2.1 Properties and Biosynthesis
ALA is a keto-amino acid with a molecular weight of 131 kDa (Fig. 12.1). ALA is known as a precursor of all porphyrins compounds such as vitamin
Fig. 12.1 Structure of 5-aminolevulinic acid (ALA)
B12, chlorophyll, heme, and phytochrome, and it is naturally found in plants, animals, algae, and photosynthetic bacteria (Wang et al. 2005; Tabuchi et al. 2009). It is generally assumed that in plants, algae, bacteria (except for the a-pro-teobacteria group), and archaea, ALA is synthesized via light-dependent C5 pathway (also known as Beale pathway) in plastids from glutamic acid via glutamyl-tRNA and glutamate-1-semialdehyde (Beale 1990; Reinbothe and Reinbothe 1996) . The enzymes involved in this pathway are glutamyl-tRNA synthetase, glu-tamyl-tRNA reductase, and glutamate-1-semial-dehyde aminotransferase. In nonphotosynthetic eukaryotes such as animals, insects, fungi, and protozoa, as well as the a-proteobacteria group of bacteria, ALA is formed by a reaction known as Shemin pathway (C4 pathway) via the condensation of succinyl COA and glycine by the enzyme ALA synthase (Beale and Weinstein 1989; Bisbis et al. 1997). It has also been proposed that exposure of plants to low and high temperatures is associated with blockage of ALA biosynthesis suggesting that ALA biosynthesis is a temperature-sensitive process (Hodgins and van Huystee 1986. Tewari and Tripathy 1998). Low temperature exposure of leaf tissues impaired ALA biosynthesis in maize seedlings resulting in reduced porphyrin synthesis and chlorosis (Hodgins and van Huystee 1986). Moreover, illumination of cucumber seedlings under chilling and heat stress conditions resulted in inhibition of chlorophyll biosynthesis by 90 and 60%, respectively, which demonstrated that inhibition of chlorophyll biosynthesis is higher under chilling stress than heat stress conditions (Tewari and Tripathy 1998). Same authors also found that reduced chlorophyll biosynthesis was partly resulted from the impairment of ALA biosynthesis since ALA biosynthesis was inhibited to a similar extent both under chilling (78%) and heat (70%) stress conditions.
It is generally accepted that angiosperms can produce chlorophyll only under the influence of light. On the other hand, evidence has been accumulating that several species of angiosperms may also be capable of forming chlorophyll in the dark and that ALA could have a stimulatory role in chlorophyll synthesis in dark (Yang et al. 2003). Treating the leaves of etiolated angio-sperm plants with ALA caused the accumulation of protochlorophyllide (Pchlide) and it was concluded that all enzymes required for Pchlide synthesis were already active and present in nonlimiting amounts and that only the activity and amount of enzymes involved in ALA synthesis limited the synthesis rate (Papenbrock and Grimm 2001) . Pchlide accumulates in the dark because angiosperms reduce Pchlide to chlorophyllide (Chlide) by a photo-enzyme, light-dependent protochlorophyllide oxidoreductase and this reaction represents a key regulatory step in the strictly light-dependent biosynthesis of chlorophyll in angiosperms (Pavlovic et al. 2009). On the contrary, some photosynthetic organisms such as cyanobacteria, algae, mosses, ferns, and gymnosperms could operate a second chlorophyll biosynthesis pathway and they are capable of synthesizing chlorophyll and bacteriochlorophlls in the dark by light-independent protochlorophyllide oxidoreductase (Armstrong 1998).
ALA formation is the rate-limiting step in chlorophyll biosynthesis in plants (Beale, 1990; Pavlovic et al., 2009) and ALA concentration is firmly controlled to less than 50 nmol/g FW in plant tissues (Stobart and Ameen-Bukhari 1984). At high concentrations (5-40 mmol/L), ALA undergoes enolization and further metal-catalyzed anaerobic oxidation at physiological pH to form reactive oxygen species (ROS) such as superoxide radical (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (HO ' ) (Kumar et al. 1999; Balestrasse et al. 2010). Thus, higher concentrations of ALA in tissues enhance ROS production leading to oxidative stress, acting as a herbicide. When green plants are exposed to light after ALA treatment in darkness, excess chlorophyll intermediates such as Pchlide and protoporphyrin IX
are photosensitized and consequent photodynamic reactions destroy the plant by oxidizing the unsaturated fatty acids in the cell membranes (Rebeiz et al. 1984; Chakraborty and Tripathy 1992). ALA, therefore, can be used as a safe substitute for highly toxic herbicides such as paraquat (Sasaki et al. 2002). Similar mechanism was also observed when ALA was used as a biodegradable insecticide to combat cabbage looper (Trichopusia ni) (Rebeiz et al. 1988) . On the other hand, low ALA concentrations (0.06-0.6 mM) appear to promote rather than damage growth of several crops and vegetables (Watanabe et al. 2000; Hotta et al. 1997a). In addition, ALA is known to induce tolerance to various abiotic stress conditions in a variety of crop species (Korkmaz et al. 2010; Naeem et al. 2010) . Furthermore, ALA has low mammalian toxicity (Kennedy et al. 1990) and it is biodegradable in soil (Hotta et al. 1997b) . All of these properties suggest that ALA may have a great application potential in agriculture as a new nontoxic endogenous substance (Wang et al. 2003).
