5-Aminolevulinic acid (ALA) is a natural plant growth regulator that promotes plant freezing tolerance. The WRKY family consists of plant-specific transcription factors (TFs) associated with abiotic stress responses. Up to now, whether WRKYs are involved in ALA-induced plant freezing tolerance and the underlying mechanism is not clear. In this study, we found that pretreatment with 50 mg·L⁻¹ ALA one week earlier significantly increased the freezing tolerance of nectarine (Prunus persica var. nectarina) pistils with higher antioxidant enzyme activity and osmotic solutes when the floral twigs were stressed by −3 ℃ for 6 h. ALA also enhanced the expression of PpWRKY18, PpCBF1, PpCOR1, and several genes encoding antioxidant enzymes (such as superoxide dismutase, peroxidase, and catalase) and pyrroline-5-carboxylate synthase (P5CS). When PpWRKY18 was overexpressed in tobacco, the transgenic plants exhibited greater freezing tolerance, which was further promoted by exogenous ALA. Y2H, Pull-down, BiFC, and LCI analyses revealed that PpWRKY18 interacts with PpCBF1, promoting the latter transcriptional activity. Additionally, Y1H experiments showed that PpWRKY18 directly binds to the promoter of PpPOD41 while PpCBF1 binds to the promoters of PpP5CS1 and PpCOR1, activating the target gene expressions. Furthermore, we established a yeast library using the promoter of PpWRKY18 as the bait to screen the upstream regulatory factors. By library screening, Y1H, DLR, and EMSA, we found that PpC3H37, a zinc finger protein, was responsive to chilling and ALA treatment, and as a transcription factor, it activated PpWRKY18 expression by directly binding to the promoter. Taken together, we reveal a regulatory network where ALA induces upregulation of PpC3H37 expression, which positively regulates PpWRKY18 expression. Subsequently, the regulatory pathway diverges into two branches. The first is CBF-dependent, where PpCBF1 interacts with PpWRKY18, binding the promoters of PpP5CS1 and PpCOR1. The second is CBF-independent, where PpWRKY18 directly binds the promoter of PpPOD41 to upregulate the gene expression and increase the antioxidant enzyme activity and freezing tolerance. These findings provide a novel insight of the mechanism of ALA in regulating the cold hardiness of nectarine pistil.

5-Aminolevulinic acid (ALA), is a novel plant growth regulator that can enhance plant tolerance against salt stress. However, the molecular mechanism of ALA is not well studied. In this study, ALA improved salt tolerance of apple (Malus × domestica 'Gala') when the detached leaves or cultured calli were used as the materials. The expression of MdWRKY71, a WRKY transcription factor (TF) gene was found to be responsive to NaCl as well as ALA treatment. Functional analysis showed that overexpressing (OE)-MdWRKY71 significantly improved the salt tolerance of the transgenic apple, while RNA interfering (RNAi)-MdWRKY71 reduced the salt tolerance. However, exogenous ALA alleviated the salt damage in the RNAi-MdWRKY71 apple. When MdWRKY71 was transferred into tobacco, the salt tolerance of transgenic plants was enhanced, which was further improved by exogenous ALA. Subsequently, MdWRKY71 bound to the W-box of promoters of MdSOS2, MdNHX1, MdCLC-g, MdSOD1, MdCAT1 and MdAPX1, transcriptionally activating the gene expressions. Since the genes are responsible for Na⁺ and Cl⁻ transport and antioxidant enzyme activity respectively, it can be concluded that MdWRKY71, a new TF, is involved in ALA-improved salt tolerance by regulating ion homeostasis and redox homeostasis. These results provided new insights into the transcriptional regulatory mechanism of ALA in enhancing apple salt tolerance.

