Regulation of 5-Aminolevunilic Acid and Its Application in Agroforestry

Abstract

The review briefly introduces the natural occurrence, physicochemical properties, and biosynthesis of 5-aminolevuinic acid (ALA) and highlights a variety of applications in the planting industry and its possible mechanisms. It has been known that ALA can be used as biological pesticides, fungicides, and herbicides when the concentrations are higher than 838 mg L⁻¹ (about 5 mmol L⁻¹). When ALA concentrations are 100–300 mg L⁻¹, it can be used to thin surplus flowers in the spring of orchards and promote fruit coloration before maturation. When the concentrations are lower than 100 mg L⁻¹, especially not higher than 10 mg L⁻¹, ALA can be used as a new plant growth regulator to promote seed germination, plant (including root and shoot) growth, enhance stress tolerance, increase crop yield, and improve product quality. In photosynthesis, ALA is involved in the regulation of the whole process. In stress tolerance, ALA induces plant preventive and protective systems through the NO/H₂O₂ signaling network. In secondary metabolism, ALA regulates many gene expressions encoding transcription factors or function proteins to promote anthocyanin and flavonol biosynthesis and accumulation. In general, ALA promotes plant health and robustness, reduces the use of chemical fertilizers and pesticides—which is conducive to improving the ecological environment, human production, and living conditions—and has a broad application prospect in agroforestry production. As a new plant growth regulator with multiple and powerful functions, the underlying regulatory mechanisms need more study.

1. Introduction

5-Aminolevulinic acid (ALA) is a natural compound that occurs in all living organisms. It has long been known to be the essential biosynthetic precursor of tetrapyrroles, such as chlorophylls, heme, vitamin B12, and phytochrome chromophore [1]. Therefore, it should be involved in photosynthesis, respiration, and many other physiological processes [2,3,4]. In recent decades, more and more studies have revealed that ALA is not only a common biochemical metabolite but can also be used in human pharmacology [5], animal nutrition [6], and plant production [2,3]. In 2003, Wang et al. [7] proposed the potential application of ALA in agriculture as a new plant regulator. In 2009, Jahn and Heinz induced progress in ALA biosynthesis [8], while Tanaka et al. [9] summarized the advance of tetrapyrrole metabolism in Arabidopsis thaliana. Then, Akram and Ashraf [10] assessed the regulation of ALA in promoting stress tolerance in plants, while Wu et al. [11] reviewed ALA biosynthetic and metabolic pathways and its role in higher plants. Recently, Tan et al. [12] estimated advances in ALA priming to enhance plant tolerance to abiotic stress. All these review papers presented the characteristics of ALA, its physiological regulatory functions, and its different applications in plant growth and development, which are of great significance for understanding the applications of ALA in planting production. However, ALA has become a hot topic in modern agroforestry practice, and dozens of research works are published annually. Based on the previous reports and the recent achievements in our lab, we present a review here to briefly introduce the basic characteristics, biosynthesis, and bioproduction of ALA and highlight its application as an insecticide, fungicide, herbicide at high concentrations, and plant growth regulator at low concentrations. We suggest that ALA can not only promote plant growth but also improve plant resistance to biotic and abiotic stresses, enhance crop yield, and modify product quality. Additionally, we introduce the possible regulatory mechanisms of ALA in improving plant photosynthesis, salt tolerance, drought tolerance, and apple coloring. We hope it may be helpful for further study in the field and ALA application in modern green agroforestry.

2. Natural ALA Occurrence

ALA is an oxygen- and nitrogen-containing hydrocarbon with the formula C₅O₃NH₉ and a molecular weight of 131.2. ALA is a δ-amino acid, possessing an amino group and a carboxyl group, which cannot participate in the biosynthesis of proteins. Thus, it is often called as non-protein amino acid. Conversely, ALA is an essential precursor for the biosynthesis of all porphyrin compounds, where chlorophylls are necessary for photosynthesis, while heme is an essential prosthetic group of cytochromes for the electron transport in many redox reactions, including photosynthetic and oxidative phosphorylation, as well as many enzymatic reactions [1]. Heme also plays a key regulatory role in the expression of several genes by acting as a ligand for transcriptional factors [13]. In higher plants, mosses, some algae, and bacteria (such as Escherichia coli and Salmonella arizona), ALA is biosynthesized in the stroma of plastids (including chloroplasts, leukoplasts, and chromoplasts), and then converted into chlorophyll in the thylakoid grana or heme in the stroma (Figure 1) [9]. In animals, fungi, and some bacteria (such as Bradyrhizobium japonicum), ALA biosynthesis occurs in the inner mitochondria, which is exported to the cytosol by a threonine/homoserine transporter, RhtA, and converted into coproporphyrinogen III (CPG III). The latter returns to the mitochondria and is converted into different porphyrin compounds, including heme [14].

3. Physicochemical Properties and Toxicity of ALA

The pure product of ALA is a white, odorless powder crystal with a melting point of 155–157 °C and a packing density of 0.565–0.689 g cm⁻³. The solubility in 100 g of water at 20 °C is 151.1 g, which is easy moisture in air. It is easy to decompose when exposed to light, alkalinity, or strong oxidants. Thus, the compound should be stored at a low temperature and in darkness. An oxygen-free environment is also suggested for preservation.

The ALA commodity is mostly present in the form of hydrochloride, a strong acid whose Chemical Abstracts Service (CAS) number is 5451-09-2. Therefore, the molecular weight of the commercial ALA used in regular experiments or agroforestry production is 167.59. It is an irritant to the human skin, eyes, digestive and respiratory tracts, etc. If the skin is accidentally contaminated, a timely rinse with tap water (more than 15 min) to avoid dermatitis is necessary. Contamination with ALA on food, shoes, or socks should be fully cleaned and exposed to sunlight to promote light decomposition. If the eyes inadvertently contact ALA, one should break open the eyelids with hands and rinse with running water for more than 15 min. If one accidentally swallows something in the mouth, it is necessary to rinse with clean water. If inadvertently inhaled into the respiratory tract, one should immediately transfer to fresh air conditions.

The toxicity of ALA has not been reported in detail. The data on ALA’s teratogenesis, mutagenesis, and toxicity to the ecological environment are hitherto difficult to query. Yet, since it is a natural substance, many indicators are exempt from inspection. Our unpublished study showed that acute oral rats with a dosage of 5 g kg⁻¹ body weight of ALA did not show any other discomfort except for mild inflammation on the first day, which recovered on the next day. In the other rat toxicity tests, including acute transcutaneous, acute transoral, and acute inhalation, the safe dosages of ALA were all higher than 5 g kg⁻¹. In environmental biological tests, our data showed that the half-lethal concentrations (LC₅₀) of ALA were 1.63 g L⁻¹ for silkworms, >2.04 g L⁻¹ for honey bees, >102 mg L⁻¹ for fish, >72.5 mg L⁻¹ for algal growth inhibition, and >42.3 mg L⁻¹ for adventitia activity, respectively. All these suggest that ALA is a naturally biodegradable and environmentally friendly biochemical amino acid whose toxicity both to humans and the environment is so low that it can be ignored and safely used in agroforestry production.

4. ALA Biosynthesis and Biological Production

4.1. The Pathways of ALA Biosynthesis

All organisms can synthesize ALA, but it is rapidly catabolized in living organisms with very limited remains. More ALA can be biosynthesized and accumulated only in some specific genetic engineering organisms, especially microbes, under optimized fermentation conditions, which offers the possibility of ALA bioproduction.

ALA is synthesized by two different pathways in different organisms, which are called the C4 and the C5 pathways, respectively (Figure 1). In mammals, birds, yeast, fungi, protozoa, and some non-sulfur photosynthetic bacteria such as Rhodobacter sphaeroidescoccus, Pseudomonas swamp, and Bradyrhizobium japonicum, ALA synthesis is through the C4 route, dependent on the catalytic reaction of ALA synthase (ALAS). ALAS, encoded by HemA, condenses succinyl CoA and glycine into ALA in the presence of pyridoxal phosphate. On yeast, HemA consists of 1647 nucleotides, encoding a polypeptide containing 549 amino acids. Due to the comparative simplicity of microbial ALA synthesis, many genetic engineering studies are based on the C4 pathway for HemA gene transformation. In higher plants, moss, algae, and a few bacteria (such as E. coli and Corynebacterium glutamicum), ALA biosynthesis is through the C5 route, which is transformed from glutamate catalyzed by glutamate tRNA^Glu synthase (GluRS), glutamyl tRNA^Glu reductase (GluTR), and glutamate-1-semialdehyde-2, 1-aminotransferase (GSA). Among them, GluRS is not a specific enzyme for ALA synthesis. Instead, it is necessary for protein synthesis from glutamine. In fact, GluTR is the first enzyme necessary for ALA synthesis, whose activity is inhibited by negative feedback from metabolites such as heme and regulated by light to control ALA synthesis. In Arabidopsis, a GluTR gene called HemA1 is controlled by a photosensitive promoter, which drives gene expression only under light conditions [15]. Therefore, ALA biosynthesis occurs during the day and stops at night with a circadian rhythm. GSA, encoded by HemL and also regulated by light, is another key enzyme in ALA synthesis. These two genes are the main targets for plant genetic engineering to regulate ALA biosynthesis [7].

4.2. Biological Production of ALA

In the early days, ALA products were chemically synthesized. It is not only expensive but also contains a variety of difficult-to-determine impurities that may cause potential harm to agroforestry production. Therefore, the biological production of ALA is receiving more and more attention. Methanogens such as Methanosrcina [16] and photosynthetic bacteria such as Rhodobacter [17] were first selected for ALA bioproduction, whereas E. coli and C. glutamicum have been most often used recently [18,19].

