Planta DOI 10.1007/s00425-015-2289-1

ORIGINAL ARTICLE

Genetic reduction of inositol triphosphate (InsP3) increases tolerance of tomato plants to oxidative stress Mohammad Alimohammadi1 • Mohamed H. Lahiani1 Mariya V. Khodakovskaya1

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Received: 30 January 2015 / Accepted: 27 March 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Main conclusion We demonstrate here that the reduction of InsP3, the key component of the phosphoinositol pathway, results in changes in ROS-scavenging machinery and, subsequently, increases the tolerance of tomato plants to light stress. Different plant stress signaling pathways share similar elements and, therefore, ‘cross-talk’ between the various pathways can exist. Links between the phosphoinositol signaling pathway and light signaling were recently found. Tomato plants expressing InsP 5-ptase and exhibiting reduction in the level of inositol 1,4,5-triphosphate (InsP3) demonstrated enhanced tolerance to stress caused by continuous light exposure. To understand the molecular basis of observed stress tolerance in tomato lines with decreased amount of InsP3, we monitored the expression of enzymatic antioxidants as well as important factors in light signaling associated with non-enzymatic antioxidants (secondary metabolites). Here, we demonstrated that InsP 5-ptase transgenic plants accumulate less hydroxide peroxide and maintain higher chlorophyll content during stress caused by continuous light exposure. This observation can be explained by documented activation of multiple enzymatic antioxidants (LeAPX1, SICAT2, LeSOD) at levels of gene expression and enzymatic activities during continuous light exposure. In addition, we noticed the up-regulation of

photoreceptors LePHYB and LeCHS1, key enzymes in flavonoid biosynthesis pathway, transcription factors LeHY5, SIMYB12, and early light-inducible protein (LeELIP) genes in transgenic tomato seedlings exposed to blue or red light. Our study confirmed the existence of a correlation between phosphoinositol signaling pathway modification, increased tolerance to stress caused by continuous light exposure, activation of ROS-scavenging enzymes, and up-regulation of molecular activators of non-enzymatic antioxidants in InsP 5-ptase expressing tomato lines. Keywords Oxidative stress  Phosphoinositol pathway  Light signaling  Secondary metabolism  ROS-scavenging enzymes Abbreviations InsP3 Inositol 1,4,5-triphosphate InsP 5-ptase Inositol trisphosphate 5 phosphatase SOD Superoxide dismutase CAT Catalase APX Ascorbate peroxidase LeCHS1 Lycopersicon esculentum chalcone synthase LeHY5 Lycopersicon esculentum long hypocotyl 5 transcription factor LePHYB Lycopersicon esculentum photochrome B SICRY1 Solanum lycopersicum cryptochrome 1 LeELIP Lycopersicon esculentum early lightinducible protein

Electronic supplementary material The online version of this article (doi:10.1007/s00425-015-2289-1) contains supplementary material, which is available to authorized users. & Mariya V. Khodakovskaya [email protected] 1

Department of Biology, University of Arkansas at Little Rock, Little Rock, USA

Introduction The production of reactive oxygen species (ROS) during photosynthesis and respiration is an undeniable part of plant life cycle (Asada 2006). ROS molecules have the

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Fig. 1 Schematic of mechanism used by antioxidant enzymes for the detoxification of hydrogen peroxide produced during photosynthesis and in response to environmental factors in chloroplast

potential to cause oxidative damage to DNA, proteins, and lipids (Apel and Hirt 2004). Plants developed multiple enzymatic and non-enzymatic mechanisms that use antioxidants to neutralize the toxic effects of ROS. ROSscavenging enzymes include superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) (Fig. 1). SOD converts highly toxic superoxide to a less toxic molecule, hydrogen peroxide (H2O2). Eventually, CAT and APX convert H2O2 to non-toxic molecules such as H2O and O2 (Pandhair and Sekhon 2006). Non-enzymatic antioxidants, such as ascorbic acid, flavonoids, and carotenoids, further remove the ROS from the cells (Ahmad et al. 2008). In addition to the ROS produced in everyday photosynthetic and respiration activities, environmental stress factors including drought, cold, and light can also induce the formation of ROS and cause oxidative damages (Elstner 1991; Malan et al. 1990; Prasad et al. 1994; Tsugane et al. 1999). Among these environmental factors, light is especially an important stress factor. Light has a dual role in plant life. It is the primary source of energy for plants and induces several developmental processes (Fankhauser and Staiger 2002; Chen et al. 2004). On the other hand, continuous light exposure causes extreme changes in the gene expression pattern in the cells and has the potential to cause severe damages (Mittler et al. 2004; Vandenabeele et al. 2004; Sagar and Briggs 1990). The mechanisms by which plants sense and respond to the stress caused by continuous light exposure are not clearly understood (Kleine et al. 2007). It is known, however, that the expression level of antioxidant enzymes SOD, CAT and APX is regulated by light. These enzymes are the primary line of ROS elimination and the reduction of adverse effects due to continuous light exposure (Karpinski et al. 1997; Kimura et al. 2003). Biosynthesis