2.2 Effects of ALA on Chlorophyll Content and Crop Productivity
The trend in present day agricultural crop production is to increase the economic yield through more efficient use and partitioning of photosyn-thates as well as by improving net photosynthesis in crop plants under ever-changing conditions (Bindu Roy and Vivekanandan 1998a; Wardlaw 1980). Yield of crop plants can be improved through a number of ways such as by promoting branching, increasing leaf area index, enhancing tolerance to various biotic and abiotic stress conditions, and by manipulating partitioning of pho-tosynthetic assimilates. PGRs alter plant growth and development by triggering numerous physiological responses, and ALA is listed among these PGRs that are used to manipulate plant growth and yield (Watanabe et al. 2006; Hotta et al. 1997c). ALA applied through different methods such as seed soaking, root drenching, or foliar application is known to enhance plant growth and productivity.
ALA has a variety of physiological effects on chlorophyll biosynthesis, photosynthesis, and plant growth. The efficacy of ALA application on overall plant growth and yield depends on ALA concentration used, application method as well as application time and crop species (Kantha et al. 2010). Soaking the seeds of several legume species in ALA solutions at concentrations ranging from 10 to 100 g m-3 resulted in enhanced chlorophyll accumulation and photosynthetic rate which, in turn, caused an increase in yield (Bindu Roy and Vivekanandan 1998a). The authors concluded that the action of ALA cannot be simply explained by the fact that ALA is the precursor of chlorophyll and that the increment in leaf photo-synthetic rate and plant leaf area significantly contributed to plant photosynthetic carbon assimilation and total biomass production. Similar confirmatory findings were also reported by others that ALA stimulates photosynthesis and decreases respiration in the dark promoting the yield of several crops such as kidney beans, garlic and spinach (Tanaka et al. 1992), and pakcohoi (Memon et al. 2009) . Although positive effects of ALA treatment in low concentrations on growth, chlorophyll content, and photosynthetic rate of angio-sperms has been well documented by several studies, the effects of exogenously applied ALA on chlorophyll formation in seedlings of gymno-sperms is somewhat ambiguous. Treating pine (Pinus nigra Arn.) seedlings with ALA at 10-5 M concentration did not have a notable effect on chlorophyll accumulation in the light whereas chlorophyll formation was significantly enhanced in dark-grown seedlings (Drazic and Mihailovic 1998). It was presumed that in the light, ALA was synthesized endogenously in optimal amounts which may explain the lack of response to exogenous ALA application. On the other hand, ALA application promoted chlorophyll accumulation in darkness probably due to compensation of insufficient production of endogenous ALA. On the contrary, application of 1 mM ALA to the Norway spruce (Picea abies) seedlings in light resulted in increased chlorophyll accumulation while higher concentrations of ALA had negative effect on growth and chlorophyll accumulation (Pavlovic et al. 2009).
Treating 2-year-old grapevines with ALA through foliar application (30-300 mg L-1) or root drench (0.1-10 mg L-1) significantly increased photosynthetic rate by up to 22%, fruit sugar content by 2.7%, and cluster weight by 53% (Watanabe et al. 2006). Enhanced photosynthesis was reported to cause higher sugar content in berries, and increased berry weight in the cluster may have given the cluster a stronger sink strength which could be an indirect cause for the enhanced photosynthesis besides increased sto-matal aperture. It was also reported that ALA applied at 0.06-6 mM through root soaking improved the growth of rice seedlings in light while ALA at 0.06 mM elicited chlorophyll accumulation in addition to increased photosynthetic rate in pothos lime (Epiprenunum aureus) plants (Hotta et al. 1997c). Additionally, in another study where wide range of ALA concentrations were tested in order to identify the optimum concentration for different application methods, it was found that up to 50% increase in plant mass of rice seedlings was observed when the optimum concentrations were applied at the rate of 0.1-1, 30-100, and 10-100 ppm for root soaking, foliar spray, and soil treatment, respectively (Hotta et al. 1997b) . However, when concentrations above the optimum were used, deleterious effects were reported.
The most comprehensive results on the effects of exogenous ALA application on photosynthetic activity and crop's yield were published by Hotta et al. (1997a) . They found that ALA positively affected the growth and yield of several crops and vegetables at concentrations lower than those eliciting herbicidal effect (i.e., less than 1.8 mM). Foliar application of ALA at the range of 0.061.8 mM significantly increased dry weight of radish roots but injured radish seedlings at 6 mM, while concentrations of 0.18 and 0.6 mM increased CO2 assimilation in light and decreased the release of CO2 in darkness. ALA application at low concentrations (0.18 and 0.6 mM) increased the growth and yield by 10-60% over non-ALA-treated plants on kidney bean, barley, potato, and garlic. In addition, ALA applied at the range of 10-50 ppm increased the yield of wheat by about 15% over the control after foliar application, and it was found that the effects of ALA depended on the timing of application and its concentration (Bingshan et al. 1998).