Both E. coli and C. glutamicum biosynthesize ALA through the C5 pathway, which requires the key enzymes GluRT and GSA as well as the encoding genes HemA and HemL. Yet, the biosynthetic precursors of the C4 pathway, including succinyl-CoA and glycine, are naturally present in the bacteria. What they lack is the gene and ALAS. The transformation of the genes either in the C4 or C5 pathway can promote ALA synthesis in the cells. van der Werf and Zeikus [20] first transformed the HemA of R. sphaeroides (a C4 route species) into E. coli DH1 to cause the C4 and C5 routes to work simultaneously, resulting in an extracellular ALA accumulation of 3.3 g L⁻¹, much higher than methanogens or photosynthetic bacteria. Up until now, many strategies have been established to improve ALA yield. The first is to overexpress HemA in the C4 pathway [20]. It can also be achieved by the transformation of HemA or HemL in S. arizona [21]. To avoid injury from reactive oxygen species (ROS) induced by high ALA concentrations accumulated in the bacterial cells, a RhtA gene, encoding a threonine/homoserine exporter, was overexpressed in C. glutamicum to export the intracellular ALA to release the growth inhibition of cells for more ALA production. Secondly, ALA catabolism should be reasonably weakened to accumulate more ALA. The condensation of ALA into porphobilinogen is catalyzed by the dehydrase encoded by HemB. Either the addition of the enzyme inhibitor levulinic acid (LA) or downregulation of HemB expression can promote ALA accumulation in cells [22]. The third strategy is to modify the genes involved in the biosynthesis of ALA precursors, such as glutamate, succinyl-CoA, glycine, and pyridoxal phosphate [23]. Knocking out NCgl1221, LysE, and PutP (encoding the efflux proteins of glutamate, arginine, and proline, respectively) promoted glutamine accumulation [24]. Mutating OdhI decreased the α-ketoglutarate dehydrogenase activity, leading to more carbon flow from the tricarboxylic acid cycle (TCA) into glutamate synthesis [25]. All the gene modifications cause more ALA accumulation in C. glutamicum cells. Additionally, the addition of substates, the modification of carbon or nitrogen metabolic flux [26,27,28,29], the release of ALAS inhibition by heme [30], and the optimization of fermentation conditions such as temperature or light all facilitate bacterial growth and cheap ALA bioproduction [31]. Recently, synthetic sRNA-based repression was used to systematically regulate ALA biosynthesis, metabolism, and extra flux, and ALAS was rationally engineered to release the inhibition of heme and improve the catalytic activity. ALA export and antioxidant defense systems were targeted to enhance cellular tolerance to intra- and extra-cellular ALA. Consequently, the engineered strain produced 30.70 g L⁻¹ of ALA in bioreactors with a productivity of 1.02 g L⁻¹ h⁻¹ and a yield of 0.53 mol mol⁻¹ glucose [19]. Due to hard work, the mass production of biological ALA has become a realistic and feasible technology.

5. Multiple Applications of ALA in Agroforestry

5.1. As an Herbicide or a Toxicide of Herbicides

Rebeiz et al. [32] first reported the weeding function of ALA. When cucumber seedlings were treated with a 15–20 mM ALA solution at dark and overnight, then exposed to high light, the abnormally accumulated tetrapyrrole intermediates at dark induced a strong photodynamic reaction under light, stimulating a great amount of singlet oxygen to kill the seedlings. It is well known that ROS attacks the cellular membrane and oxidizes the unsaturated fatty acids, resulting in rapid and severe dehydration, bleaching, and collapse of the plant tissues. Therefore, ALA is a so-called photodynamic herbicide, or tetrapyrrole-dependent photodynamic herbicide (TDPH), deregulating normal tetrapyrrole metabolism to injury plant cells. 2,2′-Dipyridine can enhance the photosensitivity effect of ALA and reduce the effective concentrations. Fe²⁺ chelator phenanthroline also promotes herbicide activity [33]. Therefore, the compounds can be used to improve the herbicide activity of ALA. Furthermore, different species are sensitive to the TDPH. It was reported that alfalfa was the most tolerant, while Chinese cabbage was the most sensitive to ALA treatment [34]. Dicots are more sensitive to ALA than monocots [1]. Then, ALA can selectively kill dicot weeds in wheat, corn, or other grass fields. However, the concentrations needed for ALA to act as an herbicide are still very high (≥5 mmol L⁻¹). ALA in such high concentrations often injures rice leaves and deactivates physiological functions [35,36]. Therefore, whether ALA can be used in agroforestry as a photooxidative herbicide needs more study.

On the other hand, ALA, at low concentrations, can act as a toxicide in chemical herbicides. For example, 500 mg L⁻¹ ALA promoted the herbicidal activity of ZJ0273, a new herbicide, propyl 4-9(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate on Brassica napus seedlings. However, when 10–100 mg L⁻¹ ALA was combined, the herbicidal injury of ZJ0273 on the plants was significantly attenuated [37]. Either pre- or post-treatment with low concentrations of ALA improved the growth of Malachium aquaticum L. seedlings. However, when the concentration of ALA was 100 mg L⁻¹, it aggravated the herbicidal effect of ZJ0273 on plants [38]. Similar results were reported for canola [39]. Therefore, ALA may be used as a toxicide of chemical herbicides at low concentrations but as an herbicide at high concentrations. It was observed that when ALA at 6 mmol L⁻¹ was applied to radish, the leaves were partially damaged, which was attributed to the herbicidal properties [2]. Therefore, ALA cannot be used at too high dosages in agroforestry production unless it is intended to be used as an herbicide.

The mechanism for ALA’s use as an herbicide is dependent on their conversion into tetrapyrrole and its photooxidative properties. Conversely, its use as a toxicide in chemical herbicides may be dependent on the H₂O₂ signal. Methyl viologen (MV, i.e., paraquat) is a strong photooxidant, while 100 mg L⁻¹ ALA can alleviate the leaf injury of pear (Pyrus ussuriensis) from MV [40]. When the PuRbohF gene, responsible for H₂O₂ generation, was transformed into tobacco, the PuRbohF-overexpressing leaves became more tolerant to MV. Thus, high dosages of ALA induce a great amount of ROS to kill plants, but low dosages can induce ROS generation to act as a toxicide of herbicides mediated by Rboh-derived H₂O₂ signaling, which activates antioxidant enzymes to prevent stress injury.

5.2. As an Insecticide or a Toxicide of Insecticides

It was also Rebeiz et al. [41] who proposed that ALA caused the biochemical and metabolic imbalance of insects in Lepidoptera and Diptera, such as trichoplusia ni, fruit flies, and cockroaches, leading to spasms and even death. In Chinese rice locusts (Oxya chinensis), when ALA solution was applied on the back in the dark for 7–10 h, and then switched to natural light conditions, many locusts were killed [42]. The killing activity of ALA was ascribed to inducing PP IX accumulation, like the weeding effect of ALA. When injected into the locust bodies, 750–1000 mmol L⁻¹ ALA killed insects in 48 h because of the reduction of acetylcholinesterase and glutathione-peroxidase activities [43]. However, the application of the locust nymphs with 10–1000 mg L⁻¹ ALA solutions through saturated filter paper showed that ALA in a wide concentration range induced a photodynamic reaction and killed the insects without significant dose-dependent toxic effects [44]. Additionally, ALA can effectively control many species of plant-parasitic nematodes, such as Caenorhabditis elegans, Heterocleru glycines, Bursaphelenchus xylophilus, and Meloidogyne incognite, among which the effects of ALA on inhibiting the infection of the root-knot nematode M. incognite in tomato or cucumber were much better than avernectin-triazophos [45].

Although there are very few reports, ALA can alleviate the harmful effect of deltamethrin on Phaseolus vulgaris seedlings [46]. It was observed that 0.5 mg L⁻¹ deltamethrin induced DNA hypermethylation and DNA damage in P. vulgaris, while 20 mg L⁻¹ ALA alleviated the adverse effects of the insecticide. It seems that ALA has the potential to be an antigenotoxic agent against chemicals, which provides a new insight into finding alternatives to reduce genetic damage in plants under chemical stresses. Nevertheless, the mechanism needs more attention.

5.3. Bidirectional Regulation on Probiotics and Pathogens

ALA induces porphyrin synthesis and photodynamically inactivates E. coli; therefore, it can be used as a food surface disinfectant [47]. Soaking seeds with 6 mmol L⁻¹ ALA for 4 h significantly inhibited the fungal proliferation on the surface of wheat seeds, acting as a fungistatic agent [48]. These fungi included Mucor spp., Penicillium spp., Rhizopus oryzae, Fusarium avenaceum, Aspergillus flavus, Trichothecium roseum, Acremonium spp., Alternaria spp., and Mycelia sterilia. Therefore, ALA can be used to promote seed germination and healthy seedling growth. Recently, we found that ALA inhibited the growth of pathogenic fungi, including Fusarium oxysporum f. sp. fragaria (Fof), Botrytis cinerea, and Aspergillus fumigatus, and promoted the growth of probiotic Trichoderma harzianum in culture media. Furthermore, ALA enhanced the antagonism of T. harzianum against Fof and B. cinerea in confrontation culture. When the fungi were inoculated to sound strawberry plants with or without exogenous ALA, the results showed that Fof and B. cinerea induced strawberry Fusarium wilt and gray mold, respectively, while T. harzianum alleviated the disease incidence, and ALA not only alleviated the diseases but also promoted plant growth and fruit development [49]. Therefore, ALA promotes probiotic fungal growth and in the meantime, prohibits pathogenic fungal proliferation.

5.4. Plant Growth Substance

5.4.1. Application in Plant Tissue Culture

ALA is not a plant hormone, but it has regulatory activity over any classic plant hormone. It was found that 5–10 mg L⁻¹ ALA induced adventitious bud formation and rhizogenesis from calli of Vigna unguiculata L., exhibiting that ALA had both functions of IAA and CTK [50]. In Laminaria japonica, 50–500 mg L⁻¹ ALA was effective for the stable propagation of callus-like cells [51], which was ascribed to promoting cell division under white, blue, and red lights. Pentakeep-V, a fertilizer with ALA as the main active component, can be used to increase the transfer survival and growth during the acclimatization of tissue culture-derived date palm plants [52]. In Cymbidium, 0.1–1 mg L⁻¹ ALA induced protocorm-like bodies (PLBs) and shoot formation in vitro micropropagation [53,54]. Therefore, ALA can be used in plant tissue culture and propagation. However, its mechanisms need to be elucidated. Additionally, ALA can be used in the cell culture of Cyclocarya paliurus to promote polyphenol production [55]. It means that ALA can, in vitro, promote secondary metabolite accumulation.

