (Bio Rad, Raleigh, NC).20µg of protein was mixed with 1µL of Bromophenol blue and volume completed to 15 µL of with Laemmli buffer containing β-mercaptoethanol.Running buffer was 1X Tris-HCL-Glycine at 120 V for 60 min.Blot with 1X transfer buffer Tris HCL-Glycine with 10% methanol at 70 V for 60 min.Blocked with 5% non-fat milk in 1x TBST 1 hr at RT. Incubated with the Santa Cruz anti-14-3-3 rabbit polyclonal antibody as a primary antibody (Santa Cruz Biotechnology Inc, Dallas, TX), 1:1000 dilution in 1x TBST with 5% non-fat milk overnight at 4 °C.Incubate blot in cell signaling HRP-anti-rabbit antibody (Cell Signaling Technology, Danvers, MA) as the secondary antibody at 1:10 000 dilution in 1x TBST with 5% non-fat milk 1 hr at RT. Mix Thermo Scientific chemi substrate solutions in a 1:1 v/v ratio and apply to blot, incubate for 1 min and Image blot.

Real time PCR to Determine Expression of 14-3-3λ in CHS and CHI Mutants
The RNA was isolated using Trizol methods (Chomczynski & Sacchi, 1987), 0.1 gm of tissue was flash frozen in liquid nitrogen and grounded into fine powder, 0.5 mL of Trizol added.After thawing, the mixture was transferred to an Eppendorf tube and centrifuged at 13,000 RCF for 5 minutes, add 0.2 ml of chloroform and vortex briefly and incubate at room temperature for 10 minutes before centrifuging at 13,000 RCF.The top layer was moved into a new tube and 0.25 ml of RNA precipitation solution (0.8 M Sodium citrate/1.2M NaCl) and 0.25 ml of isopropanol added, centrifugate at 13,000 RCF.The RNA pellet washed twice with 75% ethanol before air drying.RNA pellets dissolved in RNAse free water.The RNA was converted to cDNA using a RETRO Script kit (Ambion, Foster city, CA).The primers were designed specific to Arabidopsis 14-3-3λ cDNA (AT5G10450) and a PCR was conducted for 25 cycles.Primers targeting a specific region of 14-3-3λ were amplified using the forward primer 'TGCTGGAGCGAGTGAGTCTA' and reverse primer 'AGCCTGTTT GGCCATGTTAC'.An actin primer for gene ACT2 (AT3G18780) 'TCCAGTGTTGTTGGTAG GCCA' and TCTCAGCACCAATCGTGATGAC' was run at the same time to control for loading differences.

Protein Immuno Precipitation and Co-Immuno Precipitation with Magnetic Beads
Immuno precipitation (IP) of 14-3-3 and co-IP of binding partners was carried out with a Thermo IP/co-IP magnetic bead kit, catalog #88805 (Waltham, MA) as described in the kit method.Briefly an antibody specific to 14-3-3λ was conjugated to the magnetic beads then crosslinked to permanently bind the antibody to the beads.The beads were exposed to 0.5 mL of protein extracts from wild type and KO plants for 1 hour at room temperature (RT) with gentle shaking to keep beads suspended.The captured protein along with binding partners was eluted with 100 µL the low pH elution solution and 5 minutes of incubation at RT with gentle shaking.The eluate solution was transferred to a clean tube and neutralized with 10 µL of neutralizing buffer as described in the kit manual.A 50 µL aliquot of the eluate was diluted with 100 µL 0.5M bicarbonate buffer digested with 50 µL of 1 mg mL -1 trypsin and 37 °C overnight, then analyzed by LC-MS.

Co-Localization of 14-3-3λ with CHS Using Confocal Microscopy
Microscopic imaging was performed on a spinning disk (Yokagawa CSU-X1; Andor Technology) confocal microscope using a 60x 1.4 NA oil immersion objective lens on a TiE microscope equipped with Perfect Focus System (Nikon) equipped with an electronic shutter (Sutter Instrument) for transmitted illumination, a linear encoded X and Y, motorized stage (ASI Technologies), and a multi-bandpass dichromatic mirror (Semrock) and bandpass filters (Chroma Technology Corp.) in an electronic filter wheel for selection of BFP, GFP, or RFP emission.405-, 488-, and 561-nm laser illumination was provided by a high-powered (20 mW 405-nm; 50 mW 488-and 561-nm) monolithic laser combiner module (MLC 400B; Agilent Technologies) that were shuttered with electronic shutters and directed to a fiber-coupled output port with an Acousto optic tunable filter and to the confocal scan-head via a single mode polarization-maintaining fiber coupled delivery system (Agilent Technologies).The exposure times for the images were 500 milliseconds for 405 and 488 nm and 600 ms for 561 nm.The primary antibody for 14-3-3λ was anti-mouse kindly gifted by Dr. Robert Ferl, University of Florida.A final dilution of 1:20 was followed according to the protocol of Pasternak et al. (2015).The secondary antibody for 14-3-3λ was tagged with Cy5 and a final dilution of 1:500 was used.The primary antibody used to probe chalcone synthase was from Agrisera (AS 12 2615).The secondary antibody for chalcone synthase was tagged with Cy3.Primary and secondary dilutions were 1:20 and 1:500 respectively (Pasternak et al., 2015).

