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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 titretable 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 titretable 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 (Raphanussativus) 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, Tanaka and Hotta 1995).
Plants themselves can synthesize phytohormones, 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 Rhodopseudomonaspalustris and Rhodobactersphaerides 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 hours 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 inoculating soil and straw products with different strains of Rhodopseudomonaspalustris 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, Koh and Song 2008), increased mushroom (Agariscusbisporus) yield (Han 1999) and controlled the root rot on rice seedlings (Kobayashi and Kobayashi 2000).
Iwai, Takeuchi and Kuramochi (2003) found that response to exogenously-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 (Smolen 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 callus-like 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 photoautotrophically growing cells of Spirulinaplatensis causing intracellular accumulations of phycocyanin and chlorophyll followed by enhancement of the photosynthetic activities of photosystems I and II (Sasaki et al. 1995). In vitro studies with Vignaunguiculata L. confirmed the hormonal role of ALA by striking proliferation of callus and paripassu 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 callusing and rooting and shooting, respectively (Bindu Roy and Vivekanandan 1998b). Also, ALA-based fertilizer 'Pentakeep' applied at the rate of 0.04 to 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-1increased 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 oC) (Bunke at al. 2000; Gadmar et al. 2002). It was also suggested that the final solution of pH 5.5-7.4 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 oC 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). Therefore, the definition of chilling stress would be the number of degrees that the environmental temperature is below optimum for the plant activity being measured (e.g., growth). 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 oC for temperate fruits, 8 oC for subtropical fruits, and about 12 oC for tropical fruits such as banana (Lyons 1973). Amongst the highest volume world food crops, maize (Zea mays), cotton (Gossypiumhirsatum) 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 oC resulting in yield loss and crop failure.
Sudden exposure to low temperatures especially to temperatures around or below 0 oC may result in extensive and irreversible damages on plant tissues since it causes the membranes to lose their semi permeability 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, Hoekstra and Crowe 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 pre-treatment of rice seedlings at 3-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 pre-treated with 1 ppm had 85% survival rate and 111.8 mg dry weight per aerial part of seedling while the untreated plants had 65% survival rate and 65 mg dry weight, thirty days after the cold treatment at 5 oC 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 pre-treatment 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 (Cucumismelo), grown under low light conditions which mimics the typical growing conditions in the greenhouses in the northern hemisphere during the winter, melon seedlings at 4-leaf stage were treated with 10 or 100 mg L-1 ALA after which they were exposed to chilling stress at 8 oC for up 6 hours (Wang et al. 2004b). Although there were no significant differences between the control and ALA-treated plants after chilling treatment at 8 oC for 2 hours, the control plants were completely dehydrated and dead after chilling at 8 oC for 6 hours, 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 hours, 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 hours 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, Korkmaz and Demirkiran 2010). Before exposing to chilling stress at 3 oC for 2 days, pepper seedlings were treated with ALA in a range of 1 to 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 2.2). 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 (Cucumissativus L.) seedlings pre-treated with 0.5 mg L-1 ALA before chilling stress at 5 oC for 4 days compared to control plants (Yin et al. 2007). Treating soybean (Glycine max L.) seedlings with ALA in low concentrations (5-10 µM) prior to a cold stress at 4 oC for 2 days resulted in elevated levels of tolerance to cold stress (Balestrasse et al. 2010). ALA pre-treatment increased chlorophyll content, relative water content and catalase and heme oxyganase-1 enzyme activities and prevented membrane damage by reducing the thiobarbituric acid reactive species. The highest cold tolerance was obtained with 5 µM ALA pretreatment, while higher ALA concentrations (15-40 µM) resulted in about 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 oC (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. Pre-soaking 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 hours after which they subjected to emergence tests under chilling (15 oC) and optimum (25 oC) conditions, emergence was significantly enhanced by ALA treatment (Fig 2.3a). ALA pretreatment of seeds also enhanced seedling shoot fresh weight (Fig 2.3b) and chlorophyll a content (Fig 2.3c). Seedlings raised from seeds treated with 50 ppm ALA had significantly lower levels of H2O2 (Fig 2.4a) and MDA contents (Fig 2.4b) and elevated SOD enzyme activity (Fig. 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 ppm 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 one month at 4 °C or 25 oC (Korkmaz and Korkmaz 2009).