5.4.2. Promotion of Seed Germination

Numerous studies show that ALA can improve plant seed germination. When wheat seeds were immersed in a 5 mg L⁻¹ ALA solution for 4 hrs, germination was promoted, not only in the germination rate but also in the growth of young roots and shoots [48]. In pakchoi, 0.01–10 mg L⁻¹ ALA promoted seed germination at normal or salt stress conditions [4], which was attributed to the conversion of ALA into porphyrins. In watermelon, 15–30 mg L⁻¹ ALA can improve seed germination under salt stress [56]. The effect might be related to its improving antioxidant enzyme activities such as superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT) while reducing ROS, lipid peroxidation, and malondialdehyde (MDA) content. Our unpublished data showed that the development of root hairs and lateral roots of watermelon seedlings was significantly improved when seeds were soaked in an ALA solution, indicating that ALA can promote seed germination and seedling growth. In Silybum marianum, ALA in the 40–160 mg L⁻¹ can improve salt tolerance during seed germination and seedling growth [57]. Under PEG 6000 stress, 10 mg L⁻¹ ALA promotes seed germination and seedling growth in Glycyrrhiza uralensis [58] or alfalfa [59]. Priming with 8.5 mmol L⁻¹ ALA promoted seed germination of rice under low-temperature conditions [60]. It was found that ALA significantly improved α-amylase activity to promote starch hydrolysis into soluble sugars for seed respiration and better growth under chilling stress in two rice cultivars. Priming the accelerated aged rice seeds with 0.5 mg L⁻¹ ALA significantly enhanced seed germination and seedling growth [61]. Priming with 4–16 mg L⁻¹ ALA promoted seed germination of Cajanus cajan and attenuated UV-B radiation-induced damaging effects on seedlings [62]. All these results suggest that ALA has the potential to be used in the seed industry of agroforestry. However, the mechanisms by which ALA promotes seed germination need more study.

5.4.3. Improvement of Plant Growth and Crop Yield

ALA at low concentrations can promote growth and yield in many species of crops [2]. It was found that the dry weight of rice plants increased by 14% after roots were soaked in a 0.1 mg L⁻¹ ALA solution. In radish plants, the taproot yield was increased by 20%–26% after foliar application of 30–100 mg L⁻¹ ALA. Barley yield was increased by 41% when 30 mg L⁻¹ ALA was sprayed twice before and after flowers. The tuber production was increased by 63% when potatoes were foliar sprayed with 100 mg L⁻¹ ALA. After garlic was treated with 30 mg L⁻¹ ALA, bulb production was increased by 40%. After broad beans were treated with 100 mg L⁻¹ ALA at the primary, first leaf, and fruit-set stages, the production was increased by 19%, 30%, and 8%, respectively. In wheat, seed soaking with 0.5–50 mg L⁻¹ ALA increased tiller number by 15%–20%, and the total yield was increased by approximately 5%. During the growth period, foliar spray with 10–50 mg L⁻¹ ALA improved grain yield by 15%. However, 100 mg L⁻¹ ALA diminished crop yield [63]. It seems that the timing and concentrations of ALA application are very important for yield increase. For many fruit species, foliar spraying at the flowering stage is not recommended because it may affect pollination and fertilization (see later sections). In pakchoi, 50 mg L⁻¹ ALA solution increased the dry weight of roots by 35% but did not affect the shoot weight [64]. Obviously, species of crops are different in their responsiveness to ALA treatment. For example, ALA promotes especially those with underground as the product organ, such as radishes [65], carrots [66,67], and potatoes [68]. In wheat, the application of ALA with 25–100 mg L⁻¹ increased grain yield by 18%–41% [69]. In barley, 100 mg L⁻¹ ALA increased yield by 51% [70], seeming to be more responsive to ALA than rice and wheat, with a greater increase in fresh weight. In date palm, 100 mg L⁻¹ did not affect yield but significantly increased fruit weight and volume [71]. Using the ³²P tracer, ALA was found to promote phosphate uptake and transport in rice, with grain yield increasing by 15% [72], which is similar to Yang et al. [73]. In tomatoes, ALA increased the biomass and fruit yield by about 14% [74]. In spinach, ALA increased yield by 13% [75]. In jujube, ALA-induced fruit yield increases were near 25% [76]. Consistent with this, 5–20 mg L⁻¹ ALA increased jujube yield by 12%–44% [77]. In soybean, a foliar fertilizer containing ALA as the main active substance can increase yield by 22%–64% [78]. Similarly, the foliar fertilizer increased strawberry yield by 15%–18% [79], while in rice, the fertilizer partially substituted urea to increase rice yield by 16% [80]. In maize, 100 mg L⁻¹ ALA increased yield by 4.9% and 9.7% under different planting densities [81]. In potatoes, ALA increased yield by 34.75% [68]. In carrots, ALA promoted root weight increases of 18% [66] or 73% [67]. In a two-year consecutive trial with sweet potatoes, we found that ALA increased the yield of root tubers by 34%–71%. The promotive effect of ALA not only exhibits itself on the fresh weight but also on the dry weight (Wang et al., unpublished), which may be the result of promoting leaf photosynthesis, carbohydrate transport, and accumulation from leaves to tubers. Recently, our study with C¹³ feeding on peach leaves showed that ALA promoted C¹³ transport from leaves to fruits, suggesting that ALA coordinated the relationship between sink and source, promoting photosynthate accumulation in sink organs [82]. However, why ALA promotes photosynthate distribution from leaves to product organs needs to be studied.

The possible mechanisms for ALA to promote shoot growth have rarely been studied. However, ALA-induced root growth has been reported recently. It is found that ALA induces PIN expression and PIN protein accumulation in the root tips of Arabidopsis and strawberries, which not only improves acropetal transport of auxin but also basipetal transport to control cell division and elongation [83]. Abscisic acid (ABA) is often accumulated when plants encounter stressful conditions [84], which tend to inhibit root growth [85]. Nevertheless, ALA alleviates ABA-induced root growth inhibition in strawberries [86]. It is found that ABA downregulates PIN1 expression, while ALA reverses the inhibition by ABA. When FaPIN1 of strawberries was overexpressed in Arabidopsis, ABA-induced and ALA-reversed root growth inhibition disappeared. Thus, the alleviation of ALA on ABA-inhibiting root growth is attributed to its promotion of IAA polar transport in the root tips.

5.4.4. Improvement of Plant Stress Tolerance

The biological functions of ALA to improve plant stress tolerance are the most striking among all its physiological effects. The functions seem similar to the stress hormone ABA; however, they are quite different. In fact, ALA antagonizes the activity of ABA in many aspects (see the above section). Therefore, it is an important scientific issue and has great practical significance for agroforestry production.

Low Temperature Stress

Hotta et al. [87] first proposed that when the roots of rice seedlings at three leaves old were soaked in a 1 mg L⁻¹ ALA solution and then transferred to 5℃ for 5 d, the survival rate and dry matter were increased by 30% and 72%, respectively, compared to the control. Foliar application of 30–100 mg L⁻¹ ALA on manilagrass (Zoysia metrilla) in early October prevented discoloration of the turfgrass in early winter and promoted sprouting and greening in the following spring, suggesting that ALA increased the overwintering ability and prolonged the greening period of the turfgrass [88]. Melon seedlings all died after 4 °C chilling for 6 hrs. However, if they were pretreated with 10–100 mg L⁻¹ ALA, all plants survived except for a few leaves with margin dehydration [89]. In watermelon, seedlings treated with ALA survived when overnighted at the minimum natural night temperature of −4 °C [90]. In tomatoes, 30 mg L⁻¹ ALA effectively improved the chilling resistance [91]. In cucumbers, 0.5 mg L⁻¹ ALA treatment improved chilling tolerance, with increases in SOD and POD activity, proline, and soluble sugar content, as well as higher root vitality, lower leaf electrolyte conductance, and the MDA content [92]. Additionally, ALA has been reported to increase the chilling tolerance of many other species, such as peppers [93,94,95], soybeans [96,97], summer squash [98], bananas [99], eggplant [100], canola [101], Elymus nutans [102], Rhododendron simsii and Cinnamomum camphora [103], tomatoes [104,105], Scenedesmus obliquus [106], cucumbers [107,108], tea [109,110,111], sweet cherries [112], and peaches [113]. The most important mechanism for ALA to increase chilling tolerance has been ascribed to antioxidant systems [114]. However, H₂O₂ and NO have been suggested to be the signals of ALA in oxidative stress tolerance induced by low temperatures in tomatoes [115]. It is a good beginning toward the cellular regulatory routes of ALA-inducing plant stresses. More molecular regulatory mechanisms need to be explored.

High Temperature Stress

On the other hand, ALA also protects plants against heat stress [116]. When watermelon seedlings were treated with a series of increasing temperatures from 30 °C to 49 °C, the leaf prompt fluorescence at I and P steps in the chlorophyll rapid induction fluorescence kinetic curves gradually decreased as the temperature increased. Treatment at 46 °C for 0.5 h harmed the leaf photochemical efficiency. Pretreatment with 20 mg L⁻¹ ALA significantly protected photosystem reaction centers under heat stress. The protective effects of ALA covered the activity of the oxygen-evolving complex (OEC), the photosystem II (PSII) reaction center, and the electron transfer chain in the thylakoid membrane. Therefore, the ALA-treated watermelon leaves maintained higher levels of the maximum fluorescence (F_m), the maximum photochemical efficiency (φ_Po), the energy flux per reaction center for electron transport (ET_o/RC), and the photosynthetic performance index (PI_ABS) than the control. In date palms, 200–250 mg L⁻¹ ALA application promoted the yield, fruit quality, and chlorophyll content under a hot arid climate with two-year experiments [117]. In cucumbers, heat stress (42/38 °C, day/night) inhibited the growth of seedlings, whereas 0.5 mg L⁻¹ ALA pretreatment significantly alleviated the inhibition by heat stress [118]. It was found that heat stress elevated the H₂O₂ and MDA content and the superoxide radical (O₂^˙¯) production rate in the leaves of plants, which were depressed by ALA pretreatment. Heat treatment induced the antioxidant enzyme activities, the proline, and the soluble sugar content, while ALA treatment induced much more than heat stress alone. Up to now, ALA-promoting heat tolerance has been reported in Ficus carica [119], Avena nuda [120], Qinling alpine rhododendrons [121], Ligustrum Japonicum and Spiraea japonica [122], and Medicago sativa [123]. Relatively, studies of ALA on plant heat tolerance are much fewer than those on chilling stress.

