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Conventional photomixotrophic micropropagation system has many inconveniences due to the presence of sucrose in Murashige and Skoog medium (MS). A large number of micropropagated plants suffer damages and even die when transferred from in vitro to ex vitro conditions. The presence of sugar in the culture medium during the rooting stage is suggested to be the major cause of this problem. Classical approaches, relying on biochemistry and enzymology, have been used to understand the problem, but have provided no definitive answers to the causes. To investigate the central role of the sucrose supply on the regulation of the plantlets morphology and biochemistry, we measured growth parameters and used a comprehensive nonbiased analysis providing a thorough image of the metabolic profile of in vitro potato leaf tissues subjected to different tissue culture conditions. Using gas chromatography coupled to mass spectrometry (GC-MS), we identified a set of 51 differing metabolites in leaf tissues produced from two contrasting culture medium treatments during the rooting stage: (1) MS medium without sucrose (photoautotrophic conditions) and (2) MS medium with 3% sucrose (photomixotrophic conditions). Most of the growths parameters like shoot height, leaf weight, leaf number and leaf area/plant were significantly lower under photomixo- than photoautotrophic conditions, also photosynthesis was inhibited due to partial stomatal closure under photomixotrophic conditions. Metabolomic methods and bioinformatics tools, Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA), revealed distinct metabolic phenotypes for the different treatments and led to the identification of marker metabolites for each. Photoautotrophic leaves were characterised by the accumulation of urea and erythritol. Moreover, photomixotrophic leaves were characterised by an accumulation of primary metabolites and catecholamines, and by compounds related to abiotic stress, including proline, hydroxyproline, asparagine, GABA and myo-inositol. In addition, large accumulation of soluble sugars in leaf tissue has been observed. Metabolomic tools were instrumental in obtaining comprehensive informations on plant metabolites and their physiological role demonstrating the usefulness of this technique as a diagnostic tool for the tissue culture problems.
Micropropagation is being predominantly utilized for the rapid propagation of many plant species and cultivars. However, despite its potential, this technique has not lived to expectations and is still plagued with many problems. One of its principal limitations is the poor survival of plantlets once transferred from in vitro conditions to the natural uncontrolled environment (Pospisilova et al. 1999). These problems have limited the application of this technology to the mass production of valuable plants like fruit trees and some other flower crops.
Plantlets or shoots growing in vitro are continuously exposed to unique cultural conditions, depicted by high relative humidity, poorly ventilated vessels, low level of light, low CO2 concentration in the culture vessel during photoperiod, presence of high sugar, nitrogen and phytohormones in the culture medium. These nutritional and physical conditions are responsible for abnormal morphology, anatomy, and physiology of in vitro grown plantlets (Desjardins 1995). In many cases, these aberrations complicate the acclimation to ex vitro conditions.
One of the principal causes for the poor acclimatization successes of ex vitro plantlets is their low photosynthetic rates caused most probably by the presence of sucrose in the culture medium (Desjardins et al. 1988; Hdider and Desjardins 1994; Lees et al. 1991). For instance, the activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was reduce by adding sugar in the medium (Grout 1988). Furthermore, Premakumar et al. (2001) found that micropropagated oak and strawberry plantlets had a reduced quantity of the both the large and the small Rubisco subunits. In avocado, Rubisco content was noticeably diminished in leaves of plantlets cultured with high sucrose supply (87.6 mM) and maximum photosynthetic rate was significantly decreased when plants grew in presence of both high sucrose concentration and elevated CO2 (de la Viña et al. 1999). However, micropropagated olive plantlets did not show any difference in the Rubisco levels. Others have also demonstrated that sugar caused end-product inhibition of many photosynthetic enzymes (Sheen 1994; Koch 1996). Besides, many authors have shown that growth of tissue cultured plantlets can be improved by using the sugar-free media, and by elevating photosynthetic photon flux (PPF) and the CO2 concentration in the vessel in a culture system designated as photoautotrophic (PAT). PAT plants are defined as those that use CO2 as their only carbon source for growth and development (sugar-free medium) whereas photomixotrophic (PMT) plants use sucrose (3% in this experiment), but may satisfy some of their carbon requirements by photosynthetic fixation of CO2. For example, Kubota et al. (2001) reported that the dry weights of tomato plantlets when cultured photoautotrophically (under high light, high CO2 concentration and higher number of air exchanges) were more than twice as large as those of plantlets cultured photomixotrophically (under low light intensity, low CO2 concentration, and low number of air exchanges). In addition, Nguyen et al. (1999) found that, the fresh weight, shoot length, root length, leaf area and photosynthesis ability of coffee plantlets when cultured on Florialite with sugar-free medium and under high ventilation rate were greater than those cultured in MS agar medium with 20 g lâˆ’1 sucrose.