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of non-enzymatic antioxidants, flavonoids, carotenoids, and ascorbic acid is also affected by light irradiation at the level of gene expression (Fini et al. 2011; Tattini et al. 2004; Pe´rez et al. 2002). Recently, we found connections between phosphoinositol metabolism, light signaling, and the production of antioxidants in tomato plants overexpressing human type I InsP 5-ptase (Khodakovskaya et al. 2010; Alimohammadi et al. 2012). Particularly, genetic reduction of a key secondary messenger, inositol-(1,4,5) triphosphate (InsP3), in InsP 5-ptase transgenic tomato lines resulted in the modification of light signaling and the activation of light-inducible enzymes associated with the synthesis of secondary metabolites (lycopene, flavonoids) with antioxidant activity. For example, the transcription level of light-signaling transcription factor LeHY5, a positive regulator of photomorphogenesis, was up-regulated in transgenic tomato plants with reduced InsP3. The increase in LeHY5 expression enhanced biosynthesis of flavonoids in transgenic lines by upregulating the expression of chalcone synthase (LeCHS1), a key enzyme in flavonoid pathway and transcription factor SIMYB12 (Alimohammadi et al. 2012). As a result of modifications in light-dependent branches of plant metabolism, production of lycopene, phenylpropanoids (rutin, chlorogenic acid), and ascorbic acid (vitamin C) was significantly increased in InsP 5-ptase tomato fruits (Khodakovskaya et al. 2010; Alimohammadi et al. 2012). We also found that transgenic lines with reduced InsP3 can maintain their chlorophyll content for a longer time compared to wild-type plants under continuous light exposure (Alimohammadi et al. 2012). In this paper, we made an attempt to identify the link between the reduction of InsP3 in established transgenic tomato lines and changes in the response of InsP 5-ptase plants to oxidative stress caused by continuous light exposure. Particularly, we monitored the generation of hydrogen peroxide in correspondence to changes in expression of the genes encoding antioxidant enzymes such as LeSOD, SICAT2, LeAPX1 during continuous light exposure. These changes were followed by changes in the activity of correspondent enzymes in wild-type tomato leaves and leaves of InsP 5-ptase lines exposed to prolonged light stress. Established results demonstrated a positive correlation between the expression of enzymatic antioxidants and the enhanced tolerance of InsP 5-ptase tomatoes to oxidative stress. Furthermore, the significance of molecular perturbations in light-perception machinery in tomato plants with a reduced level of InsP3 was proved by demonstration of changes in expression of the key light-signaling transcription factors (LePHYB, SICRY1, LeHY5, SIMYB12, LeELIP) in wild-type and InsP 5-ptase lines exposed to red or blue light.

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Materials and methods Plant growth and stress experiment Both transgenic lines expressing the human type I inositol polyphosphate 5-phosphatase (Ins 5-ptase) gene (Line 6, Line 7) were generated and characterized in our previous work (Khodakovskaya et al. 2010). Tomato (cv. MicroTom) seeds of wild-type (WT), empty vector (EV), and of InsP 5-ptase transgenic (L6 and L7) were surface-sterilized and germinated on Murashige and Skoog (MS) medium in a growth chamber with 50 lmol m-2 s-1 light intensity at 12-h daylight and 12-h dark for 10 days. For continuous light exposure experiment, seedlings were transferred to a growth chamber with 50 lmol m-2 s-1 light intensity and continuous light exposure (24 h of light) at 35 °C. Samples were collected at three different time points after 7, 14, and 21 days of continuous light exposure. For real-time quantitative RT-PCR (qRT-PCR), leaf samples were snap-frozen in liquid nitrogen and immediately ground to a fine powder. The ground samples were directly used for total RNA extraction or stored at -80 °C for further analysis. For the light exposure experiment, tomato (cv. Micro-Tom) seeds of wild-type (WT), empty vector control (EV), and of transgenic lines 6 and 7 (L6 and L7) were surface-sterilized and germinated on MS medium in dark. After 10 days, seedlings were exposed to red or blue light for 3-h after which samples were snap-frozen in liquid nitrogen. Samples were immediately used for total RNA extraction or stored at -80 °C for further analysis. Determination of chlorophyll and hydrogen peroxide in tomato plants exposed to continuous light Plant samples were obtained from seedlings grown in day/light conditions for 10 days and then exposed to continuous light (50 lmol m-2 s-1 light intensity at 35 °C, only day regime) for 7, 14, and 21 days. The specific chlorophyll concentration was calculated as described by Chory et al. (1994). Two leaves from each sample were blotted dry, weighed, and placed in a 1.5 mL microfuge tube. The samples were suspended in 80 % acetone, after which they were ground with a disposable pestle and then incubated in the dark for 30 min. Total chlorophyll (Chl lg mL-1) was determined according to the equation: 20.2A645 ? 8.02A663. Hydrogen peroxide was quantified using a protocol described by Velikova et al. (2000). Briefly, leaf tissues (200 mg) were homogenized in 2 mL of 0.1 % (w/v) trichloroacetic acid (TCA) solution on ice. The homogenate was then centrifuged at 12,000g for

