Effects of copper and manganese elements on the growth and accumulation of three phenolic compounds in tissue-cultured seedlings of Vaccinium dunalianum

Objective This study aimed to investigate the effects of two trace elements copper （Cu） and manganese （Mn） on the growth of Vaccinium dunalianum tissue-cultured seedlings and the accumulation of three major phenolic compounds 6'-O-caffeoyl arbutin （CA），β-arbutin （Arb），and chlorogenic acid （CGA）， so as to provide a reference for elucidating the mechanism by which trace elements regulate the growth of tissue-cultured seedlings and targeted accumulation of phenolic compounds in Vaccinium dunalianum.Method Tissue-cultured seedlings of Vaccinium dunalianum were used as experimental materials. Based on the concentrations of Cu and Mn trace elements in the 1/4 MS medium， five concentration treatments （0， 0.5， 1.0， 1.5， and 2.0×） for each trace element were established. After 70 d of cultivation，growth indicators including plant water content，plant height，root length，rooting rate，and number of roots per plant，as well as the contents of Arb，CGA， and CA were measured. The relative expression of genes associated with the biosynthesis of these three phenolic compounds were determined by real-time fluorescence quantitative PCR.Result With increasing Cu concentration in the media， the rooting rate and root length of Vaccinium dunalianum tissue-cultured seedlings exhibited a gradual decreasing trend. The rooting rate， root length， and number of roots per plant in the Cu（2.0） treatment were significantly lower than those in the Cu（1.0） treatment （P<0.05， the same below）. Except for the Cu（2.0） treatment， the plant height was significantly lower in all other treatments compared with Cu（1.0） treatment， and the Cu（1.0） treatment exhibited the highest water content at 89.39%. The Mn（2.0） treatment showed significantly higher rooting rate， root length， and number of roots per plant than the other three treatments did， except for the Mn（1.5） treatment. Under both elements treatments， Vaccinium dunalianum tissue-cultured seedlings exhibited optimal growth performance in the 1.0× groups， with the three phenolic compounds ranked as CA>Arb>CGA. Under the Cu（2.0） treatment， the highest Arb and CA contents in Vaccinium dunalianum were observed， at 2.71 and 30.81 mg/g， respectively. The Mn（1.0） treatment yielded the highest CGA content， which significantly exceeded that of the other four treatments. Under the Cu（2.0） treatment and compared with the Cu（1.0） treatment， the relative expressions of VdAS-1 and VdAS-2 genes were significantly decreased and the relative expression of VdAS-4 gene was significantly increased； the relative expression of VdAT-3 gene was significantly decreased under Cu（0）， Cu（1.5） and Cu（2.0） treatments， while only the relative expression of VdCCoAOMT-2 gene was significantly increased under Cu（2.0） treatment. Under Mn（2.0） treatment and compared with the Mn（1.0） treatment， the relative expressions of VdAS-1 and VdAS-2 genes were significantly decreased， the relative expression of VdAS-4 gene was significantly increased， and the relative expression of VdAT-1 gene was significantly decreased. However， under the Mn（2.0） treatment， the relative expression of VdAT-3 gene was significantly increased， the relative expressions of the VdC3H-2， VdC3H-3， VdCCoAOMT-1 and VdHCT genes were significantly decreased， while those of VdCCoAOMT-2 and VdCCoAOMT-3 were significantly increased.Conclusion Excessive or deficient Cu inhibits the growth of Vaccinium dunalianum tissue-cultured seedlings. A 2.0× Mn concentration promotes roots development but suppresses the growth of above-ground parts of Vaccinium dunalianum tissue-cultured seedlings. The two phenolic compounds CA and Arb play crucial roles in Vaccinium dunalianum defence against Cu and Mn stresses.