Over-expression of the Hybrid Aspen Homeobox PttKN1 Gene in Red Leaf Beet Induced Altered Coloration of Leaves

PttKN1 (Populus tremula × tremuloides KNOTTED1) gene belongs to the KNOXI gene family. It plays an important role in plant development, typically in meristem initiation, maintenance and organogenesis, and potentially in plant coloration. To investigate the gene functions further, it was introduced into red leaf beet by the floral dip method mediated via Agrobacterium tumefaciens. The transformants demonstrated typical phenotypes as with other PttKN1 transformants. These alterations were very different from the morphology of the wild type. Among them, morphological modification of changed color throughout the entire plant from claret of wild type to yellowish green was the highlight in those transgenic PttKN1-beet plants. The result of spraying selection showed that the PttKN1-beet plants had kanamycin resistance. PCR assay of the 35S-Promoter, NPTII and PttKN1 gene, PCR-Southern analysis of the NPTII and PttKN1 gene showed that the foreign PttKN1 gene had successfully integrated into the genome of beet plant. Furthermore, the results of RT-PCR analysis showed that the gene was ectopic expressed in transgenic plants. These data suggested that there is a correlation between the ectopic expression of PttKN1 gene and morphological alterations of beet plants. Pigment content assay showed that betaxanthins concentrations shared little difference between wild type and transgenic lines, while betacyanins content in transgenic plants was sharply decreased, indicating that the altered plant coloration of the transgenic beet plants may be caused by the changed betacyanins content. The tyrosinase study suggested that the sharply decreased of betacyanins content in transgenic plants was caused via the decreased tyrosinase level. Therefore, the reason for the altered plant coloration may be due to partial inhibition of betacyanin biosynthesis that was induced via the pleiotropic roles of PttKN1 gene.

Transgenic lines of Begonia maculata generated by ectopic expression of PttKN1

KNOX (KNOTTED1-like homeobox) genes encode homeodomain-containing transcription factors which play crucial roles in meristem maintenance and proper patterning of organ initiation. PttKN1 gene, isolated from the vascular cambium of hybrid aspen (Populus tremula × P. tremuloides), is a member of class I KNOX gene family. In order to understand the roles of PttKN1 gene in meristem activity and morphogenesis as well as to explore the possibility to generate novel ornamental lines via its ectopic expression, it was introduced into the genome of Begonia maculata Raddi by Agrobacterium tumefasciens-mediated gene transformation here. Four types of transgenic plants were observed, namely coral-like (CL) type, ectopic foliole (EF) type, phyllotaxy-irregular (IP) type and cup-shaped (CS) type, which were remarkably different from corresponding wild type and were not also observed in the regenerated plantlets of wild type plant. Among these four types of transgenic plants, the phenotype of coral-like was observed for the first time in the transformants ectopically expressed KNOX genes. The observation of scanning electron microscope (SEM) showed ectopic meristems on the adaxial leaf surface of the transformants. Interestingly, the plantlets with ectopic foliole could generate new ectopic folioles from the original ectopic folioles again, and the plants regenerated from the EF-type transformants could also maintain the original morphology. The same specific RT-PCR band of the four types of transgenic plantlets showed that PttKN1 was ectopically expressed. All these data demonstrated that the ectopic expression of PttKN1 caused a series of alterations in morphology which provided possibilities producing novel ornamental lines and that PttKN1 played important roles in meristem initiation, maintenance and organogenesis events as other class I KNOX genes.