Light Stresses

Low light is a common stressor for plants grown under dense planting conditions, especially in facility cultivation systems. Wang et al. [89] first found that the net photosynthetic rate (P_n) of melon grown under low light conditions was different between leaf positions, where the lowest was at the base of the plant and the highest was at the top. Treatments with 10–100 mg L⁻¹ ALA significantly increased leaf P_n, especially in the base leaves. It seems that ALA prevented low-light-induced leaf senescence and maintained leaf photosynthetic capacity. In watermelon, treatment with 100 mg L⁻¹ ALA alleviated the shade-induced reduction of leaf photochemical efficiency. When the SOD activity inhibitor DDC (diethyl dithiocarbamate sodium) was added, the promotion of ALA on the photochemical activity of PSII reaction centers was not found to be directly related to the PSII itself but to the enhancement of antioxidant enzymes such as SOD near PSI reaction centers. Therefore, ALA-induced elimination of ROS was the key mechanism for plants stressed by shading [124,125,126]. In cucumbers, 0.5 mg L⁻¹ ALA spraying improved the leaf P_n and photochemical efficiency when seedlings were cultured at a sub-suitable light temperature condition (PFD 300 μmol m⁻² s⁻¹, 18/12 °C, day/night) [108]. The activity of RuBPCase (ribulose-1, 5-bisphosphate carboxylase) and FDPase (fructose-1,6-diphosphatase) was improved by ALA, suggesting the protection of ALA on plants against low light stress not only depended on photosynthetic pigments but also photosystem activity as well as dark reaction activity. In tomatoes, ALA improved leaf photosynthesis under shade conditions [127]. In Isatis indigotica, ALA promoted plant growth and secondary metabolite accumulation under artificial shading conditions [128]. Since Chinese traditional medicine such as I. indigoticai is often cultivated in the interplant mode with low light intensity, the pharmaceutical ingredients such as indigo and indirubin tend to be lower than those under natural light conditions. It is meaningful that ALA does not only increase vegetative growth but also improves medicine quality. In an apple orchard covered with black shading nets to reduce the light intensity, the leaf antioxidant enzyme activity was significantly decreased while MDA was increased with the decrease in light intensity [129]. Exogenous ALA increased the leaf antioxidant enzyme activities and decreased the MDA content, maintaining higher levels of leaf photosynthetic energy utility. When cucumber was grown under low-temperature and weak-light stress, ALA significantly alleviated the inhibition of plant growth and enhanced leaf area and the fresh and dry weight of seedlings [130].

On the other hand, ALA protects plants against high light stress. Liu et al. [131] first reported the relationship between ALA and the strong light tolerance of strawberries. They found that spraying 100 mg L⁻¹ ALA significantly increased the leaf P_n when strawberry was cultured under high light intensity (1500 μmol m⁻² s⁻¹), compared with that under 800 μmol m⁻² s⁻¹. The ALA-treated leaves contained more total chlorophyll with higher carboxylation efficiency. ALA reduced the initial fluorescence (F_o) but increased the maximum fluorescence (F_m) and the variable fluorescence (F_v). ALA also improved the actual photochemical efficiency of PSII (Φ_PSII), photochemical fluorescence quenching (q_P), non-photochemical fluorescence quenching coefficient (NPQ), apparent photosynthetic electron transfer rate (ETR), photochemical rate (PCR), and antenna thermal dissipation (D), but reduced the relative photosynthetic limit (L_PFD). When watermelon grew under strong light conditions, ALA-treated leaves had high photochemical conversion ability, with higher Φ_PSII, q_P, ETR, and PCR compared to the control [116]. Comparing the chlorophyll fluorescence light response curves of watermelon leaves treated with 50–200 mg L⁻¹ ALA, it was found that many fluorescence parameters, such as F_v’/F_m’, Φ_PSII, q_P, NPQ, ETR, PCR, and P_c, were almost the same when leaves were kept under low light intensity; however, when leaves were exposed to strong light conditions, the parameters varied greatly, where ALA-treated leaves had higher photochemical conversion capacity under high light than the control [132].

In addition, ALA prevents plant damage from ultraviolet light [133]. It is known that UV-B radiation exerts an adverse effect on lettuce seedlings, and some of the negative effects can be alleviated by exogenous ALA. UV-B exposure inhibits the growth of pigeon peas, with lower levels of photosynthetic pigments and photosynthetic gas exchange characteristics (P_n, C_i, G_s, E, and WUE), accompanied by the accumulation of ROS and MDA [63], while ALA can alleviate all the damages by enhancing plant growth and growth-regulating parameters. Therefore, ALA acts as a sustainable remedy for reducing the hazardous effects of UV-B stress on crop productivity by triggering up-regulation of photosynthesis and the antioxidant system to counterbalance the ROS-mediated damage to macromolecules.

Salt Tolerance

It was Watanabe et al. [134] who discovered that ALA significantly improved cotton salt tolerance after they compared the effects of 12 kinds of plant growth regulators. All cotton plants died when stressed by 1.5% NaCl, while the damage rate was only 20–30% after 100–300 mg L⁻¹ ALA treatment. In spinach, 100 and 300 mg L⁻¹ ALA treatment improved the leaf photosynthetic efficiency when seedlings were stressed by 100 mmol L⁻¹ NaCl [135]. The connection between the promotive effect of ALA on stress tolerance and the increments in antioxidant enzyme activities was first proposed in the study. Many follow-up studies are relevant to the findings. In pakchoi, 0.01–10 mg L⁻¹ ALA enhanced seed germination and respiration under salt stress [4]. It was found that ALA increased the heme content of seedlings under salt stress, while the ALA metabolism inhibitor LA completely blocked the promotion of ALA. Therefore, the conversion of ALA into porphyrin compounds was necessary for its promotion of salt tolerance. Since heme is an important prosthetic group of respiratory enzymes, its content is associated with the respiration of germinating seeds. Up to now, the promotion of ALA on plant salt tolerance has been demonstrated in many species, such as watermelons [56], muskmelons [136], Pinus sylvestris, P. tabuliformis, and Amorpha fruticosa [137], lettuce [138], date palms [139], wheat [140], strawberries [141], oilseed rape [142], Perilla frutescens [143,144], sunflowers [145], cucumbers [146,147], Silybum marianum [57], grapes [148], rice [149], Swiss chard [150], tomatoes [151,152,153], peaches [154], Leymus chinensis [155], pumpkins [156], asparagus [157], Atropa belladonna [158], cauliflower [159], jujube [160,161], peanuts [162], Swiss chard [163], maize [164], Salvia miltiorrhiza [165], and Arundo donax [166].

In paddy fields, the application of a purple nonsulfur bacterium (Rhodopseudomonas palustris), a germ biosynthesizing ALA, can improve rice salt tolerance. The germ can reduce global warming gas methane emissions, which may be meaningful for global environmental protection [167]. In a date palm orchard, ALA increased the actual water use when the plants were irrigated with different salinities of water [168]. It is a good step toward seawater agriculture.

In addition to exogenous application, several transgenic plants that overproduce endogenous ALA by expression of yeast Hem1 controlled by the Arabidopsis HemA1 promoter were established. It is worth noting that the promoter is light-responsive [15], which drives gene expression and ALA accumulation under light conditions but not in darkness [169]. The circadian rhythm changes of gene expressions and ALA levels prevent abnormal tetrapyrrole accumulation at night; otherwise, the photodynamic reaction will kill plants under normal sunlight [170]. In transgenic Arabidopsis, overproduction of endogenous ALA can improve salt tolerance at either seed germination or seedling stage [171]. In a similar transgenic canola, increases in endogenous ALA improved salt tolerance with much higher biomass and rapeseed yield [172]. When the peanut AhHEMA1 gene is transformed into tobacco, the leaves can accumulate higher levels of ALA and chlorophyll, with higher activities of antioxidant enzymes and more salt tolerance [162]. Thus, both exogenous application and endogenous overproduction of ALA facilitate plants growth under salt stress conditions.

In addition to salinity, sodic alkalinity also affects plant growth. However, ALA application can increase survival rates and growth rates of young forest seedlings, including Amorpha fruticose, Pinus tabulaeformis, and Platycladus oriental, when they are grown in alkaline sandy soil (pH 8.41) [137]. In Swiss chard, ALA can alleviate the harmful effects of sodic alkalinity (pH 9.0, modulated by NaHCO₃ and Na₂CO₃) on growth [163]. Similarly, in soybean [173] and Populus wutunensis [174], ALA can alleviate the inhibitory effect of saline-alkali stress (pH 8.40) on young seedling growth. Therefore, ALA may be widely applied in agroforestry production, either in secondary salinized greenhouses or seawater agroforestry.

Drought Stress

When we first observed that ALA promotes leaf stomatal aperture [89], we could never have predicted that ALA could improve plant drought tolerance because a larger stomatal aperture means higher transpiration and more water loss from leaf surfaces. However, ALA was reported to promote the growth and yield of wheat [69] or barley [70] under dry conditions. In potted tobacco, rhizospheric application with an engineered bacterial fermentation broth containing ALA improved drought tolerance in the greenhouse [175]. Therefore, the effect of ALA on promoting plant drought tolerance is definite. Up to now, the effect has been reported in many species of plants, such as cucumber [176], Glycyrrhiza uralensis [58], rapeseed/canola [177,178,179], Fagopyrum dibotrys [180], Sarcandra glabra [181], wheat [182,183,184,185,186,187], Leymus chinensis [188], Gardenia jasmioides [189], Dalbergia odorifera [190], Kentucky bluegrass [191,192], date palm [193], sunflower [194], tobacco [195], maize [196,197], poplar [198], alfalfa [59], and banana [199].

The mechanism for ALA to promote plant drought tolerance has been reported in strawberries recently [200]. On the one hand, ALA promotes leaf stomatal conductivity and transpiration rates, which are beneficial for photosynthesis, biomass accumulation, and plant growth; on the other hand, ALA promotes aquaporin gene expressions, which enhance root water absorption and hydraulic conductivity in xylem. Therefore, ALA can promote roots to absorb water from high-osmotic soil or substrate and transport it to maintain water balance under water stress. We once found that the maize plants seemed to be burned after ALA application in a rainfed farming region because there was no water system available. We suggest that ALA be applied to the crops with regular water systems available. Once water is recovered, the resilient growth of plants treated with ALA is much better than that without.

Waterlogging Stress

ALA can also improve the waterlogging tolerance of plants. An et al. [201] found that ALA pretreatment significantly attenuated symptoms of potted fig (Ficus carica) cuttings during 6-day full-waterlogging stress. The control leaves were wilted and had chlorosis, curling, and abscission, while the ALA treatment kept them much better. The activities of antioxidant enzymes, alcohol dehydrogenase, and lactate dehydrogenase, as well as root vigor, were significantly improved by ALA pretreatment, which was responsible for waterlogging tolerance. In snap beans (Phaseolus vulgaris), foliar application of ALA offsets the unfavorable effects of shallow water tables on growth and yield [202]. ALA in 0.3–0.45 mmol L⁻¹ increased pod numbers compared with the stressed plants.