Exogenous application of ALA to fruits and vegetables is also reported to influence positively the quality of crop species. Spraying date palm fruits with 100 ppm ALA at different stages of fruit development increased fruit weight, fruit flesh percentage, fruit volume, and total and reducing sugar content although the positive response was dependent upon the application time or fruit developmental stage (Al-Khateeb et al. 2006). Application of 300 ppm ALA to apple fruits 20 days before harvest increased the total soluble solid content and decreased titratable acidity with no negative effect on fruit firmness and shelf life (Wang et al. 2004a) . It was also noted that no significant residue was found on the fruits which suggested that ALA could be used to improve apple quality. ALA application at the rate of 300 mg L-1 to 'Fuji' apple fruits 43 days before harvest significantly increased anthocyanin accumulation rate doubling the final anthocyanin content present in the fruit in comparison to untreated control fruits (Wang et al. 2006). Moreover, exogenous application of ALA to tomato fruits decreased respiration rate, the malondialdehyde (MDA) content, relative membrane permeability and titratable acidity and increased total soluble solid content, all of which resulted in improved fruit quality and prolonged shelf life (Wang et al. 2009). Additionally, exogenous application of ALA caused significant enhancement in glucose content and starch degrading enzyme, amylase activity in radish (Raphanus sativus) taproot (Hara et al. 2011). ALA-based fertilizer "Pentakeep" applied at the rate of 0.3% significantly increased fruit dry matter, sugar, and citric acid contents of the hydroponically grown strawberries (Iwai et al. 2005), while an increase in N content by ALA treatment has also been reported in spinach (Yoshida et al. 1995).
Plants themselves can synthesize phytohor-mones, but they can also utilize exogenous sources such as exogenously applied phytohormones by humans or microbially produced phytohormones, and this may be one of the mechanisms of plant growth promotion by microorganisms. There have been many reports on the microbial production of phytohormones. Photosynthetic bacteria (PB) which are widely distributed in nature especially in submerged conditions such as paddy fields, riverbeds, seashores, and sewage disposable plants (Kobayashi and Kobayashi 2000) are also able to synthesize tetraphyrroles. Some PB species such as Rhodopseudomonas palustris and Rhodobacter sphaerides can produce relatively large amounts of physiologically active substances such as vitamin B12, ubiquinone, and ALA (Sasaki et al. 2002), and they can be considered to be one of natural fertilizers (Kantha et al. 2010). For example, Koh and Song (2007) reported that two PB strains of Rhodopseudomonas sp. produced as much as 8.75 mg L-1 ALA within 48 h of inoculation which caused efficient growth enhancement of tomato seedlings under axenic conditions. The germination percentage of PB-inoculated tomato seeds, total length, and dry mass of germinated tomato seedlings increased by 30.2, 71.1, and 270.8%, respectively, compared to those of the uninoculated control. It was also reported that when soil and straw products were inoculated with different strains of Rhodopseudomonas palustris for 4 weeks with microaerobic-dark conditions, the ALA content increased with time to achieve levels of 2.96 mM depending on the PB strain, and it was concluded that PB could be practically applied to organic saline paddy fields and increase growth and yields of rice (Kantha et al. 2010). Moreover, application of PB also enhanced growth, fruit formation, yield and fruit quality in tomato plants grown in greenhouse (Lee et al. 2008) , increased mushroom (Agariscus bisporus) yield (Han 1999), and controlled the root rot on rice seedlings (Kobayashi and Kobayashi 2000).
Iwai et al. (2003) found that response to exog-enously applied ALA was amplified when plants were supplied with higher rates of N, which may be partially attributed to the role of N in chlorophyll synthesis and plant growth. Hydroponically grown paprika type pepper plants treated with ALA yielded up to 9% more than control plants which may have been due to the fact that ALA-treated plants utilized 16% more NO3 from the nutrient solution. Similar results were also reported in papaya (Carica papaya L.) where simultaneous application of N and ALA increased vegetative growth and reduced the time from papaya seedling emergence to the transplanting stage (Morales-Payan and Stall 2005). Increased Ca2+ content in spinach plants were also reported when ALA-based fertilizer "Pentakeep V" was applied simultaneously with N fertilizers (Smoleñ and Sady 2010).
Plant tissue culture is an important technique in plant propagation and since ALA possesses PGR properties, it is reported to play an important role in plant tissue culture. ALA treatment of explants of Laminaria japonica sporophyte was found to be useful to produce and propagate calluslike cells stably (Tabuchi et al. 2009). ALA treatment at the rate of 50-500 mg L" 1 was more effective in inducing callus formation than control (0 mg L"1" and cell division rate was the highest when explants were cultured with 500 mg L" 1 ALA. Same concentration of ALA also promoted the growth of photoautotrophi-cally growing cells of Spirulina platensis causing intracellular accumulations of phycocyanin and chlorophyll followed by enhancement of the pho-tosynthetic activities of photosystems I and II (Sasaki et al. 1995). In vitro studies with Vigna unguiculata L. confirmed the hormonal role of ALA by striking proliferation of callus and pari-passu induction of rooting and shooting with a profound effect of the former than the latter, and ALA was therefore reported to exhibit both auxin and cytokinin properties in the induction of cal-lusing and rooting and shooting, respectively (Bindu Roy and Vivekanandan 1998b). Also, ALA-based fertilizer "Pentakeep" applied at the rate of 0.04-0.08% shortened the required program to acclimatize the tissue culture-derived date palm seedlings by about 4-5 months compared to untreated plants by enhancing the growth of the seedlings via increasing nutrient uptake, chlorophyll concentration, and photosynthetic assimilation (Awad 2008) .
