Do cluster roots of Hakea actities (Proteaceae) acquire complex organic nitrogen?

Although there has been speculation that cluster roots play a role in plant nitrogen (N) acquisition there have been few experimental studies, demonstrating this. We grew the subtropical wet heathland plant Hakea actities in sand and in axenic vermiculite-sand cultures to investigate its use of different N sources. Seedlings produced greater quantities of cluster roots when grown with NO3-, glutathione or protein as N sources than with NH4+, glutamine or without N addition, suggesting that N source influences cluster root initiation and development. Axenically grown seedlings acquired more N when grown with 4.5 mM than with 0.5 mM glutathione or protein as N sources suggesting that seedlings are able to assimilate complex organic N. However, control seedlings that did not receive N other than seed storage N (approximately 0.2 mg N) acquired 2 mg N from an unknown source, so that data are not unequivocal. Peptidase activity in extracts derived from non-axenic cluster root tissue changed with age of cluster roots. Developing and mature cluster roots had higher peptidase activity than young and senescing cluster roots. To determine whether peptidases are associated with the outer surface of cluster roots, entire cluster roots were gently centrifuged and the resulting solution analysed for peptidase activity using gelatine-containing PAGE. Different peptidases were active at different times of cluster root development, suggesting that plant or microbially derived peptidases could play a role in organic N acquisition by cluster roots. Roots and cluster roots expressed a putative amino acid transporter (HaAAT4-1) and peptide transporter (HaPepT1) as determined by northern blot analysis. Expression of HaPepT1 in cluster roots increased throughout cluster root development although further studies need to confirm this.