Abnormal expression of bHLH3 disrupts a flavonoid homeostasis network,causing differences in pigment composition among mulberry fruits

Mulberry fruits with high concentrations of anthocyanins are favored by consumers because of their good taste,bright color,and high nutritional value.However,neither the regulatory mechanism controlling flavonoid biosynthesis in mulberry nor the molecular basis of different mulberry fruit colors is fully understood.Here,we report that a flavonoid homeostasis network comprising activation and feedback regulation mechanisms determines mulberry fruit color.In vitro and in vivo assays showed that MYBA-bHLH3-TTG1 regulates the biosynthesis of anthocyanins,while TT2L1 and TT2L2 work with bHLH3 or GL3 and form a MYB-bHLH-WD40(MBW)complex with TTG1 to regulate proanthocyanidin(PA)synthesis.Functional and expression analyses showed that bHLH3 is a key regulator of the regulatory network controlling mulberry fruit coloration and that MYB4 is activated by MBW complexes and participates in negative feedback control of the regulatory network to balance the accumulation of anthocyanins and proanthocyanidins.Our research demonstrates that the interaction between bHLH3 and MYB4 in the homeostasis regulatory network ensures that the fruits accumulate desirable flavonoids and that this network is stable in pigmentrich mulberry fruits.However,the abnormal expression of bHLH3 disrupts the balance of the network and redirects flavonoid metabolic flux in pale-colored fruits,resulting in differences in the levels and proportions of anthocyanins,flavones,and flavonols among differently colored mulberry fruits(red,yellow,and white).The results of our study reveal the molecular basis of the diversity of mulberry fruit colors.

Dynamic changes in transposable element and gene methylation in mulberry (Morus notabilis) in response to Botrytis cinerea

DNA methylation has been proposed to regulate plant stress resistance.However,the dynamic changes in DNA methylation in woody plants and their correlations with pathogenic responses are not fully understood.Here,we present single-base maps of the DNA methylomes of mulberry(Morus notabilis)leaves that were subjected to a mock treatment or inoculation with Botrytis cinerea.Compared with the former,the latter showed decreased mCG and mCHG levels and increased mCHH levels.DNA methylation inhibitors reduced resistance gene methylation levels and enhanced mulberry resistance,suggesting that the hypomethylation of resistance genes affects mulberry resistance to B.cinerea.Virus-induced gene silencing of MnMET1 enhanced the expression of mulberry-resistance genes,thereby increasing the plant’s resistance to B.cinerea.We also found that MITEs play a dominant role in controlling DNA methylation levels.MITEs appear to be the main sources of 24-nt siRNAs that regulate gene expression through the RNA-directed DNA methylation pathway.

Genome-wide identification and expression analyses of cytochrome P450 genes in mulberry(Morus notabilis)

Cytochrome P450 S play critical roles in the biosynthesis of physiologically important compounds in plants.These compounds often act as defense toxins to prevent herbivory.In the present study,a total of 174 P450 genes of mulberry（Morus notabilis C.K.Schn） were identified based on bioinformatics analyses.These mulberry P450 genes were divided into nine clans and 47 families and were found to be expressed in a tissue-preferential manner.These genes were compared to the P450 genes in Arabidopsis thaliana.Families CYP80,CYP92,CYP728,CYP733,CYP736,and CYP749 were found to exist in mulberry,and they may play important roles in the biosynthesis of mulberry secondary metabolites.Analyses of the functional and metabolic pathways of these genes indicated that mulberry P450 genes may participate in the metabolism of lipids,other secondary metabolites,xenobiotics,amino acids,cofactors,vitamins,terpenoids,and poiyketides.These results provide a foundation for understanding of the structures and biological functions of mulberry P450 genes.

Identification and characterization of genes involved in the jasmonate biosynthetic and signaling pathways in mulberry(Morus notabilis)

Jasmonate （JA） is an important phytohormone regulating growth, development, and environmental response in plants, particularly defense response against herbivorous insects. Recently, completion of the draft genome of the mulberry （Morus notabilis） in conjunction with genome sequencing of silkworm （Bombyx mori） provides an opportuni-ty to study this unique plant-herbivore interaction. Here, we identified genes involved in JA biosynthetic and signaling pathways in the genome of mulberry for the first time, with the majority of samples showing a tissue-biased expression pattern. The analysis of the representative genes 12-oxophy-todienoic acid reductase （OPRs） and jasmonate ZIM-domain （JAZs） was performed and the results indicated that the mulberry genome contains a relatively smal number of JA biosynthetic and signaling pathway genes. A gene encoding an＆amp;nbsp;important repressor, MnNINJA, was identified as an alternative splicing variant lacking an ethylene-responsive element binding factor-associated amphiphilic repression motif. Having this fundamental information wil facilitate future functional study of JA-related genes pertaining to mulberry-silkworm interactions.

Two homologous INDOLE-3-ACETAIVHDE (IAM) HYDROLASE genes are required for the auxin effects of IAM in Arabidopsis

Indole-3-acetamide(IAM)is the first confirmed auxin biosynthetic intermediate in some plant pathogenic bacteria.Exogenously applied IAM or production of IAM by overexpressing the bacterial iaaM gene in Arabidopsis causes auxin overproduction phenotypes.However,it is still inconclusive whether plants use IAM as a key precursor for auxin biosynthesis.Herein,we reported the isolation IAM HYDROLASE 1(IAMH1)gene in Arabidopsis from a forward genetic screen for IAM-insensitive mutants that display normal auxin sensitivities.IAMH1 has a close homolog named IAMH2 that is located right next to IAMH1on chromosomeⅣin Arabidopsis.We generated iamh1 iamh2 double mutants using our CRISPR/Cas9gene editing technology.We showed that disruption of the IAMH genes rendered Arabidopsis plants resistant to IAM treatments and also suppressed the iaaM overexpression phenotypes,suggesting that IAMH1 and IAMH2 are the main enzymes responsible for converting IAM into indole-3-acetic acid(IAA)in Arabidopsis.The iamh double mutants did not display obvious developmental defects,indicating that IAM does not play a major role in auxin biosynthesis under normal growth conditions.Our findings provide a solid foundation for clarifying the roles of IAM in auxin biosynthesis and plant development.