Toxic Metal Stresses

Noriega et al. [203] first reported the relationship between ALA and toxic metal stress. They found that cadmium (Cd) inhibited ALA dehydrase activity in the roots and leaves, as well as nodules, of soybean, leading to endogenous ALA accumulation with much less porphobilinogen content. ALA (18 mmol L⁻¹) had the same effect as Cd, which reduced the protein abundances of SOD and POX, inducing cell toxicity. However, the results of the study exhibited a harmful effect of ALA at high concentrations, like heavy metal stress. Ali et al. [204,205] found that 12.5–25 mg L⁻¹ ALA alleviated the harmful effect of Cd on rapeseed in plant growth, root development, leaf photosynthesis, ROS levels, lipid peroxidation, and tissue ultrastructure, which was considered as ALA improving mineral element absorption and attenuating oxidant stress induced by Cd [206]. In Chinese cabbage, 25 mg L⁻¹ ALA promoted plant growth, downregulating gene expressions related to Cd absorption and transport in shoots (HMA2 and HMA4) and roots (IRT1, IRT2, Nramp1, and Nramp3), which resulted in a decrease in Cd content in the whole plant [207]. In Salvia miltiorrhiza, ALA alleviated Cd stress on plant growth and repressed the tissue Cd content; however, the related gene expressions were upregulated [208]. The conflicting results await further clarification. In rice, 100 mg L⁻¹ ALA significantly reduces the Cd content in leaves, culms, and brown rice [209], suggesting that ALA may be used to increase the safe production of rice in Cd-polluted farmland. However, in sunflowers, ALA cannot only improve the growth of plants under Cd stress but also increase the Cd content in plant tissues [210], suggesting it is a cadmium accumulator. If it is true, ALA can be used in sunflowers for phytoextraction of Cd from contaminated soils. The mechanism for sunflowers to deal with the toxic metal in the plants needs to be studied.

Chromium (Cr) is another toxic element. Gill et al. [211] first reported that ALA can enhance plant chromium tolerance. They found that 15–30 mg L⁻¹ ALA alleviated the negative effects of Cr on growth inhibition, cell structural damage, and lipid peroxidation in Brassica napus. ALA upregulated the antioxidant enzyme activities and gene expressions to relieve oxidative stress-induced Cr stress, with less Cr accumulation in the leaves and roots of the stressed plants. Similarly, ALA decreased the Cr content in cauliflower plants, which was the key mechanism for ALA to minimize Cr toxicity to plants [212]. Nevertheless, the ALA-induced Cr content did not decrease but instead increased in sunflower plant tissues. ALA alone or combined with citric acid improved biomass, photosynthesis, and gas exchange attributes when plants grown on Cr-contaminated soil [213,214]. Therefore, ALA can promote Cr uptake and transport in sunflowers, which may be used for phytoremediation of heavy metal-contaminated soils.

Lead (Pb) is also an important toxic heavy metal that pollutes farmland more and more as a result of industrialization. Tian et al. [215] and Ali et al. [216] proposed that ALA alleviated the toxic effect of lead on rapeseed plants. It was suggested that the ALA improvement might be associated with the TCA cycle because many species of organic acids, including fumarate, succinate, malate, and citrate, were enhanced accompanied by upregulated expression of genes like CS (encoding citrate synthase), SUCLG1 (encoding succinyl CoA ligase 1), SDH (encoding succinate dehydrogenase), and FH (encoding fumarate hydratase) after ALA treatment [217]. Additionally, ALA decreased ROS production by increasing antioxidant enzyme activities under Pb stress. Furthermore, ALA improved the uptake of mineral nutrients under Pb stress. However, the mechanism is not clear.

Arsenic (As), one of the dangerous metalloids, is regularly encountered by the environment due to human activity and environmental practices such as mining, widespread use of pesticides and fertilizers, burning fossil fuels, and increased deposition of municipal and industrial garbage. Recently, ALA was found to alleviate arsenic toxicity in pepper plants by modulating its sequestration and distribution within cell organelles of the roots [218]. Heme, converted from ALA, was considered an important regulator for the increase of antioxidant systems and cellular arsenic detoxification.

Copper (Cu) preparations are widely used in fruit production. However, excess Cu causes serious damage to photosynthetic systems, especially PSII, thus inhibiting photosynthesis. It was found that 10 mmol L⁻¹ CuSO₄ caused membrane lipid peroxidation and leaf injury in grapevines, while 50 mg L⁻¹ ALA significantly alleviated the toxic effect of Cu [219]. Therefore, ALA can be used in viticulture to prevent copper stress.

Other Environmental Pollution Stresses

Petroleum is essential for modern society. However, the broad application of petroleum causes soil and groundwater contamination all over the world. Oil contamination of soil and groundwater negatively impacts human health and plant growth. ALA can be used for bioremediation of oil-contaminated soils [220]. An ALA-contained fertilizer not only promoted plant (especially root) growth of Zinnia hybrida under stressful conditions but also reduced the petroleum hydrocarbon content of the oil-contaminated soil with increased soil dehydrogenase activity, suggesting that ALA improved the biological activity of the contaminated soil. On the other hand, city traffic roads are heavily polluted areas, alongside which the greening plants should tolerate various environmental stresses from the soil and atmosphere. It was found that spraying ALA solution on shrubs of Buxus megistophylla grown along urban roadsides increased plant survival and promoted vegetative growth [221]. Therefore, ALA can be used to improve the tolerance of road greening trees against complex stresses such as automobile exhaust, soil, or climate stresses around them.

With the rise in atmospheric CO₂ concentration, the environment for human survival is deteriorating. High concentrations of CO₂ significantly affect plant growth. Recently, our unpublished study showed that 800 μmol mol⁻¹ CO₂ inhibited tobacco plant growth, while exogenous ALA treatment significantly alleviated the inhibitory effect of high concentrations of CO₂ was on plant growth. Compared with the control (the ambient air CO₂ about 400 μmol mol⁻¹), the average daily P_n of the high CO₂ treatment was 74%, while the ALA treatment was 107%, 44% higher than that without ALA treatment. It manifests that ALA applications have wide prospects.

Possible Mechanisms for ALA to Improve Stress Tolerance

The functions of ALA to improve plant stress tolerance are amazing and extensive; however, the mechanisms involved have not been extensively explored. Here, we list the most possible mechanisms that have been proposed, while more need to be elucidated.

Antioxidant Systems

The antioxidant enzymes must be very important in ALA-induced plant stress tolerance. In spinach, the activities of CAT, APX, and GR were increased after ALA treatment, along with increases in the ratios of reduced ascorbate acid (AsA)/oxidized ascorbate acid (DHA) and reduced glutathione (GSH)/oxidized glutathione (GSSG). Meanwhile, H₂O₂ was increased manyfold, and its levels were correlated with the promotion of photosynthetic rates [222]. When the plant was stressed by NaCl, ALA induced increases in antioxidant enzyme activities [135]. It was the first finding of an ALA-inducing antioxidant system against environmental stresses. Since then, many studies have shown that antioxidant enzymes and small-molecule antioxidants such as ascorbate, glutathione, and proline are involved in ALA-induced stress tolerance in plants. In strawberries, when DDC, the inhibitor of SOD, was applied, the promotion of ALA on leaf photosynthesis was eliminated, while the MDA content was greatly increased, suggesting that the increased antioxidant enzyme activity induced by ALA is necessary for stress tolerance [131]. Under drought stress, ALA induces great increases in POD and CAT activity in tobacco leaves [175]. When rice seedlings were encountered with chilling and high light stress, ALA significantly enhanced the activities of APX and GR, while the decline of SOD activity was less than the control, suggesting a positive regulation of ALA on the antioxidant enzymes against environmental stresses. On the other hand, H₂O₂ has been considered a harmful ROS since ALA inhibits leaf H₂O₂ increase under stress, and too high levels of ROS would lead to stress injury [114]. In Brassica napus, ALA improved seedling tolerance to herbicide stress, where herbicide ZJ0273 depressed the activities of SOD, POD, and APX, while ALA reversed the effect of the herbicide, and seedlings grew well [37]. However, many stresses stimulate increases in antioxidant enzymes, including SOD, POD, CAT, APX, and GR, while ALA induces further increases in enzyme activities [10]. It seems that, although important, enzyme activity is not the limiting factor for plant survival under stress conditions. A similar situation has been found in an ALA-overproduction transgenic canola (Brassica napus) [172]. When the transgenic seedlings were treated with 150–450 mM NaCl, the activities of SOD, POD, CAT, and APX were increased as salt concentrations increased, and the enzyme activities in the transgenic leaves were all higher than those in the WT. Under lead stress, the activities of SOD and APX in Brassica napus decreased as lead concentrations increased in both leaves and roots, while the activities of POD increased in both tissues. Nevertheless, the CAT activity in the leaves decreased while that in the roots increased. It seems that these enzyme activity changes vary with the types of enzymes and with different plant tissues. However, ALA significantly increased the activities of these antioxidant enzymes regardless of the plant tissues [216]. This suggests that the promoting effect of ALA on antioxidant enzyme activities is extensive rather than specific. When tomato seedlings were stressed by salinity, the expressions of SOD, POD, and APX genes were upregulated while those of CAT were downregulated; however, ALA upregulated all these gene expressions [151]. In cucumbers, ALA promoted the AsA-GSH cycle to alleviate salt injury, where the activities of ascorbic acid oxidase (AAO), ascorbate peroxidase (APX), monodehydroascorbic acid reductase (MDHAR), dehydroascorbic acid reductase (DHAR), and GR were all improved after ALA application [223]. In tomatoes, ALA upregulated the expression of GSTU43, a member of the U class of glutathione S-transferase genes, resulting in higher protein abundance and GST enzyme activity [224]. If GSTU43 was silenced, the activities of GST, SOD, CAT, APX, and GR decreased with severe membrane injury. Thus, the ALA-induced chilling tolerance of tomatoes might be dependent on GSH signaling in the regulation of redox balance. Recently, in the apple (Malus domestica), MdWRKY71 was found to be a positive transcription factor (TF) in the ALA signaling cascade. Overexpressing MdWKRKY71 transgenic apples were more salt-tolerant than the wild type, where the expressions of CAT1, POD1, and APX1 were all upregulated. In the RNAi-MdWRKY71 transgenic apples, salt tolerance was depressed with down-regulation of all the gene expressions. Thus, ALA upregulated MdWRKY71 to enhance the salt tolerance in apples, partially dependent on its upregulation of protective system activities (Wang et al., unpublished).

