Ectopic expression of the atypical HLH FaPRE1 gene determines changes in cell size and morphology.

PACLOBUTRAZOL RESISTANCE (PRE) genes code atypical HLH transcriptional regulators characterized by the absence of a DNA-binding domain but present an HLH dimerization domain. In vegetative tissues, the function of these HLH proteins has been related with cell elongation processes. In strawberry, three FaPRE genes are expressed, two of them (FaPRE2 and FaPRE3) in vegetative tissues while FaPRE1 is fruit receptacle-specific. Ubiquitous FaPRE1 accumulation produced elongated flower receptacles and plants due to the elongation of the main aerial vegetative organs, with the exception of leaves. Histological analysis clearly demonstrated that the observed phenotype was due to significant changes in the parenchymal cell's morphology. In addition, transcriptomic studies of the transgenic elongated flower receptacles allowed to identify a small group of differentially expressed genes that encode cell wall-modifying enzymes. Together, the data seem to indicate that, in the strawberry plant vegetative organs, FaPRE proteins could modulate the expression of genes related with the determination of the size and shape of the parenchymal cells.

Cloning, expression and immunolocalization pattern of a cinnamyl alcohol dehydrogenase gene from strawberry (Fragaria x ananassa cv. Chandler).

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. By a differential screening of a strawberry (Fragariax ananassa cv. Chandler) fruit specific subtractive cDNA library, a full-length clone corresponding to a cad gene was isolated (Fxacad1). Northern blot and quantitative real time PCR studies indicated that the strawberry Fxacad1 gene is expressed in fruits, runners, leaves, and flowers but not in roots. In addition, the gene presented a differential expression in fruits along the ripening process. Moreover, by screening of a strawberry genomic library a cad gene was isolated (Fxacad2). Similar to that found in other cad genes from higher plants, this strawberry cad gene is structured in five exons and four introns. Southern blot analyses suggest that, probably, a small cad gene family exists in strawberry. RT-PCR studies indicated that only the Fxacad1 gene was expressed in all the fruit ripening stages and vegetative tissues analysed. The Fxacad1 cDNA was expressed in E. coli cells and the corresponding protein was used to raise antibodies against the strawberry CAD polypeptide. The antibodies obtained were used for immunolocalization studies. The results showed that the CAD polypeptide was localized in lignifying cells of all the tissues examined (achenes, fruit receptacles, runners, leaves, pedicels, and flowers). Additionally, the cDNA was also expressed in yeast (Pichia pastoris) as an extracellular protein. The recombinant protein showed activity with the characteristic substrates of CAD enzymes from angiosperms, indicating that the gene cloned corresponds to a CAD protein.