One of the physiological roles of ALA in plant growth was recently reported by Maruyama-Nakashita et al. (2010). They demonstrated that exogenously applied ALA at the rate of 0.3-1 mmol L-1 increased the transcript levels of sulfur transport and assimilatory genes causing significant enhancements of sulfate uptake under both sulfur-sufficient and sulfur-deficient conditions in Arabidopsis thaliana. In addition, ALA application also increased the accumulation of cysteine and glutathione, particularly in the shoots all of which suggest a new role for ALA in regulating the sulfur assimilatory pathway.
The chemical stability of ALA in aqueous solutions was reported to be a function of its concentration, pH, and storage temperature with the higher the concentration, pH, and storage temperature were, the faster the rate of ALA in aqueous solution degraded. Thus, when ALA solutions are prepared, the concentration and the pH of the solution should be as low as possible according to different application purposes, and the solution should be stored at low temperatures (<-20°C) (Bunke et al. 2000; Gadmar et al. 2002). It was also suggested that the final solution of pH 5.57.4 would have to be prepared a maximum of 1 h before use.
2.3 ALA and Plants Under Stress
2.3.1 Effects of Exogenous ALA on Plants Under Chilling Stress
Low temperature is one of the major factors limiting the productivity and geographical distribution of many species, including several important agricultural crops. Reductions in temperatures can substantially slow the velocity of many metabolic pathways, which leads to the natural deterioration and loss of crop quality. There are two types of injuries a plant faces under exposure to low temperatures. The first type of injury is called freezing injury which occurs when the external temperature drops below the freezing point of water. When a plant freezes, this causes ice formation within the tissues and ruptures cell walls causing loss of cellular integrity and ultimate death of the tissue. Freezing-tolerant plants have several strategies to reduce the probability of this phenomenon occurring, even when air temperature drops below zero, including maintaining high intracellular solute concentrations which reduces probability of freezing inside cells and encouraging ice nucleation outside the cells
(Allen and Ort 2001). Many plants, primarily young plants or seedlings that are native to tropics or warm climates, are very sensitive to low temperatures, showing abrupt reductions in the rates of physiological processes and exhibiting signs of injury following exposure to temperatures less than 15°C and they are called chilling-sensitive plants. Chilling injury can be defined as injury resulting from temperature that is cool enough to cause damage but not cold enough to freeze or to kill the plant (Levitt 1980). Chilling injury depends not only on the species and tissue type, but also on the severity and duration of exposure to low temperature (Lynch 1990) . The temperature below which chilling injury can occur varies with species and regions of origin, ranging from 0 to 4°C for temperate fruits, 8°C for subtropical fruits, and about 12°C for tropical fruits such as banana (Lyons 1973). Major crop species including maize (Zea mays), cotton (Gossypium hirsatum), and rice (Oryza sativa) are sensitive to chilling temperatures. Warm season vegetables such as those belonging to Cucurbitaceae and Solanaceae also suffer heavily from chilling stress and their growth and development can be adversely affected by temperatures below 15°C resulting in yield loss and crop failure.
Sudden exposure to low temperatures especially to temperatures around or below 0°C may result in extensive and irreversible damages on plant tissues since it causes the membranes to lose their semipermeability and thus their active ion transporting ability (Janda et al. 2007). During chilling stress, the phospholipids in the membranes start to decompose, phase transition takes place, the distribution of the membrane proteins changes and the first visible symptom of low temperature injury, wilting, occurs. Freshly imbibed seeds of chill-sensitive species tend to be also very sensitive, as does the pollen development stage. Imbibitional chilling injury occurs in sensitive seeds such as soybean or cotton during the early stages of imbibition. If soil temperatures are very low at planting, water entering into the seed disrupts membrane integrity, increases electrolyte leakage, and blocks germination. However, if chilling stress follows a brief period of imbibition at warm temperatures, then no damage occurs. The initial reorganization of the membranes from the dry to the hydrated state, therefore, is the critical cellular process (Crowe et al. 1989). Imbibitional chilling injury may also take place in the pollen of sensitive species and the lipid phase properties of membranes in pollen are sensitive to both hydration and temperature. Normally, lipids in the membranes are in a fluid or liquid-crystalline phase, but at either low moisture or low temperature, they form the more rigid gel phase. If rehydration occurs when the membrane lipids are locked in the gel phase due to low temperature exposure, they cannot reorganize and they become leaky and dysfunctional. On the other hand, if rehydration occurs when the membrane lipids are in the liquid-crystalline phase, membranes can reorganize successfully and become tolerant to a subsequent exposure to low temperatures.
One of the earliest works to report on the protective effects of ALA against abiotic stress factors dealt with low temperature stress. The pretreatment of rice seedlings at three-leaf stage by root soaking with ALA solution at 0.1-1 ppm concentration reduced the ratio of leaf rolling and tissue electrolyte leakage after cold treatment (Hotta et al. 1998). Seedlings pretreated with 1 ppm had 85% survival rate and 111.8 mg shoot dry weight per aerial part of seedling while the untreated plants had 65% survival rate and 65 mg dry weight 30 days after the cold treatment at 5°C for 5 days. It was also found that the protection obtained from ALA application was similar to that caused by brassinolide (BR) application, but differed from the ABA pretreatment in terms of leaf rolling or visual appearance of cold damage, since ABA protected younger leaves while ALA and BR were more effective on the protection of older leaves.