The presence of exogenous sugar in the culture medium may also cause osmotic stress to the plantlets. Javed and Ikram (2008) found that raising sucrose concentrations above a certain level, caused osmotic stress in the tissue culture medium of two wheat genotypes (S-24 and MH-97); plantlets accumulated organic compatible solutes such as proline and total soluble carbohydrates in greater amount and grew much less. Finally, Desjardins et al. (2007) suggested that in vitro plantlets are continually intoxicated by high concentration of sucrose and nitrogen from the medium and they have to adjust their metabolism in order to survive under these artificial stressful conditions.
Although the presence of sugar in the culture medium depressed photosynthesis and disturbed in vitro plantlets hardening (Deng and Donnelly 1993), the opposite effect has also been observed (Tichá et al. 1998). For instance, sugar appears to be essential component of the medium for many species. In some cases, independent growth could not be achieved on a medium without sugar. Wainwright and Scrace (1989) found that higher shoot height, fresh and dry weight of Ficus lyrata and Potentilla fruticosa were obtained in vivo when plantlets were cultured in liquid medium with 2 or 4% sucrose. Plantlets establishment success declined when sucrose was not used. In apple root initiation was decreased proportionally with decreasing sucrose level (Lane 1978) and shoots without sucrose did not survive their transfer to greenhouse (Zimmerman 1983). Sucrose linearly increases the level of reducing sugars, starch and total chlorophyll in citrus plantlets (Hazarika et al. 2000). Moreover, Tichá et al. (1998) reported that plant growth, dry matter accumulation and total leaf area were higher under photomixotrophic than photoautotrophic conditions. Not only biomass accumulation, but also photosynthesis ability were positively affected by exogenous sucrose.
Many investigators have studied acclimatization problem by evaluating some plant compounds one at the time, but this approach as proven incomplete to provide sufficient information about the causes of this problem. To distinguish alterations of the overall metabolism, a comprehensive, high-throughput and unbiased analysis is required. Metabolite profiling using gas chromatography-mass spectrometry (GC-MS) was first successfully applied to plant biology by Roessner et al. (2000). This method of metabolite profiling has already proven to be a suitable and powerful tool, for example in measuring broad-scale metabolites to distinguish between two culture system, in vitro and soil-grown potato tubers (Roessner et al. 2000), to characterize the response to different concentrations of nitrate in the culture solution (Okazaki et al. 2008) and to investigate developmental aspects of sink-to-source transition of quaking aspen leaf (Jeong et al. 2004). The technique has very high sensitivity for detecting both genetic and environmental effects on biological systems (Fiehn 2002).
In this study, we used a GC-MS-based metabolite profiling approach to distinguish dynamic metabolic responses exhibited by fully expanded potato leaves obtained under PAT or PMT conditions. This study of the leaf metabolome provids a global image of the physiological and biochemical status of the plants and documented compositional differences during this key stage of the tissue culture process. Thus, the aim of this work was to define a metabolic plant phenotype for two contrasting artificial growth conditions (PAT and PMT), an information that could then provide plant tissue-culturists with physiological biomarkers or signature of a suitable adapted plantlets, leading to high survival rate in the acclimatization conditions.
Materials and methods
Plant material and growth conditions
Potato plantlets (Solanum tuberosum L., cv Norland) were micropropagated by monthly subculture on 70 ml MS (Murashige and Skoog 1962) phytagel medium with 3% sucrose in magenta polycarbonate vessel (Magenta Corp., Chicago USA). For the experiments, homogeneous nodal cuttings (30 stem cuttings from the same position) were transferred to stacked polycarbonate vessels (Magenta GA-7) sealed at the mid-joint with a polypropylene coupler. Two holes (1 cm diameter) were perforated in two sides (one opposite the other) of the upper Magenta and covered with paper membrane (Millipore TM) to increase gas exchanges of the culture vessels. Potato plantlets were cultured either autotrophically or photomixotrophically with 0 or 3% sucrose, respectively in the standard MS medium. The plantlets were grown for 5 weeks under a PPF of 150 Âµmol.m-Â².s-Â¹. Light was provided by CoolWhite fluorescents, the temperature was maintained 23Â± 2Â°C, 50% Â±5% relative humidity and 16h photoperiod.