15 min, after which 0.4 mL of the supernatant was added to 0.4 mL of 10 mM potassium phosphate buffer, pH 7.0 and 0.8 mL 1 M KI. The absorbance of the solution was read at 390 nm. RNA isolation, cDNA synthesis and real-time PCR Total RNA was extracted from tomato leaf tissue using RNeasy Plant Mini Kit (Qiagen Sciences, Maryland, USA). The cDNA was synthesized according to the SuperScript III First Strand Synthesis System Kit protocol (Invitrogen Inc.) using Oligo (dT) primers. Following synthesis, cDNA was used for the PCR reaction using gene-specific primers. Amplification of 18S was carried out with the following primers: 50 -AGGCCGCGGAAGTTTGAGGC-30 (forward primer) and 50 -ATCAGTGTAGCGCGCGTGGG-30 (reverse primer); LeCHS1 gene (X55194) was amplified using 50 -TGGCTGAGAACAACAAGGGTGCTA-30 (forward primer) and 50 -ATTCACTGGGTCCACGGAACGTAA-30 (reverse primer). LeHY5 gene (AJ011914): 50 -ACCATCA GCTGGGACTCAAAGGAA-30 (forward primer) and 50 -T TCCTCTCCCTTGCTTGTTGTGCT-30 (reverse primer); for LeELIP gene (AY547273): 50 -TCCTAGCTGTTACTT GCCACGCC-30 (forward primer) and 50 -TGCACCGACC TCAGCCATACACT-30 (reverse primer); for SlMYB12 gene (EU419748): 50 -TGCCAAATTCTTGGGCAGGAC CTA-30 (forward primer) and (50 -TCACCACGTCTGGC ATAATCTCCT-30 (reverse primer); for SlCRY1 gene (LOC544219) 50 -CCGGCGGTGGCTTCCTGAAC-30 (forward primer) and (50 -GCTTGGTCACGCGTGTCTCT CC-30 (reverse primer); for LePHYB gene (DQ539438.1) 50 -GGAAGCTGTTGGGGGTGGCC-30 (forward primer) and (50 -TCCCATGGTGAGCTCCGGCT-30 (reverse primer). PCR products were separated on 1 % agarose gels by electrophoresis for 30 min at 5 V/cm. Gene expression quantification for selected genes was carried out by qRTPCR using the following primers: LeAPX1 gene (AY974805.1): 50 -GATGCCACCAAGGGCTGTGACCA30 (forward primer) and 50 -CCAAGGTATGGGCACCAG AGAGTGC-30 (reverse primer); for SlCAT2 gene (NM_ 001247257): 50 -TGCAGTGGCAAACGCGAGAAGT-30 (forward primer) and 50 -TGTCGGGTGTGAATGAGCG GT-30 (reverse primer); for LeSOD gene (AF527880): 50 -T GGCATGCGGTGTGGTTGGT-30 (forward primer) and 50 -TGGCAACCCGGAGAGGAGGG-30 (reverse primer); for TOMPRP6 gene (M69248.1): 50 -GTGTCCGAGAGG CCAAGC-30 (forward primer) and 50 -GCCCGACCACA ACCTAGTCG-30 (reverse primer); for TOMWIPII gene (K03291.1): 50 -CATCATGGCTGTTCACAAGGAA-30 (forward primer) and 50 -CCCGAACCCAAGATTACCAC AT-30 (reverse primer). The qRT-PCR analysis was done using SYBR Green PCR master mix (Applied Biosystems,

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Inc.) in a C1000 Thermal Cycler equipped with a CFX96 real-time detection system (Bio-Rad, Hercules, CA, USA). Three independent biological replicates were used in the analysis. The real-time PCR data were generated and analyzed by the ‘comparative count’ method to obtain relative mRNA expression in each tissue as described in the Thermal Cycler manual (Bio-Rad). Enzyme activity assays Fully extended leaves from seedlings germinated under day/night light conditions and then exposed to continuous light (50 lmol m-2 s-1 light intensity at 35 °C) for 7, 14, and 21 days were snap-frozen in liquid nitrogen. Sample tissue (100 mg) was immediately ground to a fine powder on ice with a mortar and pestle and homogenized in 1 mL of 10 mM potassium phosphate buffer (pH 7.0) containing 4 % (w/v) polyvinylpyrrolidone. Crude extract was centrifuged at 12,000g for 30 min at 4 °C, and the supernatant was used as enzyme extract. Superoxide dismutase (SOD) activity was determined by measuring the ability of SOD to inhibit the photochemical reduction of nitroblue tetrazolium chloride (NBT) according to the method of Dhindsa et al. (1981) with some modifications. The reaction mixture (3 mL) contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 mM NBT, 0.1 mM EDTA and 0.05 mL enzyme extract. The reaction was started by adding two mM riboflavin, and the reaction tube was placed under two 18-watt fluorescent lamps for 10 min. A complete reaction mixture without enzyme extract served as a control. The reaction was stopped by switching off the lights, and the tubes were transferred to the dark. A nonirradiated reaction mixture served as a blank. The reaction mixture lacking enzyme gave maximum color, and the intensity of the color decreased in the mixtures with enzyme extract. The absorbance was recorded at the wavelength of 560 nm in a Beckman DU-70 spectrophotometer. One unit of enzyme was determined as the amount of enzyme reducing 50 % of the absorbance reading compared with the non-enzyme tube. Catalase activity was determined by measuring the initial rate of disappearance of hydrogen peroxide as described by Velikova et al. (2000). The reaction mixture (3 mL) contained 10 mM potassium phosphate buffer (pH 7.0) and 0.1 mL enzyme extract, and the reaction was started by adding 0.035 mL of 3 % hydrogen peroxide. A decrease in hydrogen peroxide concentration was followed by a decline in optical density at the wavelength of 240 nm. The non-enzyme extract mixture served as a blank. The catalase activity was calculated using the extinction coefficient of 40 mM-1 cm-1.