To investigate the effect of exogenous application of ALA on chilling tolerance of melon plants (Cucumis melo) , grown under low light conditions which mimics the typical growing conditions in the greenhouses in the northern hemisphere during the winter, melon seedlings at four-leaf stage were treated with 10 or 100 mg L-1 ALA after which they were exposed to chilling stress at 8°C for up to 6 h (Wang et al. 2004b). Although there were no significant differences between the control and ALA-treated plants after chilling treatment at 8°C for 2 h, the control plants were completely dehydrated and dead after chilling at 8°C for 6 h, whereas plants pretreated with 10 mg L-1 ALA only exhibited some injury symptoms in a few leaves. Moreover, after the plants had recovered from the stress for 20 h, photosynthesis of ALA-treated leaves had almost recovered to the comparable levels of the control plants before chilling, whereas the photosynthesis of non-ALA-treated plants was only 37-47% of the control plants, suggesting that chilling stress lasted for 4 h led to an irreversible damage on the photosynthetic apparatus. ALA treatment also caused significant increase in soluble sugar levels of melon leaves under chilling stress, which might be helpful for elevating the chilling tolerance of melon seedlings as an important osmotic solute. It was concluded that even though the protection obtained from ALA application was similar to that caused by ABA, it differed significantly from ABA application in such a way that ALA did not inhibit but rather improved plant photosynthesis and growth as well as chilling tolerance without any adverse effect.
In a latter work conducted to identify the optimum ALA application method and concentration, the chilling tolerance of pepper (Capsicum annuum L.) seedlings was significantly increased by exogenous application of ALA (Korkmaz et al. 2010) . Before exposing to chilling stress at 3°C for 2 days, pepper seedlings were treated with ALA in a range of 1-50 ppm through three different methods (seed treatment, foliar spray, and soil drench). ALA application was very effective in reducing visual injury symptoms of pepper seedlings after the plants had recovered from the stress for 3 days and among the application methods, foliar spray resulted in the least visual damage symptoms followed by the seed treatment (Fig. 12.2). ALA application increased leaf chlorophyll, sucrose, and proline contents and improved relative water content, stomatal conductance, and SOD enzyme activity while reducing membrane permeability. Even though all ALA application methods increased chilling tolerance
of pepper seedlings, seed treatment and foliar spray provided better protection compared to the soil drench while plants treated with 25 ppm ALA had the highest chilling tolerance compared to rest of the ALA concentrations. Similar results were reported in other studies in which additional evidence for mechanisms underlying the protective role of ALA in low concentrations against cold stress was provided. For example, higher antioxidant enzyme (e.g., SOD, CAT, and etc.) activities with increased ascorbic acid, proline, and soluble sugar content were also documented in cucumber (Cucumis sativus L.) seedlings pre-treated with 0.5 mg L-1 ALA before chilling stress at 5°C for 4 days compared to control plants (Yin et al. 2007). Treating soybean (Glycine max L.) seedlings with ALA in low concentrations (5-10 mM) prior to a cold stress at 4°C for 2 days resulted in elevated levels of tolerance to cold stress (Balestrasse et al. 2010). ALA pretreatment increased chlorophyll content, relative water content, and catalase and heme oxygenase-1 enzyme activities, and prevented membrane damage by reducing the thiobarbituric acid reactive species. The highest cold tolerance was obtained with 5 mM ALA pretreatment, while higher ALA concentrations (15-40 mM) resulted in a dose-dependent increase of membrane peroxidation.
Seed germination in chilling-sensitive species is slowed or reduced at temperatures below 20°C resulting in poor stand establishment and is usually prevented totally at temperatures lower than 15°C (Korkmaz et al. 2004; Korkmaz 2005). The problem is exacerbated as the length of time to emergence increases because the probability of soil crust formation becomes greater.
Delayed emergence also increases the chances of germinating seeds and seedlings to be infected by damping-off causing pathogens such as Fusarium and Pythium (Hendrix and Campbell 1973). Therefore, obtaining ideal plant stands requires fast and uniform emergence to avoid these problems. Presoaking the seeds of chilling-sensitive species with ALA could also be an effective way of improving germination or emergence performance under chilling conditions. When the pepper (Capsicum annuum L.) seeds were immersed in ALA solutions with varying concentrations for 24 h after which they subjected to emergence tests under chilling (15°C) and optimum (25°C) conditions, emergence was significantly enhanced by ALA treatment (Fig. 12.3a) . ALA pretreat-ment of seeds also enhanced seedling shoot fresh weight (Fig. 12.3b) and chlorophyll a content (Fig. 12.3c). Seedlings raised from seeds treated with 50 ppm ALA had significantly lower levels of H2O2 (Fig. 12.4a) and MDA contents (Fig. 12.4b) and elevated SOD enzyme activity (Fig. 12.4c) in the leaves. Improvement of pepper seedling emergence performance under chilling stress conditions may have resulted from reduced lipid peroxidation and elevated SOD enzyme activity, all of which is an indication of membrane protection. The efficacy of seed treatment with ALA was also reported to last when seeds were stored after the treatment. For example, priming seeds in 25 or 50 ppm ALA incorporated into the KNO3 solution improved low temperature performance of red pepper seeds even after the pretreated seeds were stored for 1 month at 4°C or 25°C (Korkmaz and Korkmaz 2009).