Photosynthesis and growth parameters measurements
For the measurement of photosynthesis and growth parameters, nine replicates were used for each treatment. A leaf chamber fluorometer, attached to a portable photosynthesis system (LIâ€‘COR 6400) was utilized to measure the photosynthesis rates of the first fully expanded leaf (fourth leaf from the top of the plantlets). Photosynthesis was measured at 0, 20, 50, 100, 200, 500, 1000, 2000 and 2500 Âµmol.m-Â².s-Â¹ to obtain a light saturation curve. Assimilation chamber temperature (block) was fixed at, 22Â°C, 70Â±5% relative humidity and CO2 was fixed at 400 ppm. Leaf surface, number and weight, stem length and weigh and root weight were estimated at the end of the experiment.
Potato leaf metabolite sample preparation for gas chromatographyâ€‘mass spectrometry
Plant material preparation for metabolite analysis was described by Roessner et al. (2000). Briefly, for metabolites measurement, nine replicates from nine individual magenta boxes were used for each treatment. The fourth and fifth leaves of the plantlet were collected at the middle of the day and frozen in liquid nitrogen, then stored at -80 until samples preparation. 100 mg of frozen leaf tissue was ground to a fine powder by mortar and pestle in the presence of liquid nitrogen, and extracted with 1.4 mL of 100% methanol. 50 ÂµL of ribitol was added to the samples as an internal standard (2 mg of ribitol/1 mL H2O) to correct for the loss of analytes during sample preparation or sample injection. Metabolites were extracted from the sample by incubation for 15 min at 70Â°C, then, one volume of water was added to the mixture which was centrifuged at 2200g, and subsequently dried in a speed-vacuum. The residue was redissolved and derivatized for 90 min at 30Â°C (in 80 ÂµL of 20 mg.mL-1 methoxyamine hydrochloride dissolved in pyridine), followed by a 30-min treatment at 37Â°C with 80 ÂµL of MSTFA (N-methyl-N-[trimethylsilyl]trifluoroacetamide). Before trimethylsilylation, 40 ÂµL of a retention time standard mixture was added, 3.7% [w/v] for hepatonic acid, nonanoic acid, undecanoic acid, and tridecanoic acid; 7.4% [w/v] for pentadecanic acid, nonadeanoic acid and tricosanoic acid, 22.2% [w/v] for heptacosanoic acid; 55.5% [w/v] for hentriacontanoic acid dissolved in 50 mg/5mL-1 tetrahydrofuran). One ÂµL volumes of sample were injected with a split ratio of 25:1.
Samples were analyzed by a Hewlett-Packard (HP) model 5973 mass selective detector with a 6890 plus series GC fitted with a split/splitless injector port, an HP 7683 series automatic sampler. Separation was performed on a 30m SPB-50 column with 0.25mm internal diameter and 0.25Âµm layer thickness (Superlco, Bellfonte, CA). The injector temperature was maintained at 250Â°C. The carrier gas was helium at a flow-rate 1 ml. min-1. The oven temperature program was 5 min isothermal heating at 70Â°C, followed by a 5Â°C.min-1 oven temperature increment until it reached a temperature of 310Â°C, followed by a final heating stage for one additional min at 310Â°C. The system was then temperature equilibrated for 6 min at 70Â°C before the next injection. Mass spectra were recorded at 2.7 scan/sec with the range of 50 to 600 m/z. ChemStation software (Agilent Technologies) operated the system and validated chromatogram and spectrum output. Perfluorotributylamine (PFTBA), with m/z of 69, 219, and 502, was used for auto-tuning.
The substances peaks were manually identified using three means: 1) the National Institute of Standards and Technology (NIST library, version 98); 2) authentic standards (50 compounds) and 3) the calculation of retention time indexes of the metabolites followed by a comparison with the retention time indexes of the compounds found in Pol_fa library (Max Planck Institute of Molecular Plant Physiology, Golm, Germany).
Statistical analysis of Data
The area of every metabolite peak was divided by peak area of the internal ribitol standard, present in the same chromatogram, to correct for recovery differences. Log10 transformation was performed on data before statistical analysis. Student's t-test was employed to separate means by SAS software (version 9.1; SAS Institute inc., NC, 2003). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were performed on normalized datasets with the same software. PCA was used to account for the contribution of specific metabolites to build the treatments clustering. HCA classify metabolites into clusters on the basis of their abundance in the different treatments.
Comparison of growth parameters and photosynthesis under autotrophic and mixotrophic conditions.
At the end of the rooting stage (Fig. 1), photoautotrophic cultures were taller, had more leaves and had higher surface area than their photomixotrophic counterpart. However, photomixotrophic roots fresh weight was heavier (2-fold higher) than autotrophic roots (Table 1).