5-Aminolevulinic acid (ALA) is a novel plant growth regulator that has shown outstanding capability to promote stomatal opening. Starch degradation, catalyzed by β-amylase (EC3.2.1.2, BAM), plays an important role in stomatal opening. However, whether the starch breakdown is involved in ALA-regulating stomatal movement is unclear. In the current study, we found that exogenous ALA effectively stimulated the starch breakdown in guard cells, increased β-amylase activity and promoted stomatal opening in leaves of apple (Malus × domestica). Based on genome-wide identification, we identified a total of 119 members of BAM gene family in ten commonly Rosaceae crops. Analyses of gene structure, motif identification, and gene pair collinearity revealed relative conservation among members within the same group or subgroup. Among these genes, MdBAM17 and other 12 genes were identified as the orthologous genes of AtBAM1, which is responsible for starch degradation to modulate the stomatal movement in Arabidopsis. qRT-PCR analysis revealed a positive correlation between the expressions of MdBAM17 and stomatal aperture, as well as β-amylase activity, whereas a negative correlation was observed with the starch content. Subcellular localization analysis confirmed that MdBAM17 is a chloroplast protein, consistent with the AtBAM1. MdBAM17 was mainly expressed in guard cells and responsive to exogenous ALA. Overexpressing MdBAM17 increased β-amylase activity and promoted starch breakdown, leading to stomatal opening, which was further strengthened by ALA. RNA-interfering MdBAM17 decreased β-amylase activity, resulting in starch accumulation, and impairing the stomatal opening by ALA. However, modulation of MdBAM17 expression did not affect the levels of flavonols and H₂O₂ in guard cells, suggesting that MdBAM17-promoted starch degradation may function at downstream of ROS signaling in the ALA-regulated stomatal opening. Our findings provide new insights into the mechanisms of ALA-regulated stomatal movement.

Strawberry Fusarium wilt (SFW) is a systematic soil-borne disease caused by Fusarium oxysporum f. sp. fragaria (Fof), which infects the vascular bundles, blocking water and nutrient transport from roots to the aboveground. It is a severe pathogen which spreads rapidly and destroys strawberry production. Finding a way to control this disease is of great scientific value and practical importance. In this study, three fungi were isolated from the vascular tissues of sick strawberries in the field. After DNA sequencing, they were identified as Fof, Aspergillus fumigatus and Trichoderma harzianum, respectively, among which the first two are pathogens and the third is a probiotic. All fungi were controlled by thiophanate-methyl (TM), a commercial fungicide. On PDA medium, 20 mg·L⁻¹5-aminolevulinic acid (ALA), a natural non-protein amino acid, promoted T. harzianum proliferation, but inhibited Fof and A. fumigatus. In confrontation test, the growth of Fof or A. fumigatus was inhibited by T. harzianum and exogenous ALA promoted T. harzianum growth but significantly inhibited the pathogen growth. When three species of fungi were separately or combinedly inoculated on healthy strawberry plants, T. harzianum promoted plant growth and development while Fof or A. fumigatus caused growth retardation, where Fof directly caused leaf yellowing and plant wilting. When the plants inoculated with different fungus were treated with ALA, the results turned out that ALA alleviated SFW symptoms by bidirectionally promoting T. harzianum proliferation and inhibiting Fof and A. fumigatus. Thus, ALA might be used in comprehensively controlling SFW in strawberry industry.

5-Aminolevulinic acid (ALA) can inhibit abscisic acid (ABA)-induced stomatal closure. However, the molecular mechanism is unclear. In this study, we found that ALA upregulated the MdPP2AC expression and PP2A activity in the apple (Malus domestica Borkh. cv. ‘Fuji’) leaves. With the promoter of MdPP2AC as bait, a diacylglycerol kinase MdDGK3-like was selected by the Yeast One Hybrid (Y1H) from the cDNA library of the epidermis of apple leaves treated by exogenous ALA. Additional to binding the promoter of MdPP2AC, MdDGK3-like was found to inhibit the transcription activity of MdPP2AC promoter, while ALA significantly eliminated the role of MdDGK3-like. In tobacco leaves, MdDGK3-like was localized in the nucleus of stomatal guard cells. Therefore, MdDGK3-like might act as a transcription factor negatively regulating MdPP2AC expression and causing stomatal closure. To further identify MdDGK3-like functions, several transiently transgenic apple leaves (including overexpression and interference) were established. The results revealed that overexpression of MdDGK3-like promoted stomatal closure by increasing Ca²⁺ and H₂O₂ and decreasing flavonol levels in the guard cells. Conversely, MdDGK3-like (ⅰ) led the stomatal opening with lower levels of Ca²⁺ and H₂O₂ but higher flavonols. Based on these, we proposed a new hypothesis that ALA up-regulated MdPP2AC expression via negatively regulating the expression of MdDGK3-like to up-regulate PP2A expression and the enzyme activity, which improved the stomatal aperture. Since it was the first time that MdDGK3-like was showed to act as a transcription factor, the proposed model provided a new insight onto the mechanisms of ALA-induced stomatal opening.