2.3.2 Effects of Exogenous ALA
on Plants Under Salinity Stress
Soil salinity presents a major limitation to agricultural production since it restrains crop yield and restricts use of land previously uncultivated. Salinity affects almost 20% of all irrigated land and 2.1% of dry-land agriculture worldwide (FAO 2005). Each year there is a deterioration of 2 million ha (about 1%) of world agricultural lands to salinity, leading to reduced or no crop productivity (Szabolcs 1994). In addition to natural causes such as salty raining waters near and around the coasts and weathering of native rocks, low precipitation, high surface evaporation and poor growing practices have also aggravated growing concentration of salts in the rhizosphere (Mahajan and Tuteja 2005). Secondary saliniza-tion, in particular, worsens the situation where once productive agricultural lands are becoming unsuitable to cultivation due to poor quality irrigation water (Ashraf and Foolad 2007) .
Salts in the soil water may be detrimental to plant growth for two reasons. First, the presence of salt in the soil solution decreases the ability of the plant to take up water and nutrients causing significant reductions in plant growth rate. This is referred to as the osmotic or water-deficit effect of salinity. The salts in the soil solution reduce shoot growth more than root growth, and decrease stomatal conductance and thereby photosynthesis (Munns 1993) . The rate at which new leaves are produced depends heavily on the water potential of the soil solution around the roots. Second, if excessive amounts of salt are taken up by the plant via the transpiration stream, there will be injury to cells or tissues in the transpiring leaves and this may cause further reductions in growth. This is called the salt-specific or ion-excess effect of salinity (Greenway and Munns 1980). The definition of salt tolerance is usually the percent biomass production in saline soil relative to plants in nonsaline soil, after growth for an extended period of time. For slow-growing, perennial, or uncultivated species it is often difficult to determine the reduction in biomass production, so percent survival is generally used (Munns 2009).
It has been shown that ALA at low concentrations (10-100 mg L-1) has the potential to improve salinity tolerance in cotton seedlings through foliar application (Watanabe et al. 2000). Cotton seedlings treated with ALA survived in the soil containing 1.5% NaCl while untreated seedlings died. The ALA treatment significantly counteracted the negative effects of salinity and ALA pretreated seedlings weighed as much as those that were not exposed to salinity stress. The analysis of mineral composition of seedlings revealed that Na+ concentrations in the roots of plants treated with ALA were significantly lower compared to control plants, and it was presumed that
Fig. 12.3 Effect of presowing seed treatment with ALA with various concentrations of ALA for 1 day before sow-
on pepper seedling emergence percentage (a), seedling ing after which they were subjected to emergence test at shoot fresh weight (b), and Chl a content (c). Vertical bars 15°C (chilling stress) or 25°C (optimum conditions). Dry represent mean ± SE (n = 8). Pepper seeds were treated seeds are the untreated seeds
Fig. 12.3 Effect of presowing seed treatment with ALA with various concentrations of ALA for 1 day before sow-
on pepper seedling emergence percentage (a), seedling ing after which they were subjected to emergence test at shoot fresh weight (b), and Chl a content (c). Vertical bars 15°C (chilling stress) or 25°C (optimum conditions). Dry represent mean ± SE (n = 8). Pepper seeds were treated seeds are the untreated seeds the presence of ALA may cause a reduction of Na+ uptake and may suppress water deficiency caused by osmotic stress resulting from high Na+ concentration around the roots in growth media. Consistent with the results of Watanabe et al.
(2000) in cotton seedlings, foliar application of ALA at the rate of 30 mg L-1 to the oilseed rape (Brassica napus L.) seedlings grown under high salt stress (up to 200 mM NaCl) significantly reduced the accumulation of Na+ and K+, leading
Fig. 12.4 Effect of presowing seed treatment with ALA on H.O2 content (a), MDA concentration (b), and SOD enzyme activity (c) of pepper seedlings. Vertical bars represent mean ± SE (n = 8). Pepper seeds were treated with various concentrations of ALA for 1 day before sowing after which they were subjected to emergence test at 15°C (chilling stress) or 25°C (optimum conditions). Dry seeds are the untreated seeds
Fig. 12.4 Effect of presowing seed treatment with ALA on H.O2 content (a), MDA concentration (b), and SOD enzyme activity (c) of pepper seedlings. Vertical bars represent mean ± SE (n = 8). Pepper seeds were treated with various concentrations of ALA for 1 day before sowing after which they were subjected to emergence test at 15°C (chilling stress) or 25°C (optimum conditions). Dry seeds are the untreated seeds to a reduction of Na+/K+ ratio both in roots and leaves compared to control plants (Naeem et al. 2010). It was also reported that higher Na+ accumulation in the leaves compared to roots suggests that ALA does not influence Na+ transport from the roots to the shoots but it might rather suppress the uptake of Na+ from the growth media to the roots. Reduced Na+ accumulation in the leaves and reduced K+ uptake by the roots which led to a concomitant reduction in K+/Na+ ratio were reported when date palm (Phoenix dactylifera L.) seedlings exposed to 30 mS cm-1 salinity stress were treated with ALA-based fertilizer "Pentakeep-v" (Youssef and Awad 2008).