Photosynthesis and conductance
As showed in Figure 2 A and B, photoautotrophic plantlets demonstrated a typical CO2 assimilation response to increasing light intensities, also a normal stomatal conductance was observed (between 0.125 and 1.00 mol. m-2. s-1) under this type of culture. However, stomata were almost closed for plants grown under photomixotrophic conditions (between -0.07 and 0.04 mol. m-2. s-1) and the photosynthesis was clearly inhibited.
Comparison of metabolites abundance in the photoautotrophic and photomixotrophic plantlets
We next sought to uncover whether metabolites found in plantlets grown under contrasting in vitro conditions would yield biochemical signature changes consistent with the measured growth and photosynthesis parameters. When metabolic profiling approach was applied to extracts from potato leaves of photoautotrophic (sugar-free medium) and photomixotrophic (3% sucrose in the medium) plants, we were able to accurately identified 51 distinct metabolites (Table 2). Yet, there were a large number of peaks which were not found either in the commercial NIST library nor from an injected standard.
As shown in Table 2, the identified metabolites include most plant amino and organic acids, sugars, sugar alcohols, aromatic amines and nitrogen containing compounds (urea), total identified metabolites were arranged in a histogram according to decreasing normalized peaks area in mixotrophic plants (Fig. 4A).
In general, leaves of mixotrophic potato plantlets were found to contain a higher amount of most metabolites compared with leaves of autotrophic plantlets (Fig. 3). For instance, we found that 3/4 of the total identified metabolites content in mixotrophic plants were 1.2 to 40.5 times more concentrated than in autotrophic plantlets. Furthermore, 14% of metabolites were not detected at all in autotrophic plantlets. For example, the ubiquitous abiotic plant stress compounds proline, hydroxyproline, pyrroleâ€‘2â€‘carboxylic acid, ribose and maltitol. Only urea and erythritol (4% of the total identified metabolites content) were found in higher concentration in autotrophic than in mixotrophic leaves. The rest of total metabolites (6%) were present at comparable concentrations in both leaf types.
We have detected 21 free amino acids under photomixotrophic conditions and 19 under photoautotrophic conditions. The overall level of amino acids was higher in mixotrophic leaves, compared to autotrophic (Fig. 4B). Major differences between the two culture systems were found in the group of amino acids derived from Î±-ketoglutarate such as proline, hydroxyproline, gamma-aminobutyric acid (GABA) (11.7-fold), arginine (8-fold) and glutamine (4.4-fold). Very large amount was observed in the level of asparagine (40.5â€‘fold) and other amino acids derived from oxaloacetate for example aspartic acid (6-fold), isoleucine (4.5-fold) and threonine (4-fold) in potato mixotrophic leaves compared to photoautotrophic leaves. All the rest of amino acids identified in mixotrophic leaves were between 1.8 and 3.5-fold higher compared with leaves grown under autotrophic conditions. However, tryptophan level was stable under either culture conditions.
Photomixotrophic potato leaves had larger amounts of the TCA cycle intermediates citrate, malate and succinate, which were 11.6-, 7.2- and 4.2-fold higher than in autotrophic leaves, respectively. Interestingly, glycolytic intermediates 3PGA and pyruvate were not detected in photoautotrophic potato leaf, also pyrrole-2-carboxylic acid, which come from the degradation of hydroxyproline, was observed only in mixotrophic plants. There was a 5-fold higher concentration of quinic acid and slight changes in the other organic acids such as glyceric acid and benzoic acid under photomixotrophic conditions. Yet, fumaric and lactic acid levels were the same in both leaf types (Fig. 4C).
Exogenous sugar in the medium led to a strong accumulation of soluble sugars in the leaves (Fig. 4D). Most of the sugar and sugar alcohol accumulated as sucrose, myo-inositol, fructose and glucose (97% of total identified sugars). A massive difference was observed in glucose and fructose levels between the two types of plants, with larger levels in mixotrophic than autotrophic plantlets; up to 13.7-and 26.6- times respectively. The amount of myo-inositol and sorbose was about 9-fold higher in response to exogenous sugar. Overall, the other sugars and sugar alcohol showed an approximate 1.4- and 4-fold increase respectively in photomixotrophic leaves. Erythritol was found at higher levels in photoautotrophic compared to mixotrophic leaves. Finally, maltitol and ribose were detected only in mixotrophic leaves.
Aromatic amines, including octopamine, noradrenaline, dopamine, tyramine and normetanephrine were found in higher quantities under mixotrophic cultures. Surprisingly, the urea content in the autotrophic leaves was up to 3.5-fold higher compared with mixotrophic leaves.
