Fig. 5: AsA metabolic pathway genes, expression patterns, and MDHAR evolution in cointree.

A Schematic of the AsA metabolic pathway with a heatmap of gene expression in root, stem, and leaf (left to right). Key enzymes include GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), L-galactono-1,4-lactone dehydrogenase (GLDH), D-galacturonic acid reductase (GalUR), aldonolactonase (Alase), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), L-ascorbate oxidase (AO), and L-ascorbate peroxidase (APX). Expression values represent the mean of three biological replicates, log2-transformed and normalized to z-scores for heatmap visualization. Genes are grouped into four principal AsA biosynthetic pathways. B Aggregate expression levels of AsA pathway genes (n = 91) across root, stem, and leaf. C AsA content in root, stem, and leaf based on three biological replicates. Error bars represent the standard deviation (SD) of three biologically independent replicates (n = 3). For B and C, significance was assessed using Student’s t-test; pairs without labeled p-values are not significantly different. D Heatmap of pairwise log2FC comparisons of FPKM values among tissues for MDHAR and DHAR genes. Log2FC values are shown in each grid. Rows were clustered using Pearson correlation distance, and columns were clustered using Ward’s criterion. E Maximum-likelihood phylogenetic tree of MDHAR genes from 20 representative species. Six major clades are labeled; genes from different species are color-coded, with cointree genes highlighted by a red dotted line. F Collinearity plot of tandemly duplicated MDHAR genes in Rhamnaceae compared with other Rosales species and two high–AsA species (citrus and kiwifruit). MDHAR genes are highlighted in orange, and red curves link the key gene (MDHARs3) from cointree tandem clusters to its orthologs in other Rosales species.