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LED light experiment For the light exposure experiments, 10-day-old seedlings grown in dark at the conditions mentioned before were transferred to wooden boxes (40 9 45 9 45 cm) with the top side holding LED light bulbs of a specific wavelength. Seedlings were exposed to red light (650 nm, 1.2 lmol m-2 s-1) or blue light (450 nm, 0.32 lmol m-2 s-1) for 3-h from above. The light intensity was quantified with a LI-COR light meter (LI-250). Seedlings were then snap-frozen in liquid nitrogen and were used for RNA isolation and cDNA synthesis as described previously. Microarray analysis and real-time quantitative polymerase reaction The RNA was isolated from seedlings of wild-type (WT) and transgenic line 7 (L7), germinated on MS medium in a growth chamber with 50 lmol m-2 s-1 light intensity at 12-h daylight and 12-h dark for 10 days and then exposed to continuous light (50 lmol m-2 s-1 light intensity at 35 °C) for 7 days. The total RNA of 17-day-old seedlings (control and L7) was isolated as described previously. Biotinylated cRNA targets were synthesized according to Affymetrix IVT Express target labeling assay as specified in the Affymetrix GeneChip Expression Analysis Technical Manual. Hybridization reactions to the Affymetrix Tomato GeneChips were carried out by Expression Analysis, Inc (Durham, NC, USA). The validation of transcript abundance was performed using real-time RT-qPCR (SYBR Green detection) of two genes: PR Protein TOMPRP6 (Les.3408.1.S1_at) and woundinduced proteinase inhibitor II prepeptide (TOMWIPII) (Les.1675.1.S1_at). The complimentary DNA (cDNA) was synthesized as previously described. For each relative quantity determination, three independent replicates were used. Statistical analysis of microarray data Microarray raw data were normalized by column-wise normalization using a reference sample (control) and by row-wise normalization using generalized logarithm transformation. Normalized data were then visualized using TM4Ò Software, Multi Experiment viewer (Mev). T test was performed by setting-up a P value of 0.01 and assuming equality of variance between variables. Significant genes were clustered by hierarchical clustering (Euclidean distance). All known and unknown genes of upand down-regulated genes were clustered by cellular function using the Tomato Functional Genomics Database (http://ted.bti.cornell.edu/cgiin/TFGD/array/funcat.cgi). All generated microarray data were deposited in the public database GEO under accession number GSE58521.

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Results Production of hydrogen peroxide in tomato wild-type and InsP 5-ptase transgenic lines during continuous light exposure To clarify the differences in response between wild-type and InsP 5-ptase lines to oxidative stress caused by continuous light exposure, we monitored the chlorophyll content and hydrogen peroxide accumulation during 21 days of incubation under continuous light exposure. We found that the transgenic tomato plants with increased InsP3 metabolism kept their typical phenotype for a longer time when exposed to stress caused by continuous light exposure while wild-type plants showed severe photobleaching symptoms (Fig. 2a). Measurement of hydrogen peroxide content in leaf samples of stressed plants revealed that the level of H2O2 was constantly increased during photo-oxidative stress in wild-type (WT) and empty vector control (EV) plants; however, no significant increase of H2O2 was noticed in samples collected from transgenic lines (L6 and L7) (Fig. 2b). After 21 days of continuous light exposure, the H2O2 level remained stable in transgenic plants with reduced InsP3. Wild-type plants, on the other hand, showed a fourfold increase in their H2O2 content. The increase of H2O2 production in wild-type

Fig. 2 Response of control and InsP 5-ptase transgenic tomato lines on 21 days of stress caused by continuous light exposure. a Phenotype of wild-type (WT), empty vector (EV) and transgenic lines (L6, L7) after 21 days of continuous light exposure (50 lmol m-2 s-1 light, no night). b Hydrogen peroxide content as affected by photo-oxidative stress in leaves of WT, EV, L6, L7. Hydrogen peroxide level is

plants was followed by a decrease in the chlorophyll content of these plants. Wild-type plants showed a severe reduction in chlorophyll content during continuous light exposure, while the chlorophyll content of transgenic plants was only slightly reduced during the stress period (Fig. 2c). These results demonstrate that the accumulation of H2O2 in leaves can affect the ability of plants to maintain the chlorophyll content in wild-type plants and that transgenic plants with lower InsP3 content are able to keep a normal level of chlorophyll during photo-oxidative stress. Expression of enzymatic antioxidants in tomato wild-type and InsP 5-ptase transgenic lines during stress caused by continuous light exposure Enzymatic antioxidants play an important role in ROS detoxification by converting them to non-toxic compounds. The activity of these enzymes controls the cellular content of ROS during photosynthesis and protects the plant cells against accumulation of toxic compounds (Fig. 1). To investigate the role of enzymatic antioxidants during photooxidative stress in tomato lines with modified phosphoinositol metabolism, we measured the transcription level of important enzymatic antioxidants in tomato (LeSOD, SICAT2, LeAPX1) leaves of wild-type and InsP 5-ptase transgenic plants after 7, 14, and 21 days of exposure to

indicated as a relative value to wild type. Data are mean ± SE of three biological replicate for both chlorophyll and hydrogen peroxide measurement. c Chlorophyll content as affected by photo-oxidative stress in leaves of WT, EV, L6, L7 lines. Total chlorophyll content is expressed as lg/g FW after 7, 14 and 21 days of exposure to continuous light