Addition of ALA at low concentrations (0.3-3 mg L^) to the culture media promoted development and growth of potato (Solanum tuberosum L.) microtubers in vitro by increasing average number, diameter, and fresh weight of microtubers under 0.5% NaCl stress conditions (Zhang et al. 2006), but further increase in ALA concentration caused significant reduction in microtuber yield under salt stress. The microtu-bers treated with low concentrations of ALA exhibited 73% more peroxidase and 28% more polyphenoloxidase activity compared to the untreated control plants which implied that ALA functions as a protectant against oxidative damages of membranes. ALA concentrations of 30 mg L- 1 or higher induced oxidative damage probably via formation and accumulation of pho-tooxidative porphyrins compounds; therefore, it is important to determine optimal application rates and timing of ALA for growth promotion of crops. The authors concluded that it was important to clarify optimal rates for the promotion of crop growth; optimal rates might be slightly higher for crops exposed to stressful conditions and the safety margin of application rates of ALA would rather wide since ALA is rapidly metabolized in both plants and their surrounding environment (Zhang et al. 2006) .
To investigate the role of exogenously applied ALA in spinach (Spinacia oleracea) plants grown under salinity stress, seedlings exposed to two levels of salt stress (50 and 100 mM NaCl) were treated with ALA with varying concentrations (0, 0.18, 0.60, and 1.80 mM) (Nishihara et al. 2003). Plants treated with 0.6 and 1.80 mM ALA showed marked increases in photosynthetic rate under both salinity stress levels, while photosynthesis continued to decline in control (0 mM ALA) plants. With regard to antioxidant enzyme activities in the leaves, catalase, ascorbate per-oxidase, and glutathione reductase activities were enhanced significantly 3 days after ALA treatment at the rate of 0.60 and 1.80 mM. Other results also indicate that foliar application of
30 mg L-1 ALA to oilseed rape plants exposed to salinity stress improved the growth of shoots and roots and increased leaf chlorophyll content and net photosynthetic rate (Naeem et al. 2010, 2011). ALA-treated plants grown under 100 mM NaCl also maintained similar levels of leaf water potential as that of plants grown under optimal conditions. ALA treatment also triggered accumulation of osmolytes such as soluble sugars, free amino acids, and proline in the leaves of salt-stressed plants as well as enzymatic (APX, CAT, and SOD) and nonenzymatic (glutathione and ascor-bate) antioxidants activity while decreasing membrane permeability, MDA content and ROS production. On the contrary, no enhancement in chlorophyll content of rice (Oryza sativa L.) seedlings under salt stress treated with ALA was observed, suggesting that the growth recovery was not due to the increased chlorophyll content but rather due to reduced lipid peroxidation caused by increased antioxidant enzyme activity (Wongkantrakorn et al. 2009) . Augmented activities of antioxidant enzymes such as SOD, APX, CAT, and GR after ALA treatment were also reported in other species grown under various conditions such as Gingko biloba seedlings grown under optimal conditions (Xu et al. 2009), oilseed rape plants under herbicide toxicity stress (Zhang et al. 2008). and watermelon seedlings exposed to low light conditions (Sun et al. 2009).
The ability of barley plants to synthesize ALA was reported to increase in response to increasing salt concentrations and maximum amount of ALA accumulated in plants grown at 100 mM NaCl was two fold higher than in control plants grown under optimum (0 mM NaCl) conditions (Averina et al. 2010). When salt concentration was further increased to 200 mM, the rate of ALA accumulation was decreased and the reduced ability to synthesize ALA was accompanied by an increase in proline content. Thus, the impairment in ALA-synthesizing ability was reported to redirect metabolic conversion of glutamic acid from chlorophyll synthesis to the proline synthesis pathway, which would stimulate proline biosynthesis and enhance salt tolerance.
The efficacy of ALA-based fertilizer "Pentakeep-v" applied at the rate of 0.08% in inducing tolerance to salinity stress was also tested in date palm (Phoenix dactylifera L.) seedlings (Youssef and Awad 2008). Application of Pentakeep-v significantly improved chlorophyll a content of plants, leading to improved total chlorophyll content and chlorophyll a/b ratios. Pentakeep-v also enhanced the biochemical efficiency of carbon fixation of Rubisco enzyme and the rate of electron transport required for RuBP regeneration over untreated plants exposed to salinity stress at 15 mS cm-1. In addition, Pentakeep-v reduced the percentage contribution of stomatal factor (gas phase limitation) to the apparent reduction in photosynthetic gas exchange to values similar to those of control plants and lowered CO2 compensation points by reducing respiratory CO2 loss with increasing salinity to the 30 mS cm-1. It was concluded that ALA-based fertilizer improved salt tolerance of date palm seedlings by increasing photosynthetic assimilation via boosting light-harvesting capabilities of the treated plants by enhancing chlorophyll a content and by reducing stomatal limitation to photosyn-thetic gas exchange (Youssef and Awad 2008).
