Abstract:
Objective The expression of Populus class Ⅰ KNOX genes during the regeneration of adventitious shoot and adventitious root as well as in the vascular cambium were analyzed to reveal the function of class Ⅰ KNOX genes in woody plants meristem formation and differentiation.
Method The nucleic acid and amino acid sequence of Populus class Ⅰ KNOX genes were obtained through blast analysis using Arabidopsis STM protein sequence as query in the genome of Populus trichocarpa. The phylogenetic tree was constructed according to the full length protein sequences of class Ⅰ KNOX genes from Arabidopsis and Populus. The intron/exon structure and domain composition were presented along the phylogenetic tree. The regeneration of adventitious bud and adventitious root using leaf and stem explants from 84 K (Populus alba×P. glandulosa) was used to simulate the shoot and root apical meristem initiation and differentiation, respectively. Quantitative real-time PCR was carried out to analyze the expression of Populus class Ⅰ KNOX genes during the regeneration of adventitious bud/root and in the vascular cambium related region.
Result Ten class Ⅰ KNOX genes were found in the genome of P. trichocarpa through sequence alignment analysis. According to the phylogenetic relationship and gene structure similarity, class Ⅰ KNOX genes from Arabidopsis and Populus could be divided into three groups. Arabidopsis KNAT2 and KNAT6 along with their Populus homolog genes belong to group 1, Arabidopsis STM and BP along with their Populus homolog genes belong to group 2. Group 3, to be noticed, was unique to Populus. Through investigating the expression alteration of Populus class Ⅰ KNOX genes during the regeneration of adventitious buds, it was found that group 1 genes showed increased expression during the transition from bud primordium to adventitious bud, while group 2 and group 3 genes demonstrated higher expression during the transition from meristem to bud primordium. As for the regeneration of adventitious root, group 1 genes showed increased expression in the stage when root primordium differentiate to adventitious root, while group 2 and group 3 genes demonstrated higher expression in the adventitious root formation stage. In addition, all Populus class Ⅰ KNOX genes had an expression in the vascular cambium, and the expression of group 1 gene PtKNAT2/6b and group 2 genes ARK1 and ARK2 were especially high.
Conclusion Group 3 is a new group occurred during the evolution of class Ⅰ KNOX genes from A. thaliana to P. trichocarpa, which was along with group 1 and group 2 to participate in the regulation of different stages of meristem formation and differentiation. Most importantly, PtKNAT2/6b, ARK1 and ARK2 show high expression in the vascular cambium, which may play important roles in vascular cambium activity maintenance and xylem differentiation.