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continuous light. The transcript abundance was quantified using qRT-PCR for all selected genes. We noticed significant differences between transgenic and non-transgenic lines in the expression of genes coding for enzymatic antioxidants. For example, the expression of LeAPX1 was dramatically higher after 7 days of continuous light exposure in both InsP 5-ptase transgenic lines than in the wild type. The expression of LeAPX1 gene significantly dropped in transgenic lines after 2 weeks of continuous light exposure (Fig. 3a). Similarly, the expression of LeSOD gene was up-regulated during the first week of continuous light exposure and decreased after 14 days of incubation under continuous light exposure (Fig. 3c). SICAT2 expression was up-regulated in transgenic lines compared to wild type during 2 weeks of continuous light exposure (Fig. 3b). Results of antioxidants’ enzymatic activity were also in line with the expression pattern observed for these genes

(Fig. 4). Thus, activity of catalase (CAT) was higher in leaves of both transgenic lines (L6, L7) compared to leaves of wild type during 2 weeks of continuous light exposure (Fig. 4a). Superoxide dismutase (SOD) activity in InsP 5-ptase expressing lines exceeded the level of SOD in wildtype after 7 days of stress caused by continuous light exposure (Fig. 4b). However, the activity of SOD was significantly lower in transgenic lines than in wild type after 14 and 21 days of continuous light exposure. Established data suggest that the metabolism of H2O2 during oxidative stress is more intensive in transgenic tomato plants compared to wild-type. Expression of key regulators of light signaling in tomato wild-type and InsP 5-ptase transgenic lines under red and blue light To study the interaction between phosphoinositol metabolism and light-regulated induction of oxidative response genes and to further understand the mechanisms by which light regulates this interaction, we performed expression analysis on light-induced transcription factors. Response to

Fig. 3 Transcript abundance of major tomato antioxidant enzymes: a ascorbate peroxidase (LeAPX1), b catalase (SICAT2) and c superoxide dismutase (LeSOD) in wild-type and InsP 5-ptase tomato line under continuous light exposure. qRT-PCR was performed using specific primers and cDNA obtained from RNA extracts from leaves of wild-type (WT), empty vector (EV) and transgenic lines (L6, L7) after 7, 14 and 21 days of continuous light exposure (50 lmol m-2 s-1 light intensity at 35 °C). Data are mean ± SE of three independent replicates

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Fig. 4 Enzyme activity assay for tomato antioxidant enzymes. Enzyme activity was measured for catalase (a) and superoxide dismutase (b) enzymes in leaves of wild-type (WT), empty vector (EV) and transgenic lines (L6, L7) tomato plants after 7, 14 and 21 days of continuous light exposure (50 lmol m-2 s-1 light intensity at 35 °C). Enzyme activity is indicated as a relative value to wild-type for each time-point. Data are mean ± SE of three independent replicates

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light can be associated with different branches of secondary metabolite biosynthesis. Thus, light can induce flavonoid biosynthesis through the activation of HY5 transcription factor which in turn activates the MYB12 transcription factor (Stracke et al. 2010; Heijde and Ulm 2012). Together, these transcription factors induce the expression of chalcone synthase, a key enzyme in the flavonoid biosynthesis pathway (Fig. 5). Flavonoids can neutralize radicals and protect the cells from damages caused by ROS (Løvdal et al. 2010). Flavonoids also absorb UV light and induce protection against UV radiation in plants with high flavonoid content (Cle´ et al. 2008). In our experiments, the expression of light-signaling transcription factors and also chalcone synthase was studied under red and blue light influence (Fig. 6). Exposure of dark-grown seedlings to red light for 3-h resulted in higher expression of PHYB, a receptor of red light, in InsP 5-ptase lines compared to control lines (WT, EV). Exposure of seedlings to blue light slightly increased expression of CRY1 gene in transgenic lines compared to control lines. CRY1 is responsible for the perception of blue light (Lin et al. 1998; Cashmore et al. 1999). Transgenic plants with reduced InsP3 (L6 and L7) showed higher expression of transcription factor HY5 under blue and red light exposures compared with control lines (WT, EV). HY5 induces the expression of several light-regulated genes by direct interaction with their promoter sequence or by activating other proteins (Ang et al. 1998; Lee et al. 2007). In the next step, the expression level of light-regulated downstream

Fig. 5 Hypothetical model explaining signaling mechanism used by red and blue light for induction of the flavonoid pathway in tomato plants by regulating the abundance of HY5 transcription factor based on a model suggested by Quail (2002). Light signal is perceived by red and blue light receptors. Activation of phytochrome B (LePHYB) by red light increases the expression of LeHY5 at transcriptional level during the activation of cryptochrome 1 (SICRY1), by blue light acts in post-transcriptional level by reducing the degradation of LeHY5 via E3 ubiquitin ligase LeCOP1. Binding to CRY1 inactivates COP1 which in return increases level of HY5 in the cell. HY5 binds to the promoter of LeCHS1 and together with flavonol-specific regulator of phenylpropanoid biosynthesis, MYB12 activates expression of LeCHS1

