melinoe mutant uncovers the role of AtFMN/FHy enzyme as a biochemical hub for flavin synthesis in plants
Riboflavin, or vitamin B2, is the biosynthetic precursor of flavocoenzymes, which are essential for the cell. As the central component of FMN and FAD cofactors, riboflavin controls the function of flavoproteins, being indispensable for growth and cellular metabolism. These cofactors sustain photosynthesis, mitochondrial electron transport, fatty acid oxidation, photoreception and secondary metabolism. While animals depend on exogenous resources, riboflavin is mostly biosynthesized in plants and microorganisms. In the model species Arabidopsis thaliana, the key enzyme AtFMN/FHy has both riboflavin kinase and FMN hydrolase activities. Phylogenetic analysis suggests that AtFMN/FHy proteins occur in algae and land plants. AtFMN/FHy 3D structure displays two distinct domains representing the different enzymatic properties. Here, we report the characterization of a FMN/FHy mutant called melinoe (melin), which displays abnormal plant growth and leaf chlorosis. A nucleotide transition at an intron-exon junction of the FMN/FHy gene of melin homozygous plants results in alternative 3' splicing, skipping twelve amino acids within the FMN phosphatase domain. Leaf variegation was apparent during the entire life cycle of melin homozygous plant that showed a specific gradient pattern of chlorosis beginning from the center of the leaves and extending to the entire leaf lamina. The growth of melin homozygous plants ceased upon transition from the reproductive to the vegetative stage. Biochemical approaches revealed that melin mutant seedlings accumulated riboflavin to exceptionally increased levels. Translational fusion to YFP, showed subcellular localization of FMN/FHy in the cytosol and FMN/FHy was constantly expressed in various organs upon distinct developmental stages. ? holistic transcriptome analysis carried out using Next-generation sequencing of RNA isolated from gradually bleached leaves of fmn/fhy plants and bioinformatics analysis used to identify and implicate genes and signaling pathways involved in flavin metabolism in plants. Overall, the results will shed light on the regulatory role of FMN/FHy in intracellular flavins biosynthesis. Overall design: Total RNA was extracted  from three distinct leaf stages namely green, pale green and chlorotic variation using the Direct-zol RNA Miniprep (Zymo Research, Irvine CA, USA) kit with an on-column DNase treatment according to the manufacturer's instructions. The quantity and integrity of RNA were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) and agarose gel electrophoresis. RNA-seq libraries were generated using the polyA Strand-Specific Transcriptome Library method of BGI for DNBSEQ platform. Sequencing was performed on DNBSEQ platform instrument at BGI (Beijing Genomics Institute) in three biological replicates for each sample. Raw reads were filtered into clean reads and aligned to the Arabidopsis genome (TAIR10). RNA-seq data were analyzed using SOAPnuke (version 1.5.2) with parameters “-l 15 -q 0.2 -n 0.05" and the HISAT2 pipeline (version 2.0.4) with parameters “--sensitive --no-discordant --no-mixed -I 1 -X 1000 -p 8".