Increase in specific proteins associated with the desiccation of tolerant roots of a horticultural dicot, Ranunculus asiaticus

Ranunculus asiaticus is a horticultural dicot that is able to tolerate long-term desiccation during its annual life cycle. The annual cycle of desiccation and resurrection of R. asiaticus is correlated with the presence of a putative storage protein (Ra14) and stress-related proteins (dehydrins and smHSPs) in its underground storage organs, the tuberous roots. The 14 kDa protein (Ra14) as a major root protein fluctuates seasonally and increases upon drying. It is a glycoprotein and has an amphipathic character. A 492 bp full-length Ra14 cDNA sequence and a 615 bp genomic sequence were obtained by 3'-RACE and 5'-RACE PCR using oligonucleotide sequences based on the terminal amino acids of the 14 kDa mature protein. A 500 bp probe based on the Ra14 genomic sequence detected at least 4 homologs in R. asiaticus using Southern blots. The 14 kDa protein was localized in the storage vacuoles in R. asiaticus roots using an antibody raised against it. SDS-PAGE analysis of total proteins extracted from different organs of R. asiaticus revealed that the 14 kDa protein is specifically expressed and synthesized in tuberous roots but not in above-ground vegetative organs such as leaves and stems, and its homolog was only detected in Anemone corms, a desiccation-tolerant organ. Immunoblots indicated that dehydrins (LEAII D-11 family) are present in desiccated roots and their expression declined upon rehydration. A 477 bp full-length cDNA sequence of Dhn17.5 encoding a 17.5 kDa dehydrin and a 543 bp full-length cDNA sequence of Dhn20 encoding a 20 kDa dehydrin were obtained by degenerate PCR, 3'-RACE and 5'-RACE PCR. The expression of Dhn20 was induced by dehydration and ABA but not by salt. As with dehydrins, smHSPs were also detected by immunoblots in the tuberous roots during desiccation, and their expression was induced by dehydration, ABA and salt. Gel filtration chromatography showed that smHSPs may act as molecular chaperons which are able to bind to other molecules during desiccation to protect them from damage. A possible interaction between the 14 kDa protein and smHSPs was determined using S-tagged 14 kDa protein and total proteins extracted from desiccated roots, but the gel filtration chromatography showed that there is no interaction between the two in vivo.