On the other hand, ALA positively induces ROS generation, which is more rapid than the increases in antioxidant enzyme activity and plays an important role in ALA-induced stress tolerance. Tan et al. [12] depicted a regulatory network of ALA priming-mediated abiotic stress tolerance, where the increases in antioxidant enzyme activity are located downstream of the ALA-Rboh-H₂O₂-NO signaling route. It is the fact that H₂O₂ is a ROS as well as an important cellular signaling factor involved in ALA-induced stress tolerance. In strawberries, ALA induced an increase in H₂O₂ in the roots but a decrease in the leaves [225]. The increase of root H₂O₂ upregulated the expressions of genes encoding salt ion transporters (see next sections), while the depression of H₂O₂ in the leaves induced by ALA avoided ROS toxicity to photosynthesis and plant growth. It is a smart mechanism for ALA to enhance plant stress tolerance. Furthermore, the increase of H₂O₂ in the roots is dependent on NO production [226]. Thus, ALA signaling may be mediated by NO and/or H₂O₂ to upregulate salt ion transport gene expressions for ion homeostasis. In photooxidative stress, ALA relieving the MV-induced injury in detached pear leaves (Pyrus ussuriensis) was also dependent on H₂O₂ signaling [40]. When the expression of PuRbohF, the gene encoding NADPH oxidase and H₂O₂ production, was silenced, the promotive effect of ALA was abolished. When PuRbohF was transiently overexpressed in tobacco leaves, MV-induced damage was alleviated by enhancing the activities of SOD and CAT. Therefore, ALA-induced H₂O₂ is responsible for the photooxidative tolerance in pear leaves. Nevertheless, our recent data showed a more complex signaling network. Inhibiting the generation or elimination of either NO or H₂O₂ alone impaired ALA-induced salt tolerance, while simultaneously inhibiting the generation or elimination of both ROS completely abolished ALA-induced salt tolerance in strawberries. Therefore, H₂O₂ and NO signaling are reciprocally regulated by each other in ALA-induced salt tolerance in strawberries (Figure 2). Furthermore, the tissue specificity of the biosynthesis and function of H₂O₂ and NO cannot be ignored. When strawberries were stressed by PEG 6000 [200] or salinity [225,226], ALA-induced H₂O₂ and NO were accumulated in the roots but depressed in the leaves. Similarly, the ABA content was improved in the roots but inhibited in the leaves by exogenous ALA. This may explain why the stomatal conductance of leaves increased after ALA treatment under water stress. Generally, ALA-induced ROS signaling may be a warning mechanism for plants to resist various abiotic and biotic stresses. In practice, ALA should be used as a preventive rather than a therapeutic agent.

Photosynthesis and Stomatal Regulation under Stressful Conditions

It is long known that ALA can improve plant photosynthesis [3]. Firstly, ALA is not only the key precursor of chlorophyll biosynthesis but also involved in biosynthetic regulation. In Kentucky bluegrass [191], tomatoes [224], or canola [227], ALA alleviated stress caused by the chlorophyll synthesis obstacle of uroporphyrinogen III (UROIII) conversion to Proto IX and enhanced the production of chlorophyll and its precursors. Exogenous ALA treatment stimulates endogenous ALA synthesis under stress. Under osmotic stress, the endogenous ALA as well as chlorophyll synthesis genes, including HemA1, HemG1, PORB (encoding protochlorophyllide oxidoreductase B), and ChlG (encoding chlorophyll synthase), were upregulated in walnut leaves after exogenous ALA application, while CAO (encoding chlorophyll a oxygenase) expression was downregulated, which was responsible for leaf chlorophyll maintenance under stress [228]. However, ALA regulating chlorophyll synthesis in tomato leaves under chilling was not at the level of transcription [224]. Therefore, ALA’s role in regulating chlorophyll biosynthesis needs further study. Secondly, the activities of the photosystem II and I reaction centers as well as the whole electron transfer chain, including the donor side and the acceptor side of PSII and the terminal electron acceptors of the PSI reaction center, are upregulated by ALA [103,122]. The expressions of the psbA and psbD genes, encoding D1 and D2 proteins, respectively, of the PSII reaction centers of wheat leaves were upregulated under drought stress following ALA pretreatment [184]. A similar response was found in potatoes when measurements were conducted at noon and afternoon [68], suggesting that ALA promoted the turnover of the core proteins of the PSII reaction center when plants were experiencing stressful conditions. In pears, ALA depressed the noon break of leaf photosynthesis when the environment was not suitable [229]. In Kentucky bluegrass, ALA upregulated the gene expression of many photosynthetic electron transfer chains, including the ferredoxin-NADP⁺ reductase gene, resulting in weakening photoinhibition [191]. However, ALA-induced antioxidant enzymes, located near PSI reaction centers, also accept photosynthetic electrons and alleviate photoinhibition [126]. Thirdly, ALA induces gene expression of the Rubisco small subunit and the Rubisco initial activity of pear leaves throughout the day. A similar promotive effect of ALA on RuBPCase and FDPase activity was found in cucumbers under suboptimal temperature and light intensity [108]. Niu et al. [191] proposed that more than ten genes involved in the Calvin–Benson cycle were upregulated by ALA when Kentucky bluegrass was osmotically stressed. Thus, the improvement of ALA in photosynthesis has multiple aspects. Nowadays, many scholars suggest that the improvement of ALA in photosynthesis is an important mechanism for plants to tolerate environmental stresses. But according to our opinion, the increase in photosynthesis after ALA treatment under stress is a result rather than the reason. There are many molecular mechanisms that need to be studied.

Stomata are the passageways for atmospheric CO₂ into the mesophyll cells, and the stomatal aperture is an important factor for CO₂ entrance and photosynthesis. ALA can promote stomatal aperture even under stressful conditions, which facilitates alleviating the decline of leaf photosynthesis induced by stress [89]. ABA is known to be a stress hormone that is de novo biosynthesized and accumulated in plant tissues when plants encounter stressful conditions [84,85]. The accumulated ABA induces stomata closure, decreasing leaf transpiration and water loss. However, it blocks atmospheric CO₂ from entering mesophyll cells, which is unfavorable for photosynthesis and plant growth. In our lab, a series of studies show that ALA can reverse ABA-induced stomata closure [230,231,232,233,234,235,236], which may be important for ALA to improve leaf photosynthesis under stress conditions. More recently, a regulatory mechanism of ALA to reverse ABA-induced stomatal closure in apple leaves has been deciphered [237], where MdPP2A, especially the catalytic subunit MdPP2AC, is the most important component of the ALA signal route, which can catalyze dephosphorylation of MdSnRK2.6, the important component of the ABA signal, to block ABA-induced stomatal closure. Upstream, PP2A activity is regulated positively by MdPTPA [238] but negatively by MdDGK3-like [239]. Exogenous ALA upregulates the activity of PTPA and, meanwhile, downregulates DGK3-like. Both facilitate PP2A activity but depress ABA signals. The fine regulatory mechanisms of ALA cause stomatal opening after ABA treatment or under stressful conditions. More interestingly, the overexpressing MdPTPA transgenic tobacco grows better but has less water tolerance, while the overexpressing MdSnRK2.6 transgenic plants are more water-stress-tolerant but grow less. Only the overexpressing MdPP2AC transgenic plants grow better and are more water-stress-tolerant [240]. The findings suggest that ALA and its upregulated PP2AC activity improve leaf photosynthesis, plant growth, and stress tolerance. Therefore, ALA promotes plants to absorb CO₂ and convert it into organic matter, which facilitates the drop of atmospherically inorganic CO₂.

ALA-induced Ion Interception in Roots to Improve Salt Tolerance

An excess amount of Na⁺ and Cl⁻ accumulated in plant tissues is toxic. How to deal with the toxic ion/anion is essential for plants to survive under salt stress. In strawberry (Fragaria × ananassa Duch.), we found that ALA induced Na⁺ and Cl^- interception in the roots, stored mainly in the mature regions full of vacuoles, acting as inorganic osmotic solutes, enhancing water absorption, and preventing Na⁺/Cl⁻ upward transport with less accumulation in the leaves under salt stress [225,226]. Here, ALA-induced NO and H₂O₂ in roots form a signaling cascade to stimulate gene expressions of SOS1, NHX1, and HKT1 to exclude Na⁺ out of root cells, sequestrate in vacuoles, and re-extract into xylem parenchyma cells during its transport in the xylem sap, respectively. Meanwhile, CLCs, encoding Cl⁻ transporters, and expressions were also upregulated by ALA. FaMYB44, a TF, was recently found to bind the MBS motif (ACCGTA/C) of the FaCLC-c4 promoter to negatively regulate gene expression. Salt stress upregulated FaMYB44 expression, while ALA inhibited the FaMYB44 expression induced by salinity. Overexpression of FaMYB44 in tobacco increased the leaf Cl⁻ content and aggravated salt injury. Therefore, ALA-induced salt tolerance in strawberries is dependent on its downregulating FaMYB44 expression, eliminating the inhibition of FaMYB44 on FaCLC-c4 expression, and then higher FaCLC-c4 activity permitting Cl⁻ sequestration into the vacuoles of roots, re-extraction from the xylem sap, avoiding salt injury to the photosynthetic apparatus, and other physiological processes. The working model of ALA-induced Na⁺ and Cl⁻ interception in strawberry roots is depicted in Figure 2. This model may be ubiquitous because similar root interception of the salt ion has been reported in cucumber [147] and Arundo donax [166], where the NHX1 and VHA-A (encoding H⁺-ATPase in tonoplast) expressions were upregulated by ALA in cucumber roots under salinity [147]. Additionally, ALA-induced arsenic interception in the vacuoles of pepper roots is also reported [218], although whether any transcription factor is involved in regulation is not clear. Even though it should be an important mechanism for ALA to induce detoxication of harmful elements when plants encounter chemical stresses.

