Fulvic acid alleviates cadmium-induced root growth inhibition by regulating antioxidant enzyme activity and carbon-nitrogen metabolism in apple seedlings.

Introduction: Substantial previous studies have reported that fulvic acid (FA) application plays an important role in Chinese agricultural production. However, little is known about the mechanisms for using FA to increase apple trees resistance to Cd toxicity. In order to clarify the mechanism underlying FA alleviation in Cd-induced growth inhibition in apple seedlings. Methods: Herein, we treated M9T337 seedlings to either 0 or 30 µM/L Cd together with 0 or 0.2 g/L FA and analyzed the root growth, antioxidant enzyme activities, carbon (C) assimilation, nitrogen (N) metabolism, and C and N transport. Results: The results presented that, compared with CK (without Cd addition or FA spraying application), Cd poisoning significantly inhibited the root growth of apple seedlings. However, this Cd-induced root growth inhibition was significantly alleviated by FA spraying relative to the Cd treatment (Cd addition alone). On the one hand, the mitigation of inhibition effects was due to the reduced oxidative damage by enhancing antioxdiant enzyme (SOD, POD, and CAT) activities in leaves and roots. On the other hand, this growth advantage demonstrated compared to the Cd treatment was found to be associated with the strengthen of photosynthetic performance and the elevation of C and N metabolism enzymes activities. Meanwhile, we also found that under Cd stress condition, the distribution of C and N nutrients in apple seedlings was optimised by FA spraying application relative to the Cd treatment, according to the results of 13C and 15N tracing. Conclusion: Conclusively, our results suggested that the inhibitory effect of Cd on apple seedlings root growth was alleviated by FA through regulating antioxdiant capacities and C and N metabolism.

The proanthocyanidin-specific transcription factor MdMYBPA1 initiates anthocyanin synthesis under low-temperature conditions in red-fleshed apples.

In plants, flavonoids play critical roles in resistance to biotic and abiotic stresses, and contribute substantially to the quality, flavor, and nutritional quality of many fruit crops. In apple (Malus × domestica), inbreeding has resulted in severe decreases in the genetic diversity and flavonoid content. Over the last decade, we have focused on the genetic improvement of apple using wild red-fleshed apple resources (Malus sieversii f. niedzwetzkyana). Here, we found that the MYB transcription factors (TFs) involved in the synthesis of proanthocyanidins can be classified into TT2 and PA1 types. We characterized a PA1-type MYB transcription factor, MdMYBPA1, from red-fleshed apple and identified its role in flavonoid biosynthesis using overexpression and knockdown-expression transgenes in apple calli. We explored the relationship between TT2- and PA1-type MYB TFs, and found that MdMYB9/11/12 bind the MdMYBPA1 promoter. In addition, MdMYBPA1 responded to low temperature by redirecting the flavonoid biosynthetic pathway from proanthocyanidin to anthocyanin production. In binding analyses, MdbHLH33 directly bound to the low-temperature-responsive (LTR) cis-element of the MdMYBPA1 promoter and promotes its activity. In addition, the calli expressing both MdMYBPA1 and MdbHLH33, which together form a complex, produced more anthocyanin under low temperature. Our findings shed light on the essential roles of PA1-type TFs in the metabolic network of proanthocyanidin and anthocyanin synthesis in plants. Studies on red-fleshed wild apple are beneficial for breeding new apple varieties with high flavonoid contents.

The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes.

KEY MESSAGE: MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33. Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1-5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.

MYB12 and MYB22 play essential roles in proanthocyanidin and flavonol synthesis in red-fleshed apple (Malus sieversii f. niedzwetzkyana).

Flavonoids are major polyphenol compounds in plant secondary metabolism. Wild red-fleshed apples (Malus sieversii f. niedzwetzkyana) are an excellent resource because of their much high flavonoid content than cultivated apples. In this work, R6R6, R6R1 and R1R1 genotypes were identified in an F1 segregating population of M. sieversii f. niedzwetzkyana. Significant differences in flavonoid composition and content were detected among the three genotypes by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analysis. Furthermore, two putative flavonoid-related genes encoding R2R3-MYB transcription factors, designated MYB12 and MYB22, were cloned and characterized. The expression patterns of MYB12 and MYB22 directly correlated with those of leucoanthocyanidin reductase and flavonol synthase, respectively. Their roles in flavonoid biosynthesis were identified by overexpression in apple callus and ectopic expression in Arabidopsis. MYB12 expression in the Arabidopsis TT2 mutant complemented its proanthocyanidin-deficient phenotype. Likewise, MYB22 expression in an Arabidopsis triple mutant complemented its flavonol-deficient phenotype. MYB12 could interact with bHLH3 and bHLH33 and played an essential role in proanthocyanidin synthesis. MYB22 was found to activate flavonol pathways by combining directly with the flavonol synthase promoter. Our findings provide a valuable perspective on flavonoid synthesis and provide a basis for breeding elite functional apples with a high flavonoid content.