The soaking of pakchoi (Brassica campestris ssp. chinensis var. communis Tsenet Lee) seeds in ALA solution before sowing reduced the damaging effects of salinity during seed germination (Wang et al. 2005). Treating the seeds with ALA, at concentrations ranging from 0.01 to 10 mg L-1, promoted seed germination when seeds were stressed by 150 mM NaCl. However, levulinic acid, an inhibitor of ALA dehydrase, significantly prevented seed germination and seedling growth, suggesting that ALA was necessary for seed germination, and that the effect of ALA is dependent upon its conversion into porphyrin. ALA pretreat-ment of seeds also caused significant increases in respiration rates during seed germination under salt stress compared to untreated seeds which maybe further supported by the fact that salttolerant pakchoi cultivars contained higher endogenous ALA and heme under salt stress conditions compared to salt-sensitive cultivars. Additionally, promotive effects of exogenous application of ALA were also reported in watermelon (Citrullus lanatus) seed germination under salt stress (Liu et al. 2006). Treatment of watermelon seeds with
15-30 mg L-1 ALA improved seed germination and seedling growth under 125 mM NaCl stress, and the promotion of ALA treatment on germination under salt stress might be associated with the enhanced activities of antioxidant enzymes, especially POD and decreased activity of lipoxyge-nase and lowered levels of MDA in hypocotyls and radicles.
2.3.3 Effects of Exogenous ALA
on Plants Under Water Stress
Plants are exposed to a variety of environmental stresses including extreme temperatures, unfavorable chemical and physical soil conditions, and various pests and diseases. However, in the long term, water deficit affects negatively the growth and yield of crop plants more than all the other stresses combined, because it is ubiquitous. More than one-third of the earth's surface is classified as arid or semiarid because it is subjected to permanent drought. Equally important is the fact that most of the humid temperate regions, where most of the world's agricultural production takes place, are frequently subjected to periods of severe drought. Water deficit or water stress refers to situations where plant water potential and turgor are reduced enough to interfere with normal growth of plants (Kramer 1983). The exact cell water potential at which this takes place is dependent upon the crop species, the stage of development, and the process under consideration. For example, cell division and enlargement usually stops at a water potential of only -0.2 to -0.4 MPa, whereas stomatal closure does not begin until the water potential falls below -0.8 to -1.0 MPa. The first and the most obvious symptom of water deficiency is wilting due to reduced turgor, causing significant retardation of growth processes especially lengthwise growth. Photosynthesis is also reduced for a number of reasons including reduced CO2 uptake due to stomatal closure, damaged cytoplasmic ultrastructure and impaired enzyme activity, reduced canopy absorption of photosynthetically active radiation, and decreased radiation-use efficiency (Janda et al. 2007; Farooq et al. 2009). Another outcome of water stress is the disrupted nutrient uptake due to impaired root growth and reduction in water migration which results in a significant decrease in the quantity of ions transported by water.
Even though considerable amount of data has been accumulated with regard to the roles of ALA on enhancing the tolerance to chilling and salinity stresses during the last two decades, very little effort has been put in to investigate the role of ALA in plants under water stress. Among the very few, first study to demonstrate the effect of ALA on drought tolerance showed that spraying the barley (Hordeum vulgare L.) plants from tillering to milk-ripe stage with ALA enhanced tolerance to water stress (Al-Khateeb 2006) . ALA treatment promoted the yield of barley plants under optimal conditions (weekly irrigation) causing as much as 45% increase in the grain yield and 27% in straw yield. Irrigation frequency of 21 days significantly reduced grain and straw yields per hectare while ALA application at the rate of 100 ppm increased the grain and straw yields by 35 and 41%, respectively, compared to untreated plants. The increase in yield under severe water stress (3 weeks irrigation interval) in response to ALA treatment was associated with the fact that ALA treatment enhanced photosynthesis, stomatal conductance, and intercellular CO2 concentration by 41, 29 and 50%, respectively. Confirming results were also reported in wheat (Triticum aestivum L.) that spraying the plants with ALA solutions enhanced the tolerance to water stress (Al-Thabet 2006) . Wheat plants that were irrigated every 2 weeks and foliar sprayed with ALA at the rate of 50-100 ppm ha) 1 out yielded and surpassed in water use efficiency compared with untreated plants grown under normal conditions (irrigated every week). Moreover, plants treated with 100 ppm ha-1 ALA yielded more than untreated plants when irrigated every 3 weeks although yield was considerably lower than that of plants irrigated every week.
To assess the physiological and biochemical changes within the plant caused by ALA application on plants, water-stressed oilseed rape seedlings were fed with ALA containing solutions (Liu et al. 2011). ALA treatment at low concentrations (1 mg L-1) significantly improved plant biomass and chlorophyll content, but reduced
MDA content and ROS production. On the contrary, ALA applied at moderately high concentrations (10 mg L- ') hampered plant growth while higher concentrations (100 mg L-1) killed the plants. Application of ALA at low concentrations also enhanced reduced/oxidized glutathione and ascorbic acid ratios while boosting the activity of antioxidant enzymes such as APX, CAT, GR, and POD by inducing the expression of the specific antioxidant genes.
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