genes that are involved in flavonoid biosynthesis (LeCHS1 and SIMYB12) was studied (Fig. 6). RT-PCR data demonstrated an increase in the expression level of these genes in transgenic lines after exposure to red or blue light. These data support the positive role of red and blue light in the induction of the flavonoid pathway in tomato seedlings. The results demonstrate that transgenic plants with reduced InsP3 level have a more active light-signaling pathway with a higher expression level of key light-signaling transcription factors and flavonoid biosynthesis activators. We found that the expression of photoprotective agent LeELIP in InsP 5-ptase tomato seedlings exposed to red or blue light was higher compared to that of the wild-type and empty vector control (Fig. 6). Microarray analysis of tomato wild-type and InsP 5-ptase transgenic line L7 Microarray analysis was performed to understand the total gene expression pattern in InsP 5-ptase expressing transgenic tomato lines in comparison with wild-type. This powerful technology was used as a profiling tool to identify the genes with altered gene expression in InsP 5-ptase transgenic seedlings during continuous light exposure. The analysis of microarray data using unpaired t test showed that

Fig. 6 Effect of red and blue light on transcript abundance of red and blue light receptors (LePHYB, SICRY1), transcription factor LONG HYPOCOTYL 5 (LeHY5), flavonol-specific regulator of phenylpropanoid biosynthesis (SIMYB12), chalcone synthase (LeCHS1) and early light-inducible protein (ELIP). RT-PCR was performed using specific primers and cDNA obtained from seedlings of wild-type (WT), empty vector (EV) and transgenic lines (L6, L7) after 3-h of exposure to red (650 nm, 1.2 lmol m-2 s-1) or blue (450 nm, 0.32 lmol m-2 s-1) light

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the expression of a total of 181 genes had been significantly affected in seedlings of the transgenic line L7 in response to oxidative stress (Fig. S1). Among these significant genes, only 41 had a known function according to the NCBI database. The up-regulated genes (total of 24) and downregulated genes (total of 17) in the transgenic line L7 are shown in a hierarchically organized heat-map (Fig. 7). To further analyze the biological processes affected in L7 line, the Tomato Functional Genomics Database was used to categorize the differently expressed genes based on their involvement in specific biological processes. Figure 8 summarizes the different biological processes that were affected in L7 line compared to the control line. The majority of genes were involved in the cellular process (44 genes), metabolic process (40), stress responses (37 genes), and biosynthetic processes (24 genes). Among stress-related genes, certain biotic stress response genes, including lipoxygenase (Les.3632.1.S1_at), fatty acid hydroperoxide lyase (Les.3478.1.S1_at), and chitinase (Les.3406.1S1_at), were affected. Other abiotic stress genes were also upand down-regulated including allene oxide cyclase (Les.3718.1.S1_at), ripening regulated protein (Les.274.1. S1_at), and carbonic anhydrase (Les.428.1.S1_at). Sequence-specific primers were generated and quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to validate the microarray data. Two Fig. 7 Microarray data of transcripts showing differences between InsP 5-tase transgenic line L7 (L7) and with wild-type (WT). Data are shown as heatmap and hierarchically clustered (Euclidean distance). RNA samples used for microarray experiment were isolated from seedlings of wildtype (WT) and transgenic line 7 (L7), germinated on MS medium in a growth chamber with 50 lmol m-2 s-1 light intensity at 12-h daylight and 12-h dark for 10 days and then continuous light exposure (50 lmol m-2 s-1 light intensity at 35 °C, no night) for 7 days. Two independent biological replicates for each plant line are presented here. Only genes with known functions are presented at heatmap

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independent biological replicates were used for two identified up-regulated genes in tomato tissues: PR protein (Les.3408.1.S1_at) and wound-induced proteinase inhibitor II prepeptide (Les.1675.1.S1_at). The real-time PCR data confirmed the above-mentioned microarray data. Indeed, these two genes were confirmed to be up-regulated compared to the L7 line as observed in microarray data (Fig. S2).

Discussion Inositol 1,4,5-triphosphate is a key secondary messenger in plants that regulates a variety of plant responses including the expression of genes involved in stress signaling (Xiong and Zhu 2001; Munnik 2001). Details of the downstream consequences of InsP3 signaling are not very well known. Connection of phosphoinositol pathway and light signaling in planta has been demonstrated in several publications (Salinas-Mondragon et al. 2010; Alimohammadi et al. 2012). Thus, the up-regulation of key transcription factors of light signaling, such as HY5, ELIP, and MYB12, was documented in Arabidopsis and tomato plants with a genetically decreased InsP3 level (Salinas-Mondragon et al. 2010; Alimohammadi et al. 2012). Modification of lightsignaling machinery through the regulation of phosphoinositol pathway resulted in a number of phenotypical and