Principal component analysis and hierarchical cluster analysis of metabolite levels in photoautotrophic and photomixotrophic potato leaves.
GC/MS generates a large number of metabolites in every chromatogram. For this reason, it was important to apply some bioinformatic tools to the data set to determine whether the profiles contained sufficient information to metabolically distinguish autotrophic and mixotrophic leaves.
Principal component analysis (PCA) uncovered that two distinct clusters were clearly observable with photoautotrophic and photomixotrophic potato leaves. They were largely separated along the first component axis (Fig. 5).
PCA was used also to estimate the contribution of individual metabolites to the clustering of the leaf tissues (Fig. 6). Metabolites located close to the zero and cut off the axes contributed in a relatively small manner to the variance, while a number of the more distant distributed metabolites contributed to the separation of autotrophic from mixotrophic leaves clusters.
Hierarchical cluster analysis (HCA) was used to cluster the metabolites according to significant differences in distribution between photoautotrophic and photomixotrophic tissues (Fig. 7). Metabolites that clustered as (M Ëƒ A) represent all compounds that were found in higher quantity in mixotrophic leaves than those found in autotrophic leaves. In the same way, metabolites that clustered as (A Ëƒ M) comprise all compounds which increase in abundance under photoautotrophic conditions compared with photomixotrophic conditions. Finally, M = A cluster show all metabolites which were not affected by treatments.
Growth parameters and photosynthesis
Our results show that potato plantlets growing under photoautrophic conditions had higher values of leaf number, leaf weight, and leaf area compared to those grown under mixotrophic conditions. Nguyen and Kozai (2001) also reported that in vitro banana plants, cultured on Murashige and Skoog medium with no sugar added, had increased leaf number and area after 14 and 28 days of culture. However, Tichá et al. (1998) found that plant growth, dry matter accumulation and total leaf area of Nicotiana tabacum L. were higher under photomixotrophic than photoautotrophic conditions. In addition, shoot height of photoautotrophic plantlets was significantly higher than that of mixotrophic plantlets. This agrees with the report of Xiao et al. (2003) who demonstrated that the growth of sugarcane plantlets was four to seven times greater under autotrophic than mixotrophic conditions. Yet the average fresh weight of whole plants was the same for the two treatments due to greater root growth in potato grown on sugar containing medium. The only growth parameter which was significantly higher under photomixotrophic conditions was root weight. It is known that plant rooting, as any other morphogenetic process, is an energy-consuming process and therefore the presence of sugar in the culture medium increased root formation. Root initiation in some plants like apple was decreased proportionally with decreasing sucrose level (Lane 1978).
Photosynthesis of mixotrophic plants was very limited (-1.35-0.81Âµmol CO2 m-2 s-1) at the end of potato rooting stage period, supported the argument of (Hdider and Desjardins 1994) that the presence of sucrose in the tissue culture medium as the main carbon source may limit the efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the CO2-assimilation, and thus, decrease the in vitro plantlets photosynthetic capacity. Many others authors reported the inhibition of photosynthesis under photomixotrophic conditions, but they found a different reasons for this problem. For example, it has been reported that the low net photosynthesis rate of plantlets under conventional photomixotrophic culture conditions was due to the decrease of CO2 concentration in the air-tight culture vessel, low light intensity and the presence of sugar in the medium (Hdider and Desjardins 1994; Kozai 1991; Sha Valli Khan et al. 2002). However, under our conditions, the much lower photosynthesis in mixotrophic plants was caused by stomatal conductance limitations (between -0.07 and 0.04 mol. m-2. s-1). Indeed, using microporous gas filter on the culture vessels enhanced natural ventilation which led to decrease relative humidity inside the vessels and this probably caused a water stress. It is possible that, mixotrophic plants responded to the low relative humidity by closing their leaf stomata to protect plantlets from excessive dehydration. This observation led to the suggestion that, adding sucrose in the tissue culture medium was stressful for the potato plantlets during in vitro rooting stage, which exhibited reduced leaf photosynthesis and poor development, if growing under aerated culture vessel. In many species, decreased stomatal conductance has been suggested as the main regulatory factor of carbon assimilation in response to abiotic stress (Hsiao 1973). Johnson et al. (1997) reported that sucrose may play multiple roles including carbon and energy sources and an osmotic factor. The presence of sucrose in the culture medium acts as an osmotic factor that might induce osmotic stress above certain concentrations and lead to decreased growth (Kim and Kim 2002; Javed and Ikram 2008). Sundari and Raghavendra (1990) found that under in vitro osmotic stress conditions of spinach chloroplasts, the thylakoids lost about 80% of their photosystem II activity. In addition, Asai et al. (1999) reported that stomata of Vicia faba L. closed in response to osmotic stress so the rates of CO2 uptake and transpiration are controlled by regulation of stomatal aperture.
