Appl Microbiol Biotechnol (2006) 70: 470–476 DOI 10.1007/s00253-005-0084-9 APPLIED MICROBIAL AND CELL PHYSIOLOGY S. Sreenivasaprasad . D. C. Eastwood . N. Browning . S. M. J. Lewis . K. S. Burton Differential expression of a putative riboflavin-aldehyde-forming enzyme (raf ) gene during development and post-harvest storage and in different tissue of the sporophore in Agaricus bisporus Received: 18 March 2005 / Revised: 1 June 2005 / Accepted: 28 June 2005 / Published online: 30 July 2005 # Springer-Verlag 2005 Abstract Cloning and characterisation of a putative ri- boflavin-aldehyde-forming enzyme gene (raf) from the cultivated mushroom Agaricus bisporus and its expression during morphogenesis are described. Three cDNA clones were isolated following differential screening of cDNA libraries from rapidly expanding sporophores and post- harvest stored sporophores. The cDNA sequence and pre- dicted translation analysis revealed an open reading frame (ORF) of 348 nucleotides encoding a polypeptide of 115 amino acids, with three introns (56–66 bases) interrupting the genomic ORF. Blast X searches of the databases with the gene sequence showed homology (40% identity and 56% similarity) to the riboflavin-aldehyde-forming enzyme gene from Schizophyllum commune. In A.bisporus, the raf gene sequence upstream of the ORF contained a large CT- rich putative regulatory element (-64 to -24 bases) found in highly expressed genes in various mushrooms, and a 6- base motif present in the 3′ end of the genomic sequence, but not in the corresponding 3′ non-coding part of the cDNA, was identified. The raf gene transcripts increased abundantly in rapidly developing sporophores as well in post-harvest stored sporophores. Differential expression of the raf gene transcripts in different tissues of the spo- rophore was also observed, with higher levels in the stipe compared with the cap and gills. The temporal and spatial expression patterns observed suggest transcriptional regu- lation of the raf gene during A. bisporus morphogenesis. Introduction Homobasidiomycete Agaricus bisporus (white button mush- room) is globally the most widely cultivated edible mush- room. A. bisporus undergoes a series of rapid morphogenetic changes, and the early stages involve the formation of my- celial aggregates and the development of primordia which differentiate into sporophores (mushroom fruitbody). Seven distinct stages of sporophore growth have been described (Hammond and Nichols 1975), and in the latter stages, rapid expansion of the cap (pileus) and gills and elongation of the stipe take place. The cap is covered by an outer layer (skin), and the underside is differentiated into gills that bear the basidiospores. The mycelium in the substrate mobilises the necessary resources from leaf litter or compost (the source) to the sporophore (the sink). Following harvest, the im- mature sporophores continue to develop similarly to the non-harvested mushroom, nutrition being provided by the breakdown of storage compounds and the mass movement of resources from the stipe to the cap and gill tissues. The genetic regulation and molecular physiology of morpho- genesis and resource mobilisation are beginning to be understood in A. bisporus (De Groot et al. 1998; Ospina- Giraldo et al. 2000; Sreenivasaprasad et al. 2000a,b; Eastwood et al. 2001; Kingsnorth et al. 2001; Morales and Thurston 2003) as well as in other mushrooms such as Coprinopsis cinerea, Lentinula edodes, Pleurotus ostreatus and Volvariella volvacea (Kües 2000; Ohga and Royse 2001; Chen et al. 2004; Hirano et al. 2004; Penas et al. 2004). Employing differential screening of cDNA libraries as well as targeted gene cloning via PCR, we have identified up to 25 putatively up-regulated genes associated with mor- phogenesis and post-harvest development in A. bisporus (Sreenivasaprasad et al. 2000a,b, 2004; Eastwood et al. 2001). One of the genes identified showed a homology to the gene encoding the riboflavin-aldehyde-forming en- zyme which produces schizoflavins (SFs) by the oxidation of riboflavin in Schizophyllum commune (Chen and McCormick 1997a,b). The SFs and riboflavin-aldehyde/ acid-producing activity were found to be widely distrib- uted in basidiomycete fungi including A. bisporus (Tachibana and Oka 1982; Tachibana and Murakami 1983). In this paper, we describe the molecular cloning and character- isation of a putative riboflavin-aldehyde-forming enzyme S. Sreenivasaprasad (*) . D. C. Eastwood . N. Browning . S. M. J. Lewis . K. S. Burton Warwick HRI, University of Warwick, Wellesbourne, Warwickshire, CV35 9EF, UK e-mail: s.prasad@warwick.ac.uk Tel.: +44-2476-575079 Fax: +44-2476-574500