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Fig. 8 Summary of total biological processes affected in InsP 5-ptase transgenic line L7 compared to wild-type plants, after the continuous light exposure (50 lmol m-2 s-1 light intensity at 35 °C, no night)

for 7 days. The results were calculated by the unpaired statistical t test analysis. The graph is generated using the Tomato Functional Genomics Database

metabolomic changes in tomato plants. The biosynthesis of antioxidants derived from light-regulated branches of the secondary metabolite biosynthesis pathway was increased in InsP 5-ptase expressing tomatoes (Khodakovskaya et al. 2010; Alimohammadi et al. 2012). Transgenic tomato plants with a decreased level of InsP3 were also able to withstand continuous light exposure for a longer time compared to wild-type plants (Alimohammadi et al. 2012). Here, we made an attempt to further clarify the molecular mechanism that leads to the increased tolerance of InsP 5-ptase expressing tomato plants to stress caused by continuous light exposure. The ability of InsP 5-ptase lines to maintain the chlorophyll level during 21 days of continuous light exposure (Fig. 2) could indicate that transgenic lines can utilize reactive oxygen species (ROS) more actively. There are several steps in the regulation of ROS content in plants (Fig. 1). Superoxide dismutase (SOD) can convert highly toxic O 2 molecules to less toxic H2O2 and O2. Catalase (CAT) and ascorbate peroxidase (APX) have the ability to convert H2O2 to H2O and O2 and play a critical role in neutralizing ROS molecules produced

during oxidative stress (Garg and Manchanda 2009). APX has a higher affinity for H2O2 than CAT, and the upregulation of APX has been shown during stress condition in a variety of plants (Aravind and Prasad 2003; Mobin and Khan 2007; Khan et al. 2007; Nazar et al. 2008; Srivastava et al. 2005; Zlatev et al. 2006; Yang et al. 2008; Sharma and Shanker Dubey 2005). We found that the level of H2O2 was significantly increased in control tomato lines (WT, EV), while the H2O2 level was kept low in transgenic lines (L6, L7) after 21 days of continuous light exposure (Fig. 2). This trend can be explained if synthesized H2O2 is immediately utilized in enzymatic reactions by CAT/APX enzymes. Real-time PCR analysis demonstrated the upregulation in expression of LeAPX1 gene (7 days of continuous light exposure) and SICAT2 gene (7 and 14 days of continuous light exposure) in InsP 5-ptase tomato lines (Fig. 3). As expected, enzymatic activity of catalase was significantly higher in transgenic lines during 2 weeks of continuous light exposure (Fig. 4). The balance between APX, SOD, and CAT activities is very important and plays a determining role in the efficient and precise scavenging

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of different ROS species (Apel and Hirt 2004). It is interesting that the expression of LeSOD gene, as well as enzymatic activity of LeSOD product (superoxide dismutase), increased after 7 days of continuous light exposure and dropped to the level that was lower than gene expression/ enzyme activity in control lines after 14 and 21 days. This observation may indicate that the conversion of O 2 into H2O2 was very active during the first week of stress, and then the activity of catalase was down-regulated due to active utilization of synthesized hydrogen peroxide by APX and SOD. Established data proved the fact that the entire ROS-scavenging enzymatic machinery is actively involved in the conversion of highly toxic molecules to less toxic compounds in tomato lines with a reduced level of InsP3. This activity leads to the protection of transgenic plants against oxidative stress caused by continuous light exposure. Key components of light signaling [phytochromes (PHYA, PHYB), cryptochromes (CRY1, CRY2) and downstream effector (HY5)] form a bridge between the lightperception/signal transduction pathway and biosynthesis of non-enzymatic plant antioxidants (carotenoids, ascorbic acid, flavonoids) (Fig. 5). Phytochromes and cytochromes receive the signals of red and blue light, respectively, and activate HY5 by independent pathways (Sellaro et al. 2009). HY5 acts downstream of photoreceptors and induces expression of many light-regulated genes (Ang et al. 1998; Chattopadhyay et al. 1998). Involvement of HY5 in the biosynthesis of antioxidants (carotenoids and flavonoids) has also been shown earlier (Liu et al. 2003; Stracke et al. 2010). Thus, increase in expression of LeHY5 leads to an increase in the biosynthesis of flavonoids in tomato plants by up-regulating the expression of the key enzyme of the flavonoid pathway, chalcone synthase (LeCHS1), and transcription factor SIMYB12 (Fig. 5; Stracke et al. 2010). Flavonoids have various functions in plants including pigmentation and UV protection (Olsen et al. 2010). They neutralize radicals and protect the cells from damage caused by ROS (Løvdal et al. 2010). Flavonoids also absorb UV light and induce protection against UV radiation in plants with high flavonoid content (Cle´ et al. 2008). We recently reported that fruits of InsP 5-ptase expressing tomato plants produce more light-regulated metabolites, such as flavonoids, lycopene, and ascorbic acid (Khodakovskaya et al. 2010; Alimohammadi et al. 2012). In addition, increased tolerance of InsP 5-ptase lines to oxidative stress can be explained by a documented increase in the activity of both enzymatic and non-enzymatic antioxidants. ELIP protein also can protect plant leaves from photo-oxidation (Hutin et al. 2003). Induction of ELIP can be achieved by CRY1 (Li et al. 2009). The expression of ELIP is increased during thylakoid biogenesis and

