5-Aminolevulinic acid (ALA), a newly proved natural plant growth regulator, is well known to improve plant photosynthesis under both normal and stressful conditions. However, its underlying mechanism remains largely unknown. Stomatal closure is one of the major limiting factors for photosynthesis and abscisic acid (ABA) is the most important hormone in provoking stomatal closing. Here, we showed that ALA significantly inhibited ABA-induced stomatal closure using wild-type and ALA-overproducing transgenic Arabidopsis (YHem1). We found that ALA decreased ABA-induced H2O2 and cytosolic Ca2+ accumulation in guard cells with stomatal bioassay, laser-scanning confocal microscopy and pharmacological methods. The inhibitory effect of ALA on ABA-induced stomatal closure was similar to that of AsA (an important reducing substrate for H2O2 removal), CAT (a H2O2-scavenging enzyme), DPI (an inhibitor of the H2O2-generating NADPH oxidase), EGTA (a Ca-chelating agent), and AlCl3 (an inhibitor of calcium channel). Furthermore, ALA inhibited exogenous H2O2- or Ca2+-induced stomatal closure. Taken together, we conclude that ALA inhibits ABA-induced stomatal closure via reducing H2O2, probably by scavenging, and Ca2+ levels in guard cells. Moreover, the inhibitive effect of ALA on ABA-induced stomatal closure was further confirmed in the whole plant. Finally, we demonstrated that ALA inhibits stomatal closing, but significantly improves plant drought tolerance. Our results provide valuable information for the promotion of plant production and development of a sustainable low-carbon society.

5-aminolevulinic acid (ALA), a new plant growth regulator, can inhibit stomatal closure by reducing H2O2 accumulation in guard cells. Flavonols are a main kind of flavonoids and have been proposed as H2O2 scavengers in guard cells. 5-aminolevulinic acid can significantly improve flavonoids accumulation in plants. However, whether ALA increases flavonols content in guard cells and the role of flavonols in ALA-regulated stomatal movement remains unclear. In this study, we first demonstrated that ALA pretreatment inhibited ABA-induced stomatal closure by reducing H2O2 accumulation in guard cells of Arabidopsis seedlings. This result confirms the inhibitory effect of ALA on stomatal closure and the important role of decreased H2O2 accumulation in this process. We also found that ALA significantly improved flavonols accumulation in guard cells using a flavonol-specific dye. Furthermore, using exogenous quercetin and kaempferol, two major components of flavonols in Arabidopsis leaves, we showed that flavonols accumulation inhibited ABA-induced stomatal movement by suppressing H2O2 in guard cells. Finally, we showed that the inhibitory effect of ALA on ABA-induced stomatal closure was largely impaired in flavonoid-deficient transparent testa4 (tt4) mutant. In addition, exogenous flavonols recovered stomatal responses of tt4 to the wild-type levels. Taken together, we conclude that ALA-induced flavonol accumulation in guard cells is partially involved in the inhibitory effect of ALA on ABA-induced H2O2 accumulation and stomatal closure. Our data provide direct evidence that ALA can regulate stomatal movement by improving flavonols accumulation, revealing new insights into guard cell signaling.

The red color is an attractive trait of fruit and determines its market acceptance. 5-Aminolevulinic acid (ALA), an eco-friendly plant growth regulator, has played a universal role in plant secondary metabolism regulation, particularly in flavonoid biosynthesis. It has been widely reported that ALA can up-regulate expression levels of several structural genes related to flavonoid metabolism and anthocyanin accumulation. However, the molecular mechanisms behind ALA-induced expression of these genes are complicated and still far from being completely understood. In this study, transcriptome analysis identified the differentially expressed genes (DEGs) associated with ALA-induced anthocyanin accumulation. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the flavonoid biosynthesis (ko00941) pathway was significantly enhanced in the ALA-treated apple calli at 24, 48, and 72 h after the treatment. Expression pattern revealed that ALA up-regulated the expression of the structural genes related to not only anthocyanin biosynthesis (MdCHS, MdCHI, MdF3’H, MdDFR, MdANS, and MdUFGT) but also anthocyanin transport (MdGST and MdMATE). Two R2R3-MYB transcription factors (MdMYB10 and MdMYB9), which are the known positive regulators of anthocyanin biosynthesis, were significantly induced by ALA. Gene overexpression and RNA interference assays demonstrated that MdMYB10 and MdMYB9 were involved in ALA-induced anthocyanin biosynthesis. Moreover, MdMYB10 and MdMYB9 might positively regulate the transcription of MdMATE8 by binding to the promoter region. These results indicate that MdMYB10 and MdMYB9 modulated structural gene expression of anthocyanin biosynthesis and transport in response to ALA-mediated apple calli coloration at the transcript level. We herein provide new details regarding transcriptional regulation of ALA-induced color development.

Chemical fruit thinning has become a popular practice in modern fruit orchards for achieving high quality fruits, reducing costs of hand thinning and promoting return bloom. However, most of the suggested chemical thinners are often concerned for their detrimental effects and environmental problems. 5-Aminolevulic acid (ALA) is a natural, nontoxic, biodegradable, and environment-friendly plant growth regulator. One of its outstanding roles is improving plant photosynthesis and fruit quality. Here, results showed that applying 100–200 mg/L ALA at full bloom stage significantly reduced pear fruit set. Both in vivo and in vitro studies showed that ALA significantly inhibited pollen germination and tube growth. ALA decreased not only cytosolic Ca2+ concentration ([Ca2+]cyt) but also “tip-focused” [Ca2+]cyt gradient, indicating that ALA inhibited pollen tube growth by down-regulating calcium signaling. ALA drastically enhanced pollen Ca2+-ATPase activity, suggesting that ALA-induced decrease of calcium signaling probably resulted from activating calcium pump. The significant negative correlations between Ca2+-ATPase activity and pollen germination or pollen tube length further demonstrated the critical role of calcium pump in ALA's negative effect on pollen germination. Taken together, our results suggest that ALA at low concentrations is a potential biochemical thinner, and it inhibits pollen germination and tube growth via Ca2+ efflux by activating Ca2+-ATPase, thereby thinning fruits by preventing fertilization.

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