ALA-induced Root Water Absorption to Increase Drought Tolerance

As mentioned above, it was hard to imagine that ALA improved water stress tolerance when we knew that ALA promoted stomatal aperture under normal and stressful conditions [89]. However, ALA indeed improved water stress tolerance in wheat [69] and barley [70]. Therefore, the promotive effect of ALA must be objectively present. In strawberries, we found that ALA maintained plant water balance under osmotic stress by increasing transpiration and water absorption from the culture substrate. Thus, at the end of the experiments, the relative water content of the substrate in the ALA-treated pots was much lower than that of PEG stress. Meanwhile, ALA reversed the decline of gene expressions of PIPs (encoding plasmalemma intrinsic proteins) and TIPs (encoding tonoplast intrinsic proteins) in the leaves and roots by PEG stress [200]. Thus, the reason for ALA to improve water stress tolerance is not to save water by limiting water loss but to promote root growth and root activity in order to absorb water from the high osmotic substrate. Since leaf photosynthesis was promoted by ALA under drought stress, water use efficiency increased. This is why we suggest that ALA can be used to improve plant growth against water stress with regular irrigation. In tomatoes, when plants were stressed by NaCl, it was also found that ALA-induced aquaporin gene expression regulates water homeostasis [152]. The active absorption and conduction of water from soil induced by ALA can prevent leaf wilting and physiological disorders.

It is often reported that ALA induces soluble solute accumulation (such as proline, soluble proteins, and soluble sugars) to enhance osmotic adjustment when plants are under stressful conditions [92,228]. ALA upregulates P5CS and P5CR gene expressions but downregulates ProDH and P5CDH expressions in tomato seedlings under NaCl stress, which are responsible for proline accumulation and osmotic adjustment [151]. However, compared with active water absorption, we tend to consider that osmotic adjustment may be passive and limited.

5.4.5. Delaying Senescence

ALA spraying before harvest reduced the respiration of tomato fruits during the storage period, with higher hardness, soluble sugar content, and flavor quality compared with the control [241]. These suggest that ALA can be used to improve the postharvest quality of fruits. In spinach, ALA spraying at seedling growth stages can enhance vegetable quality, inhibit ethylene production, and delay the respiration peak, with less MDA accumulation during the cold storage period [242]. In pakchoi, ALA prevented leaf yellowing, and the chlorophyll decreased under low or medium nitrogen conditions [243]. ALA spraying on “Fuji” apples every 20 days during the fruit expansion stage significantly improved the fruit quality at the mature stage, and this promotive effect was maintained for 200 days under freezing storage conditions as well as shelf life [244]. Our unpublished data also showed that ALA promoted maturation and delayed senescence in apple, pear, and peach fruits. However, the mechanisms have not been well studied.

5.4.6. Promoting Fruit Coloration and Flavonoid Biosynthesis

Once we found that ALA improved plant low-light stress tolerance [89], we imagined that ALA might promote fruit coloration under low-light conditions. Many fruits with less coloration are due to a shortage of light exposure. Then, we tested the effect of ALA on apple coloration 20 days before harvest and observed that ALA significantly promoted the anthocyanin accumulation on the fruit peel, where 300 mg L⁻¹ was the most effective, and the anthocyanin content was more than one time higher than the control [245]. The effect was verified by apple slices [246] and calli [247,248,249,250]. After a series of experiments in five provinces with different ecological environments, ALA has been officially authorized as a new plant growth regulator for apple coloration [251]. Then, similar effects of ALA were reported in peaches [252], pears [253,254], grapes [255,256,257,258], litchi [259,260], pomegranates [261], and mangoes [262]. In addition to fruit coloration, soaking wheat seeds with 50 mg L⁻¹ ALA not only improves frost tolerance but also increases anthocyanin accumulation in coleoptiles [263].

The transcriptional regulatory mechanisms of ALA on anthocyanin accumulation in apples have been reported recently (Figure 3). ALA upregulates the gene expressions involved in anthocyanin biosynthesis and transport, including MdCHS, MdF3H, MdDFR, MdANS, MdUFGT, MdMATE8, and MdGSTF12 [247,248,249,250]. MYB, bHLH, and WD40, the most common TFs involved in anthocyanin biosynthesis, have been reported to be upregulated by ALA but downregulated by LA, suggesting that ALA conversion into tetrapyrrole compounds is necessary for anthocyanin biosynthesis [251]. Based on the transcriptome and proteome, MdMADS1 was found to be involved in ALA-regulated anthocyanin biosynthesis [247]. The expression of MdMADS1 was positively correlated with the expression of MdCHS, MdDFR, MdANS, and MdUFGT in apple skin under ALA treatment. Overexpression of MdMADS1 enhanced anthocyanin content in transformed apple calli, which was further enhanced by ALA. In MdMADS1-silenced calli, the anthocyanin accumulations as well as the relative gene expression were depressed. Similarly, MdMYB10 and MdMYB9 are also induced by ALA to upregulate anthocyanin biosynthesis and transport gene expression, especially the transporter gene MATE8. These effects have been verified by overexpression and RNA interference [250]. MdERF78 is another ALA-responsive TF. In the overexpressed or RNAi-MdERF78 apple peels or calli, anthocyanins were greatly upregulated or downregulated, which was further affected by exogenous ALA. MdERF78 can directly bind the promoters of MdF3H and MdANS and activate their expressions. It can also interact with MdMYB1 to enhance the transcriptional activity of the latter on its target gene promoters, including MdDFR, MdUFGT, and MdGSTF12. Thus, MdERF78 positively mediates ALA-induced anthocyanin accumulation via the MdERF78-MdproF3H/MdproANS and MdERF78-MdMYB1-MdproDFR/MdproUFGT/MdproGSTF12 regulatory networks [248]. Recently, MdMYB110a was found to play an important role in the ALA-induced anthocyanin accumulation [249], which activated MdGSTF12 expression by binding to its promoter. Additionally, two new ALA-responsive TFs, MdWD40-280 and MdHsfB3a, were found to interact with MdMYB110a. All of them are positive regulators of ALA-induced anthocyanin accumulation. MdWD40-280 and MdHsfB3a enhanced the transcription ability of MdMYB110a on the promoter of MdGSTF12. MdHsfB3a can independently bind to the promoters of MdDFR and MdANS, whereas MdWD40-280 cannot bind any structural gene promoters, whose activity depends on MdMYB110a. Thus, a complex regulatory module composed of MBY110a-HsfB3a/WD40-280 is proposed for ALA-induced anthocyanin accumulation in apple fruits. It is definite that more complex regulatory mechanisms in ALA-induced fruit coloration need to be uncovered.

In addition to anthocyanins, ALA can promote many other species of flavonoid biosynthesis. In Ginkgo biloba, 10–100 mg L⁻¹ ALA induced total flavonoids and total polyphenol accumulation in leaves [264]. In apple leaves, ALA treatment induced flavonol accumulation in guard cells [265], which was consistent with that in the Arabidopsis cotyledons [235]. In apples, rhizospheric application of ALA can increase fruit flavonoids including cyanidin-3-galactoside, proanthocyanidin B2, quercetin, and kaempferol [266]. MdSCL8, a novel TF selected from an ALA-induced cDNA library, has been demonstrated to be a negative regulator binding the promoter of flavonol synthase 1 (MdFLS1), whose expression is inhibited by exogenous ALA [267]. Conversely, the gene expressions of two other TFs, MdHY5 and MdMYB22, are up-regulated by ALA, which can interact with and upregulate MdFLS1 expression, responsible for ALA-induced flavonol accumulation in apple calli. However, neither MdHY5 nor MdMYB22 can interact with MdSCL8. Thus, ALA-induced positive regulators MdHY5 and MdMyb22 interact to activate the promoter of FLS1 at one site, while ALA-negatively regulating MdSCL8 may independently act at another site of the MdFLS1 promoter. Consequently, more dihydroflavonol is converted to flavonol.

Figure 3

A network model for ALA to transcriptionally regulate anthocyanin and flavonol accumulation in apple fruits. Both anthocyanins and flavonols are biosynthesized from phenylalanine, which is converted through several enzymatic reactions to form distinct flavonoids stored in the vacuoles. ALA has been found to upregulate gene expressions of many TFs, such as MYB9/10, MADS1, ERF78, Hsf3a, MYB110a, and WD40-280. The upregulated TFs then independently or combined with other TFs, such as ERF78 interaction with MYB1, Hsf3a interaction with MYB110a, and MYB110a interaction with WD40-280, upregulate different structural gene expressions. Among them, except CHI (encoding chalcone isomerase), the other genes beyond CHS (encoding Chalcone synthase), including F3H (encoding Flavanone 3-hydroxylase), DFR (encoding Dihydroflavonol-4-reductase), ANS (encoding Anthocyanidin synthase), UFGT (encoding UDP glucose: flavonoid-3-O-glucosyltransferase), GSTF12 (encoding Glutathione S-transferase F12), and MATE8 (encoding multidrug and toxic compound extrusion), are all upregulated by ALA signaling. Therefore, the biosynthesis and accumulation of anthocyanins in apples are promoted by ALA. On the other hand, SCL8, a new TF, negatively controls FLS1 (encoding Flavonol synthase) expression, while MYB22 and HY5 are the positive TFs. ALA downregulates SCL8 expression but upregulates MYB22 and HY5 expressions; consequently, MYB22 interaction with HY5 upregulates FLS1 expression to promote flavonol biosynthesis in apple fruits. (Combined from [247,248,249,250,251,267]).

[Figure omitted. See PDF]

5.4.7. As a Thinning Agent to Remove Excess Flowers in Orchards

Modern fruit production promotes thinning of excess flowers and fruits in order to improve fruit quality at harvest. However, manual thinning is too expensive. A stable and reliable chemical thinning agent is expected. Since ALA has been suggested to be an herbicide [1], we imagined it might be applied to kill flowers. When 600–1200 mg L⁻¹ ALA solutions were sprayed at the end of full bloom, many flowers of pear were removed, increasing the leaf photosynthetic rate, and improving fruit quality [268]. In a Petri dish, ALA at 100–200 mg L⁻¹ was effective in blocking pollen germination and tube growth [269], which might be the cause of flower abscission with a lower setting rate. It was found that ALA promoted Ca²⁺-ATPase activity in pollen to efflux Ca²⁺ and reduced Ca²⁺ concentrations in pollen [270], which was responsible for vesicle trafficking and pollen growth inhibition. In addition to pear, ALA can also thin the flowers of other fruit trees such as apple [271] and peach (Wang et al., unpublished). Since it is necessary for the ALA solution to fall on the stigma to block pollen germination and tube growth, the thinning effect of ALA is effective for the flowers pollinated within 12 h; therefore, it cannot thin the unopened flowers and those pollinated the day before ALA spraying [269]. This feature allows ALA to be safely used in fruit production without excessive thinning.