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oxidative stress (Bruno and Wetzel 2004). Together with antioxidants, ELIP protects plants from oxidative damage caused by continuous light exposure. It was demonstrated that plants with the ability to synthesize higher levels of antioxidants and ELIP demonstrate higher tolerance to photo-oxidative stress (Mittler 2002; Hutin et al. 2003). To prove changes in the expression of several key genes of light signaling in transgenic lines, we exposed dark-grown control tomato seedlings (WT, EV) and seedlings expressing InsP 5-ptase (L6, L7) to red and blue lights using specific LEDs for 3-h and performed RT-PCR. As result, we noticed changes in the expression of a number of key genes associated with plant response to oxidative stress (Fig. 6). For example, the expression of phytochrome B (LePHYB), which is involved in the perception of red light in tomato plants, was up-regulated in transgenic lines during red light exposure. No significant changes were observed in the expression of blue-light-induced tomato cryptochrome (SICRY1) under blue light exposure. SICRY1 is a negative regulator of LeCOP1 that can negatively regulate the HY5 gene (Fig. 5). As expected, transcription factors LeHY5 and SIMYB12 that are responsible for the activation of chalcone synthase (Fig. 6) were upregulated under both red and blue lights. As a possible consequence of such up-regulation, expression of the LeCHS1 gene was up-regulated, as well (Fig. 6). Similarly, LeELIP gene expression was higher in transgenic lines compared to control lines under both blue and red light. These observations are in a good correlation with our previous data (Alimohammadi et al. 2012) suggesting that transgenic plants with lower InsP3 demonstrate enhanced tolerance to stress caused by continuous light exposure resulted by the increase in expression of LeELIP, HY5, and LeCHS1. To understand the influence of reduction of InsP 5-ptase in tomato plants exposed to continuous light in total tomato transcriptome, we performed microarray analysis. The generated microarray data can serve as a tool to study the role of the InsP3 metabolism on overall stress signaling responses in InsP 5-ptase transgenic plants. It also provides a broader image of the possible interactions between InsP3 reduction and other primary and secondary metabolism pathways that determine the stress responses during oxidative stress. We found that the majority of changes in the gene expression pattern in InsP 5-ptase transgenic line were associated with stress responses, as well as cellular and metabolic processes (Fig. 8). However, it was interesting to see that, among the stress response genes that are affected due to the continuous light exposure, the genes that are normally responsible for the biotic stress, such as wound-induced proteinase inhibitor and protein oxidase A, were also affected. Biotic stress responses in tomato are mainly controlled by a complex interaction between the

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jasmonic acid (JA) and abscisic acid (ABA) pathways (Pen˜a-Corte´s et al. 1995; Bergey et al. 1996). JA signaling during biotic stress results in an increase in the level of extracellular Ca2? (Sun et al. 2006). It has been speculated that the increase in the level of the extracellular Ca2? during biotic stress is linked to the release of the Ca2? from the sources inside the cell. The role of the secondary messenger InsP3 in regulation of the Ca2? release in response to stress conditions was experimentally proved (Berridge 2005). Studies also demonstrated that the level of InsP3 is increased after mechanical wounding and that this increase can be induced by the JA pathway (Mosblech et al. 2008). In plants, the JA pathway is activated by metabolism of products of Lipoxygenase pathway. Plant lipoxygenase (LOX) is induced by biotic stress. Our microarray data showed that tomato lipoxygenase LOXD and allene oxide cyclase (AOC) were down-regulated in transgenic plants with reduced InsP3 levels during oxidative stress. This fact may be associated with a reduction in JA-mediated signaling in InsP 5-ptase tomato plants. Reduction of JA signaling in InsP 5-ptase tomato plants is in agreement with the previous findings that show a direct connection between the level of InsP3 and JA signaling (Mosblech et al. 2008). Thus, the induction of woundinginducible genes was attenuated in InsP 5-ptase expression Arabidopsis plants compared with wild-type (Mosblech et al. 2008). We noticed that a number of genes associated with plant response to biotic stress (chitinase, PR protein, wound-induced proteinase inhibitors) were up-regulated in InsP 5-ptase tomato plants (Fig. 7). It is interesting to consider how such genes could be up-regulated if genes associated with the JA pathway are down-regulated. We can suggest that higher expression of important biotic stress-related genes in InsP 5-ptase overexpressing plants is most likely related to the JA-independent pathway of biotic stress responses. There is evidence that the biotic stress responses in tomato can be regulated by a JA-independent pathway. It includes the expression pattern of biotic stress response genes that cannot be explained by a single JAdependent signaling pathway (Dalkin and Bowles 1989; Lightner et al. 1993) followed by expression of the woundinduced genes that are not controlled by JA signaling pathway (O’Donnell et al.1998). Further gene expression and hormonal studies are required to clarify the mechanism behind the activation of these genes.

Conclusion Taken together, our results proved the existence of active ‘‘cross-talk’’ between light-signaling, phosphoinositol metabolism, and oxidative stress response. Specific components of such cross-talk are still unknown in planta and

have to be discovered. However, we were able to demonstrate here that the reduction of InsP3, an important second messenger and the key component of phosphoinositol pathway, resulted in changes in ROS-scavenging machinery and, subsequently, increased the tolerance to oxidative stress caused by continuous light exposure. Our data demonstrated the significance and biotechnological potential of genetic engineering of phosphoinositol pathway for improvement of tolerance to oxidative stress in plants. Author contribution The experiments designed by MVK. Experiments were performed and data analyzed by MA, MHL, MVK. Manuscript was written by MA, MHL, MVK. Acknowledgments Funding from EPSCoR-NSF-P3 Center (Grant P3-202 to MVK) and NASA-EPSCoR (Grant to MVK) is highly appreciated. Authors thank EPSCoR-NSF-P3 Center, Graduate Institute of Technology and College of Science, UALR for providing graduate assistantship to Mohammad Alimohammadi. We are grateful to Dr. Julian Post for designing and building of LED light boxes used for light exposure experiment. The editorial assistance of Dr. Marinelle Ringer is also acknowledged. Conflict of interest interests.

The authors declare no competing financial

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