In vitro potato plantlets were stressed by the presence of sucrose in the culture medium
Results of metabolic profiling clearly demonstrated a significant shift in nitrogen and carbon metabolism as consequence of the presence of sucrose in the culture medium. Generally, mixotrophic conditions increased nitrogen assimilation to amino acids especially proline, hydroxyproline, asparagine, glutamine and GABA compared to autotrophic plantlets. These amino acids play an important role in stress damage protection and nitrogen detoxification. In addition, mixotrophic conditions increased carbon metabolism and led to an accumulation of many metabolites related to abiotic stress such as sugars and sugar alcohols and other metabolites associated with amino acids biosynthesis like organic acids. In the following paragraphs the most important compounds and their roles in plant physiology are discussed:
Accumulation of a wide-spectrum of amino acids in mixotrophic plantlets
Many amino acids accumulated in very high concentrations under mixotrophic compared to autotrophic conditions (Fig. 4B). The most important differences between the two culture systems were found in the group of amino acids derived from Î±-ketoglutarate such as proline, hydroxyproline, GABA, arginine and glutamine.
Proline which was only detected in photomixotrophic plantlets is a reliable indicator of the response of plants to environmental stress; its accumulation is a common metabolic response of higher plants to abiotic stress, and has been the subject of numerous reviews (Claussen 2005; Saglam et al. 2008; Wang and Han 2009). Several functions are proposed for the accumulation of proline in tissues submitted to abiotic stress: osmotic adjustment of stressed tissues (Kishor et al. 1995); carbon and nitrogen reserves; detoxification of extra ammonia; stabilization of proteins and membranes and free radical scavenging (Alia and Pardha 1995; Solomon et al. 1994). Moreover, the biosynthesis of proline may be associated with the regulation of cytosolic pH or production of NADP+ for the stimulation of the pentose phosphate pathway (Lutts et al. 1999). We believed that, the accumulation of proline in the photomixotrophic plantlets provided some measure of protection against osmotic stress caused by exogenous sucrose.
GABA is another very important amino acid which was present in higher concentration (11.7 fold) in mixotrophic leaf tissue than in photoautotrophic leaves. Our results are in agreement with numerous reports showing that high levels of GABA accumulate rapidly in plant tissues exposed to a variety of environmental stresses including hypoxia (Aurisano et al. 1995; Roberts et al. 1992; Miyashita and Good 2008), low temperature (Wallace et al. 1984), heat shock (Mayer et al. 1990), drought (Raggi 1994), salt (Bolarin et al. 1995) and low pH (Carroll et al. 1994).
Asparagine (derived from oxaloacetate) levels were considerably higher in PMT leaves than PAT leaves. Accumulation of asparagines in this work might be the indication that protein synthesis is reduced due to stress conditions. It is known that asparagine accumulates in plant organs when they are experiencing low rates of protein synthesis under otherwise abundant nitrogen supply. Also, asparagine is believed to act as an ammonia detoxification product produced when plants encounter high concentrations of ammonia (Sieciechowicz et al. 1988; Givan 1979). Furthermore, asparagine accumulation occurs under many stress conditions like drought, salt, mineral deficiencies, toxic metals and pathogen attack (Lea et al. 2007). In our experiment, asparagine accounted for 42% of total amino acids detected in mixotrophic leaves (40% of the total amino acid and aromatic amines) and was 40.5-fold higher compared with the concentration found in autotrophic leaves. The observation thus confirm the suggestion made by Desjardins et al. (2007) that mixotrophic culture conditions are stressful for plant growth in vitro conditions. Although, there was the same concentration of N in both culture media of the two treatments, only mixotrophic plantlets accumulated several times more of asparagine and other amino acids. This explains the large effect of exogenous sugar on amino acids biosynthesis. In fact, carbohydrate status has a large influence on the nitrogen metabolism. For instance, Morcuende et al. (1998) reported that sucrose increased activation of nitrate and ammonium assimilation and amino acid biosynthesis in detached tobacco leaves grown under low light conditions (100 Âµmol m-2 s-1). However, decreasing levels of sugars in plants tissues by growing them in unfavourable light regimes or in transformants with low Rubisco activity, lead to decreased nitrate assimilation, perhaps to an inhibition of GOGAT activity, and result in a general amino acid metabolism inhibition (Stitt et al. 2002).
