Do polyamines contribute to plant cell wall assembly by forming amide bonds with pectins?

Two new reducing glycoconjugates [ N - d -galacturonoyl-putrescinamide (GalA–Put) and N , N ′-di- d -galacturonoyl-putrescinamide (GalA–Put–GalA)] and homogalacturonan–putrescine (GalA n –Put) conjugates were synthesised as model compounds representing possible amide (isopeptide) linkage points between a polyamine and either one or two pectic galacturonate residues. The amide bond(s) were stable to cold acid and alkali (2 M TFA and 0.1 M NaOH at 25 °C) but rapidly hydrolysed by these agents at 100 °C. The amide bond(s) were resistant to Driselase and to all proteinases tested, although Driselase digested GalA n –Put, releasing fragments such as GalA 3 –Put–GalA 3 . To trace the possible formation of GalA–polyamine amide bonds in vivo, we fed Arabidopsis and rose cell-cultures and chickpea internodes with [ 14 C]Put. About 20% of the 14 C taken up was released as 14 CO 2 , indicating some catabolism. An additional ∼73% of the 14 C taken up (in Arabidopsis ), or ∼21% (in rose), became ethanol-insoluble, superficially suggestive of polysaccharide–Put covalent bonding. However, much of the ethanol-inextractable 14 C was subsequently extractable by acidified phenol or by cold 1 M TFA. The small proportion of radioactive material that stayed insoluble in both phenol and TFA was hydrolysable by Driselase or hot 6 M HCl, yielding 14 C-oligopeptides and/or amino acids (including Asp, Glu, Gly, Ala and Val); no free 14 C-polyamines were released by hot HCl. We conclude that if pectin–polyamine amide bonds are present, they are a very minor component of the cell walls of cultured rose and Arabidopsis cells and chickpea internodes.