An Apple MdBT2 Protein Regulates Plant Growth And Anthocyanin Accumulation In Response To Nitrate

Nitrogen is an important nutrient element for the growth and development of fruit trees,and it is one of the important factors affecting the plant growth and fruit quality.It is mainly absorbed by plants from the soil in the form of nitrate.Plants have evolved a variety of mechanisms during their growth and development to cope with the fluctuation of nitrate in the external environment.Nitrate is absorbed in the roots and transported to other organs through nitrate transporters.In addition to its nutritional function,nitrate may act as a type of signal molecule to trigger local and systemic signal transduction,and then regulate the growth and development processes of plants,such as seed germination,leaf development,branche growth,stomatal opening,and flowering time.In addition,nitrate signal transduction and its downstream physiological responses are closely related to plant hormones,such as IAA,CTK,and GA.Nitrate signals can be integrated into a complex regulatory network with various environmental and hormonal signals to regulate plant life processes.BTB-TAZ protein BT2,as a scaffold protein,can interact with different target proteins and participate in a variety of signal transduction pathways.Recent evidence also shows that BT2 plays a key role in nitrogen use efficiency regulation network,and is also involved in the regulation of plant growth and anthocyanin accumulation.However,the mechanism of MdBT2 regulating plant growth in response to nitrate and the molecular mechanism of regulating anthocyanin accumulation in response to nitrate deficiency in apple are still unclear.In order to further analyze the regulatory mechanism of MdBT2,we performed a Y2H screening assay with the MdBT2 protein as a bait to identify its interacting proteins in an apple c DNA library.As a result,two positive colonies that contains a c DNA fragment encoding the MdRGL3a and MdGRF11 were obtained.Here,it was found that MdBT2regulated plant growth and anthocyanin accumulation via genetic transformation and molecular biology techniques.The results are as follows:1.Nitrates promoted the growth of apple plants.After external application of GA₃,the inhibition of plant growth by nitrate deficiency was partially rescued,whereas exogenous application of GA biosynthesis inhibitor PAC noticeably inhibited nitrate-induced plant growth.2.Nitrate induced the transcript level of MdBT2,and MdBT2 was found to act as a positive regulator of plant growth.Under sufficient nitrate conditions,overexpression of MdBT2 promoted plant growth,whereas MdBT2 suppression transgenic apple lines exhibited a growth-inhibited phenotype.Exogenous GA₃ treatment partially rescued the growth inhibition caused by suppressing MdBT2,whereas PAC treatment partially eliminated the improved plant growth by the overexpressing MdBT2,indicating that MdBT2 may mediate GA-related pathways to regulate plant growth.3.Furthermore,Y2H,protein pull-down and BiFC assays verified the interaction between MdBT2 and DELLA protein MdRGL3a.The N-terminal BTB and BACK domains of MdBT2 and the C-terminal GRAS domain of MdRGL3a were responsible for their interaction.Nitrate treatment induced ubiquitination and degradation of MdRGL3a protein.However,suppression of MdBT2 in MdBT2-Anti13 transgenic apple plantlets significantly inhibited nitrate-induced MdRGL3a degradation,indicating that nitrate induced the degradation of MdRGL3a at least partially dependent on MdBT2.Moreover,MdBT2-mediated degradation of MdRGL3a protein did not depend on the GA pathway.4.Phenotypic assays indicated that BT and DELLA proteins regulated plant growth in a conserved way among different plant species.Ectopic overexpression of MdBT2 in Arabidopsis partially rescued MdRGL3a-mediated growth inhibition,indicating that MdBT2negatively regulates MdRGL3a-mediated growth inhibition.5.Nitrate deficiency not only inhibited the expression of MdBT2 at the transcriptional level,but also promoted the degradation of MdBT2 through the 26S proteasome pathway.Under nitrate-deficiency conditions,MdBT2 overexpression transgenic apple plantlets accumulated less anthocyanins,whereas MdBT2 suppression lines accumulated more anthocyanins.6.Y2H,protein pull-down and BiFC assays verified that MdBT2 interacted with 14-3-3protein MdGRF11,and BTB and TAZ domains of MdBT2 were necessary for the interaction with MdGRF11.Nitrate deficiency induced the transcript level of MdGRF11.Phenotypic assays found that overexpression of MdGRF11 promoted the nitrate deficiency-induced anthocyanin biosynthesis in transgenic calli and Arabidopsis,whereas MdGRF11-Anti transgenic calli,which inhibited the expression of multiple MdGRFs genes,accumulated less anthocyanin.7.MdGRF11 promoted the ubiquitination of MdBT2 protein in vivo,and then MdBT2was degraded through the 26S proteasome pathway,thereby improving the stability of MdMYB1 protein.Phenotypic assays indicated that overexpression of MdGRF11 partially inhibited the MdBT2-mediated anthocyanin accumulation and the expression levels of anthocyanin biosynthesis-related genes MdF3H,MdDFR,MdANS and MdUF3GT,whereas suppression of MdGRF11 aggravated the phenotype of decreased anthocyanin accumulation in MdBT2-OX transgenic calli.The results indicated that MdGRF11 negatively regulates MdBT2-mediated anthocyanin biosynthesis.Moreover,the fruits transient injection assay and leaves transient infection assay also proved the same result.In conclusion,our study indicated that MdBT2 negatively regulates the stability of MdRGL3a protein,thereby promoting nitrate-induced plant growth.Meanwhile,MdGRF11enhances the stability of MdMYB1 protein by promoting the ubiquitination degradation of MdBT2,and then participates in nitrate deficiency-induced anthocyanin accumulation.