Photomixotrophic conditions increased organic acids biosynthesis
Sugar are precursors for organic acids synthesis thus, exogenous sucrose lead to a large and generalized increase in organic acid including, citrate, malate and succinate. Similar changes in organic acids intermediates were found in other studies when tomato plants were grown in nitrate-saturated media under high-light conditions (it is known that, high-light intensity leads to increase photosynthesis and thus increase the sugar concentration in leaf tissue) (Urbanczyk-Wochniak and Fernie 2005). Morcuende et al. (1998) also found that sucrose feeding of detached tobacco leaves led to increases in organic acids and amino acids levels.
Photomixotrophic leaves accumulated large concentration of sugars and sugar alcohols
Remarkably, mixotrophic leaf contained high level of sugars and sugar alcohols and specially sucrose, glucose, fructose and myo-inositol. We believe that the presence of sucrose in the culture medium causes an osmotic stress which leads to the build-up of compatible solutes such as sugars and sugar alcohols in plant leaves, allowing the plantlets to absorb water under these conditions. The accumulation of soluble sugars specially fructose has been reported in mixotrophic potato leaves when plants were supplied with radioactive asymmetric sucrose (marked on fructose) (Badr and Desjardins 2007). Mixotrophic plantlets demonstrate a high capacity to absorb and metabolize sucrose present in the MS medium, yet, this large quantity of soluble sugars accumulating in photomixotrophic leaves may also inhibit photosynthesis during the rooting stage. Interestingly, Klages et al. (1999) found that kiwifruit plants subjected to salt stress accumulated myo-inositol and sucrose in their leaf tissues; the quantity of myo-inositol increased linearly with increasing salt levels and decreased rapidly once the stress was removed.
Photomixotrophic leaves accumulated large quantity of catecholamines
Catecholamines accumulated to high levels under mixotrophic conditions. The abundance of these molecules in mixotrophic leaves can be explained by a possible role in the adaptation of plantlets to a changing environment under tissue culture conditions and these compounds might have a role in the acquisition of protection against in vitro stress. Catecholamines are neurotransmitters in mammals. They are sympathomimetic (substances that mimic the effects of the sympathetic nervous system) "fight-or-flight" hormones released by the adrenal glands in response to stress and they are derived from the amino acid tyrosine. They have been found to accumulate in many plants like potato (Szopa et al. 2001) and others (Smith 1977), but no fundamental role has yet been recognized for these molecules in planta. They are believed to act as precursors for benzo[c]phenanthridine alkaloids, which are the active principal ingredients of many medicinal plant extracts. The synthesis of catecholamine is regulated by stress conditions and they have a possible role of nitrogen detoxification (Kulma and Szopa 2007). It is reported that, the catecholamine dopamine is a powerful water-soluble antioxidant like ascorbic acid and stronger than glutathione. It has faster radical-scavenging rates than catechins and was similar to gallocatechin gallate (Kanazawa and Sakakibara 2000). Swiedrych et al. (2004) found that, plant catecholamines are involved in plant responses towards biotic and abiotic stresses in potato. In addition, catecholamines are involved in key processes like plant tissue growth, somatic embryogenesis, flowering, inhibition of indole-3-acetic acid oxidation and stimulate ethylene biosynthesis (Kuklin and Conger 1995).
High accumulation of urea in response to photoautotrophic conditions
In autotrophic leaves, glutamic acid and urea were the main form of nitrogen accumulating in plantlets, comprising nearly 39% of the total nitrogen compound (amino acid and aromatic amines). A most remarkable finding was the high levels of urea accumulating in photoautotrophic leaves. Urea has a very high nitrogen-to-carbon ratio (N:C 2:1) and in comparison to ammonia, urea is a rather nontoxic compound which may be used as a storage compound in plants. Urea was at its highest level in photoautotrophic leaves and this is consistent with a surplus of available nitrogen in the MS medium (65 Âµmol l-1) of photoautotrophic plants relative to the limited photosynthetic capacity or available carbon in photoautotrophic leaves. Stitt and Schulze (1994) reported that, in tobacco transformants with low Rubisco activity had low levels of sugars, low nitrate reductase activity, low levels of amino acids and accumulated large amounts of nitrogen compounds (nitrate). Urea accumulation in photoautotrophic leaves is thus a good biomarker of insufficient light or carbon fixation found in vitro. It is known that, detoxification through the urea cycle is the mean by which mammalian organisms dispose of their excess ammonia. Interestingly, most of the enzymes involved in the urea cycle have been characterized in plants (Goldraij and Polacco 2000; Tischner et al. 2007).