Statistical Analysis
For total Flavonoid, Phenolic, Antioxidant and Free radical scavenging capacity, the differences between drought-treated versus untreated plants were analyzed with a paired t-test, in Microsoft Excel.Metabolite profiling analyses were carried out with MetaboAnalyst.Data was first transformed by log normalization and jas.ccsenet.analyzed u significanc

Results
Analyses o between w (Figure 1) and dry c significant 14-3-3λ kn     When specific metabolites were observed to have high fold change differences between the groups, and were compared against the Kegg anthocyanin pathway nodes, we find that the likely nodes of interaction for production of these metabolites would be in the cyanidin and delphinidin nodes (Table 2).Anthocyanin metabolites such as 3-p-coumaurolysinapoylsophoroside-5-diglucoside and 3-caffeoylferuloy lsophoroside-5-succinoylglucoside when compared in wild-type and 14-3-3λ knockout plants, were 500-fold different between Col and 14-3-3λ wet and 69-fold change between Col and 14-3-3λ dry (Table 1, panel 2).The fold changes between wet wild-type and the 14-3-3λ knockout suggests that 14-3-3 is important in production of specific anthocyanins such as 3-p-coumaurolysinapoylsophoroside-5-diglucoside, 3-caffeoylferuloylsopho roside-5-succinoylglucoside, 3-caffeoylferuloylsophoroside-5-malonyldiglucoside, 3-feruloylsophoroside-5succinoylglucoside, Petunidin-3,5-O-diglucoside and Malvidin-3-O-p-coumarylmonoglucoside (Table 1, panel 1 to 4).The genes that regulate the metabolites shown in Table 1 are in the synthetic pathway from 4-coumaryl-coA to naringenin chalcone which is then converted to naringenin.These two steps in Pp pathway are regulated by chalcone synthase (CHS) and chalcone isomerase (CHI).Figure 4 represents a heat map distribution of the top 25 anthocyanin metabolites showing that the groups are clustered, demonstrating up or down regulation of the metabolites with consistency amongst the groups and providing confidence in the data which are listed in Table 2.       es, bind to mu horylated, whi prasad, Rakesh that both can tes if either i 14-3-3λ, co-im KO, 14-3-3λ K lyses of pepti des suggesting he 14-3-3λ KO (-) value based as not higher t 3λ overexpress HI.Whereas t response was analyses.

Conclusion
These results demonstrate that 14-3-3λ plays a role in the Pp pathway in production of anthocyanins and shows that knockout of CHS and CHI diminishes expression of 14-3-3λ during drought conditions.These data also suggest that PAL-1, PAL-2, CHS and CHI are interacting with 14-3-3.These observations demonstrate that knocking out 14-3-3λ results in decreased 14-3-3 expression under drought conditions, consequently resulting in differential accumulation of anthocyanin metabolites in the knocked out line compared to wild-type.Over-expressing 14-3-3λ leads to increased tolerance to drought in these transgenic plants.The plants showed increased accumulation of anthocyanins in the over-expressing transgenic plants providing additional evidence that 14-3-3 is interacting with the chalcone synthase gene and is involved in anthocyanin production.Hence, 14-3-3λ does play an important role in drought tolerance in A. thaliana.

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Table 1 .
Summary of LC/MS metabolite profiling for normal hydration and drought treated wild-type (Columbia-0) Arabidopsis thaliana, and 14-3-3λ mutant.Compounds showing significant changes amongst the group are shown.Positive ionization mode LC/MS data (top frame, panel 1 and 2).Negative ionization LC/MS data (bottom frame, panel 3 and 4)

Table 2 .
Anthocyanin metabolites and associated genes, location on Kegg Anthocyanin Biosynthesis pathway