5.4.8. Promoting Nitrogen Absorption and Conversion with Less Nitrate Accumulation in Plant Tissues

Many vegetables belong to nitrophilic crops, which do not grow well under nitrate shortage conditions. However, excessive application of nitrate will not only lead to nitrate accumulation in the soil but also cause excessive nitrate accumulation in vegetables. Since nitrate can be converted into nitrosamines, which are potential carcinogens, the nitrate content in vegetables is an obligatory limit for food safety. How to control nitrate amounts is important not only for plant growth but also for environmental protection and food safety. Mishra and Srivastava [272] first observed that ALA could improve the nitrate reductase activity in isolated maize leaves. In pakchoi, ALA increased the total nitrogen content by about 18%, suggesting that it promoted nitrate absorption [242]. Nevertheless, ALA decreased the nitrate content of plant tissues because it upregulated the nitrate reductase (NR) gene expression with higher NR activity, which reduced the nitrate content but improved the free amino acid and soluble protein content. Therefore, ALA can be used for high-quality, efficient, and safe production of vegetable crops. A recent study showed that both endogenous and exogenous ALA can promote the leaf chlorophyll content and chlorophyll fast fluorescence characteristics of canola grown under different nitrate levels, suggesting that ALA promotes nitrogen metabolism and photosynthesis [273].

6. Conclusions and Prospects

The potential application of ALA in agricultural biology was originally proposed by scholars of the USA [32,41], which was further promoted in plant production by Japanese scholars [2,3,134]. Nowadays, many research groups in different countries, especially China, are contributing to the study. In addition to several reviews [7,10,11,12], hundreds of research articles have been published. Thus, we know better than before that ALA has a wide range of physiological regulatory activities and has important application prospects in agroforestry at different dosages. ALA is essential for all lives. It is natural, non-toxic, and environmentally friendly. It can be applied to almost all green plants, from spirulina and kelp to herbs and tall trees. In our lab, we have tested nearly 100 crops/plants. It can be sprayed on the leaves or fruits, watered to the roots, or drip-irrigated through an integrated water and fertilizer system. It can be used to treat seeds, young seedlings, developing plants, or preharvest products. It can be mixed with common acidic or neutral agents without increasing the cost of use, making it popular with users. Yet, the amount of ALA does not require too much. Too much will kill lives (such as herbs, insects, and pathogen microbes), while the appropriate dosage application can play an important regulatory role in modifying life status and improving life vitality. In plant production, ALA can promote primary and secondary metabolism, root and shoot growth, vegetative and reproductive growth, yield and quality, growth, and survival. ALA promotes nutritive element absorption but limits toxic or harmful ion take-in and transport to the sensitive organs. The plants treated by ALA grow healthier and stronger, which facilitates a decrease in the use of chemical fertilizers and pesticides in production. ALA can promote stomatal opening under stressful conditions, which permits atmospheric CO₂ entrance into leaves for photosynthesis under stress conditions, which is conducive to biological carbon drop. All of these have never been imagined before. ALA can be biologically produced at low cost; however, the use of high concentrations as an herbicide or insecticide is still not mature. On the contrary, the application of low concentrations of ALA as a plant growth regulator in agroforestry production has become a realistic possibility. Therefore, we summarized the actual application of ALA in Figure 4.

ALA is a magical substance. However, many of the underlying regulatory mechanisms are not well understood. It can promote gene expression, including structural genes or TFs. It can also promote protein synthesis and phosphorylation/dephosphorylation. It must have its signal routes to regulate plant physiological and biochemical processes. However, up until now, we did not know how ALA works. Someone guessed whether ALA possesses its specific receptors [12], but nothing is known up to now. Our studies have shown that it is not ALA but its conversion into tetrapyrroles to improve salt tolerance [4] or fruit coloration [250]. Then how exactly does ALA work? More studies in this direction need to be conducted. ALA is not a plant hormone. However, its regulatory activity is stronger than that of any hormones (such as IAA, CTK, and ABA). It has been reported that 24-epibrassinolide [199,274] or jasmonic acid [115] is involved in ALA regulation. However, the underlying mechanisms are seldom known.

Nowadays, microbial fermentation to produce ALA is getting cheaper. We can apply it to agroforestry as we study. We can discover the problems in the application and solve them through research. We prospect for this simple non-protein amino acid with non-toxic, no residue, and environmental-friendly properties to improve plant stress resistance, promote crop growth, increase yield, improve quality, extend the storage period of fruits and vegetables, and play a more and more important role in agroforestry production and environmental protection.

Author Contributions

Conceptualization and writing original draft: L.W.; drafting: J.Z. (Jianting Zhang); providing materials, review, and editing: Y.Z., L.Z., H.Y., L.L., J.Z. (Jiayi Zhou), M.M.I. and X.G. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The data presented in this study are available upon request from the corresponding authors.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

AAO—Ascorbic acid oxidase; α-KG—α-Ketoglutaric acid; ALA—5-Aminolevulinic acid; ALAS—ALA synthase; APX—Ascorbate peroxidase; AsA—Ascorbate acid; CAS—Chemical Abstracts Service; CAT—Catalase; C_i—Intercellular CO₂ concentration; CPG III—Coproporphyrin III; CTK—cytokinin; D—Antenna thermal dissipation; DDC—Diethyl dithiocarbamate sodium; DHA—Oxidized ascorbate acid; DHAR—Dehydroascorbic acid reductase; E—Transpiration rate; ETR—Apparent photosynthetic electron transfer rate; FDPase—Fructose-1,6-diphosphatase; F_m—Maximum fluorescence; F_o—Initial fluorescence; F_v—Variable fluorescence; GluRS—Glutamate tRNA^Glu synthase; GluTR—Glutamyl tRNA^Glu reductase; GR—Glutathione reductase; G_s—Stomatal conductance; GSA—glutamate-1-semialdehyde; GSH—Reduced glutathione; GSSG—Oxidized glutathione; HMB—Hydroxymethylbilane; IAA—Auxin; LA—Levulinic acid; L_PFD—Relative photosynthetic limit; MDA—Malondialdehyde; MDHAR—Monodehydroascorbic acid reductase; MV—Methyl viologen; NO—Nitric oxide; NPQ—Non-photochemical fluorescence quenching; NR—Nitrate reductase; OEC—Oxygen-evolving complex; PBG—Porphobilinogen; P_c—Energy distribution in photochemistry; PCR—Photochemical rate; PEG—Polyethylene glycol; PFD—Photon flux density; PI_ABS—photosynthetic performance index; PIP—Plasmalemma intrinsic proteins; P_n—Net photosynthetic rate; POD—Peroxidase; PP IX—Protoporphyrin IX; PS—Photosystem; q_P—Photochemical fluorescence quenching; ROS—Reactive oxygen species; RuBPCase—Ribulose-1, 5-bisphosphate carboxylase; SOD—Superoxide dismutase; TCA—Tricarboxylic acid; TDPH—Tetrapyrrole-dependent photodynamic herbicides; TIP—Tonoplast intrinsic proteins; TF—Transcription factor; URO III—Uroporphyrinogen III; UV-B—Ultraviolet-B; WUE—Water use efficiency; Φ_PSII—Actual photochemical efficiency of PSII.

Footnotes

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Figures

Figure 1. The pathways of ALA biosynthesis and metabolism, where the left is the C4 pathway, which occurs in mitochondria, while the right is the C5 pathway, which occurs in plastids. D-glucose is considered the foremost precursor for biosynthesis and is converted to acetyl CoA through the glycolytic pathway, entering mitochondria and participating in the tricarboxylic acid cycle. On the one hand, the obtained α-ketoglutarate is converted to glutamate, which becomes the initial precursor in the C5 pathway. On the other hand, it is converted to succinyl CoA, one of the precursors of the C4 pathway. Succinyl CoA is further condensed with glycine to synthesize ALA, catalyzed by ALA synthase (ALAS, encoded by HemA). Then, ALA can be transmembrane transported out of the mitochondria by a transporter called RhtA to transform CPG III through several step reactions, which then return to the mitochondria and continue to form Protogen IX and other porphyrin compounds, including heme. The heme can exert negative feedback on ALAS to inhibit ALA synthesis. In the C5 pathway, glutamate is converted to ALA catalyzed by Glu-tRNA synthetase (encoded by GltX), Glu-tRNA reductase (encoded by HemA), and GSA aminotransferase (encoded by HemL) in the stroma of plastids. Then, ALA is in turn metabolized by ALA dehydratase (encoded by HemB), porphobilinogen deaminase (encoded by HemC), uroporphyrinogen III synthase (encoded by HemD), uroporphyrinogen decarboxylase (encoded by HemD), coproporphyrinogen III oxidase (encoded by HemE), protoporphyrin oxidase (encoded by HemG), and ferrochelatase (encoded by HemH) to form heme. More enzymes and genes are needed for chlorophyll biosynthesis. During the process, CPG III enters the thylakoid grana to convert to protogen IX and PP IX, then further synthesize chlorophylls in situ but heme in the stroma. Thus, ALA biosynthesis is involved in the stroma of mitochondria or plastids, but its metabolism is additionally involved in the membrane or cytosol. (Drawn according to [9,14]).

Figure 2. A working model for ALA to improve salt tolerance by interception of salt ion/anion in the roots of strawberries to prevent leaf injury. For Na+, ALA induces reciprocal accumulation of NO and H2O2 to upregulate SOS1, NHX1, and HKT1 expression to promote Na+ exclusion out of root cells, sequestration into vacuoles, and re-extraction to parenchyma cells from xylem sap. For Cl−, MYB44 is a negative regulator for CLC genes. Salt stress induces MYB44 expression to block CLC expression, while ALA downregulates MYB44 expression. Therefore, when ALA is present, the CLC gene is expressed, and the uniport transports Cl− into vacuoles or re-extracts it from xylem sap. Therefore, a great amount of Na+ and Cl− is intercepted in the roots, with less accumulation in the leaves. The intercepted ion and anion in roots facilitate water absorption while preventing leaf injury (improved from [220,221]).

Figure 4. Application of ALA in promoting growth, increasing production, improving product quality, enhancing stress tolerance, and reducing pesticides and chemical fertilizers used in green agroforestry production.

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