The presence of sugar in the culture medium considerably alters plant biochemistry, physiology and morphology during in vitro stage and is a source of abiotic stress for the plantlets. We used metabolite profiling technique and bioinformatics tools as a comprehensive and accurate way to compare intracellular metabolic perturbations caused by exogenous sucrose. Using this method, we were able to obtain a metabolic signature of both photoautotrophic and photomixotrophic potato plantlets.
In this case, photoautotrophic plantlets are characterized by large accumulation of urea in their leaves as a result of the excessive amount of nitrogen found in the MS medium and the low amount of carbon in leaves tissue. Adding sucrose to the culture medium has wideâ€‘ranging effects on leaf metabolism and leads to large accumulation of sugars while strongly activating nitrogen assimilation, amino acid biosynthesis, and stimulating the synthesis of organic acids that are needed during nitrogen metabolism. In addition, stress-related metabolites such as proline and GABA were only observed in mixotrophic plantlets. This is consistent with the inhibition of growth and photosynthesis observed under photomixotrophic condition. These stressful in vitro conditions are most probably the principal causes of the poor establishment of plantlets during the acclimatization stage. The data presented here confirm that GC-MS and metabolomic approaches are useful diagnostic tools to detect metabolic disorder in plantlets under different tissue culture conditions. Further studies are needed to investigate potato leaf metabolism alteration during in vitro to ex vitro transition.
Thanks to the Egyptian Higher Education and its Missions General Administration for their financial assistance. We wish to thanks NSERC discovery grant program for its financial support to YD.
List of Tables
Table 1 Rooting stage growth parameters of potato plantlets under photoautotrophic and photomixotrophic conditions. For treatment codes, PAT photoautotrophic plantlets (without sucrose in the medium); PMT photomixotrophic plantlets (with 3% sucrose in the medium). NS, **, ***: nonsignificant, significant and highly significant different between the two treatments at P â‰¤ 0.05, respectively, according to Student's t-test. Values represent means Â± SE (n=9 observations).
Table 2 List of 51 metabolites identified from a methanol potato leaf tissue extraction.
List of figures
Fig. 1 Photoautrophic plantlets (growing on sugar-free MS medium) and photomixotrophic plantlets (growing on MS medium with 3% sucrose) of potato (Solanum tuberosum L., cv Norland) at the end of rooting stage (5 weeks).
Fig. 2 (A) The light response curve of net photosynthesis (Pn) and (B) the stomatal conductance for leaves of autotrophic (closed circles) and mixotrophic (open circles) potato plantlets (Solanum tuberosum L., cv Norland) with increasing light intensity. Each value is the mean of 9 replicates Â± SE.
Fig. 3 Comparison of GC/MS chromatogram of potato (Solanum tuberosum L., cv Norland) leaves extract produced under photoautotrophic and photomixotropic conditions. By visual inspection of GC/MS chromatograms we can observe dramatic differences between photoautotrophic and photomixotrophic potato leaves which indicate the strong effects of exogenous sucrose on overall leaf metabolism.
Fig. 4 Changes in metabolite levels of potato (Solanum tuberosum L., cv Norland) grown under photomixotrophic or photoautotrophic conditions in vitro. (A) show all polar metabolites detected in the leaf, (B) amino acids, (C) organic acids, (D) sugars and sugar alcohols, (E) aromatic amines and urea. Error bars represent SE of means of n = 18 determinations. **Thr AL: trans-Threonic acid-1,4-lactone
Fig. 5 The principal component analysis (PCA) scores plot demonstrates a distinct separation between photoautotrophic (A) and photomixotrophic (M) potato plantlets. PCA factor1 and factor2 represent more than 70% of the total variance.
Fig. 6: PCA loadings plot representation of the contribution of individual metabolites to principal component clustering of photoautotrophic and photomixotrophic potato plantlets samples. Metabolites marked M Ëƒ A were most abundant in mixotrophic tissues and vice versa for A Ëƒ M. However, those marked A = M were at the same levels under the two conditions.
Fig. 7 HCA dendrogram grouping of metabolites based on significant differences in relative abundance in autotrophic (A) and mixotrophic (M) potato leaf tissues. Student's t-test was used to rank the tissue abundance of each metabolite.
Sigmaplot 11 was used to create Fig. 2A and Fig.2B.
GraphPad 5 Prism was used to create Fig. 4 A to E.
Adobe Illustrator (CS4) was used to create Fig 5, Fig. 6 and Fig. 7.