Molecular cloning of yieldins regulating the yield threshold of cowpea cell walls: cDNA cloning and characterization of recombinant yieldin

Abstract

ABSTRACTcDNA for yieldin of Vigna unguiculata L. was cloned with reverse transcriptase-polymerase chain reaction using synthetic oligonucleotides as primers. The primers were designed on the basis of the N-terminal amino acid sequence of the yieldins isolated from the wall preparation of cowpea hypocotyls. The 1·2 kbp cDNA for yieldin contained an open reading frame of 981 base pairs, encoding 327 amino acids including 23 amino acids as a putative signal sequence. An homology search of the deduced amino acid sequence revealed that the yieldin was homologous to acidic class III endochitinases (EC 3·2.1·14) and concanavalin B. A cDNA fragment containing the yieldincoding region was introduced to Escherichia coli cells using an expression vector to express the recombinant protein. The recombinant yieldin was obtained from the recombinant E. coli and its effect on the wall mechanical properties was examined by reconstitution experiments. The recombinant yieldin fully restored the acid-induced change of the yield threshold tension (y) of heat denatured glycerinated hollow cylinders (GHCs) of cowpea hypocotyls. Northern hybridization analysis revealed that the yieldin mRNA was expressed mainly in the rapid and moderate elongation region of the etiolated hypocotyl of Vigna unguiculata L.

A brief note on the study of yieldin, a wall-bound protein that regulates the yield threshold of the cell wall

“…The reconstitution experiments on the heat-denatured GHC using recombinant yieldin showed that recombinant yieldin only fully restored the pH-dependent shift in y (Okamoto-Nakazato et al 2000b;Fig. 4).…”

“…cDNA for yieldin was cloned with RT-PCR using synthetic oligonucleotides designed on the basis of the N-terminal amino acid sequence of purified yieldin 30 as a primer and total RNA extracted from the elongation region of cowpea hypocotyl as a template (Okamoto-Nakazato et al 2000b). The isolated 1.2-kbp cDNA for yieldin contained an open reading frame of 1,183 base pairs encoding 327 amino acids with a molecular mass of 35,647 Da.…”

Chitinases in root nodules

“…In embryogenic cultures of both D. carota and Picea abies, bacterial Nod factors can substitute for chitinases in their effect on early somatic embryo development (De Jong et al 1992;Egertsdotter and von Arnold 1998;Dyachok et al 2005). An acidic class III endochitinase (glycoside hydrolase family 18) from cowpea (Vigna unguiculata) was found to represent a protein that is required for cell wall loosening during acid growth (yieldin; OkamotoNakazato et al 2000a;2000b). Sasaki et al (2006) compared the substrate specificity of class I and class III chitinases from rice and found that the class III enzyme might act towards an endogenous complex carbohydrate containing a GlcNAc residue, but not against a GlcNAc oligomer or polymer, while the substrate of the class I enzyme was probably a consecutive GlcNAc sequence, maybe from the cell wall of a fungal pathogen.…”

Brassinolide as a modulator of the activities of cell wall loosening proteins

“…They are most active under acidic conditions. Recently, y proteins named yieldins were isolated from Vigna unguiculata L. hypocotyls (Okamoto-Nakazato et al 2000a) and their genes were cloned (Okamoto-Nakazato et al 2000b). The acidic environment is thought to activate cell wall loosening enzymes including f and y proteins (Taguchi et al 1999), resulting in cell wall loosening and hence cell wall expansion under the effective turgor pressure (P-y).…”