Isolation Of TaAS2 And TaLOB1 Genes And Function Analysis In Triticum Aestium

The above-ground structure of higher plants can be divided into two parts: the stem and the lateral organs that are mainly composed of foliaceous structures. Lateral organs are formed from the shoot apical meristem and the relationship between the lateral organs and the apical meristem is basis for the definition of three primordial axes: the proximal-distal, the left-right and the adaxial-abaxial axis. Amomg the three axes, establishment of adaxial-abaxial axis is perhaps the most basic and is important to the pattern fomation and function of leaves. Analysis of mutants and identification of related genes on lateral organs provided important clues to understand the molecular mechanism of the polarity establishment. Previous studies on the establishment of the abaxial-adaxial axis were mainly focused in the dicots. Owing to the differences in development and structure between lateral organs of the dicots and the monocots, their polarity establishment may be under distinct mechanism. Wheat is one of the important cereals in the world and the molecular mechanism of polarity establishment in this crop still remains uncovered. In this study, we isolated two genes TaAS2 and TaLOBl from the spikelets and the seeds at day five after pollinatin of wheat, respectively and further analyzed their functions. Our data provide some information on the molecular mechanism of the abaxial-adaxial polarity determination. Main results and conclusions are as follows: 1. Isolation and characterization of TaAS2 and TaLOBlTo investigate the molecular mechanism of abaxial-adaxial polarity in wheat, we isolated full length cDNAs of TaAS2 and TaLOBl from the young spikelets and the seeds at day five after pollinatin, respectively. The full length cDNA of TaAS2 is 1220bp, encodeing protein with 259 amino acid residues. The full length cDNA of TaLOBl is 1003bp, encoding 248amino acid residues. Both of the proteins have LOB domain. Sequences analysis indicated that TaAS2 and TaLOBl fall into the class I in LOB-Domain gene family.2. TaAS2 and TaLOBl expression patternsWe examined the expression of TaAS2 and TaLOBl by reverse transcriptase-mediated (RT) PCR. Both of the two genes were expressed in plant vegetative and reproductive organs. However, the overall expression profiles of TaAS2 and TaLOBl were distinct. TaAS2 was more abundant than TaLOBl in some tissues and vice versa. The expression pattern of TaAS2 showed high similarity with that of AS2.3. Phenotype analysis of ectopic expression of TaAS2 and TaLOBl in ArabidopsisTo understand the roles of TaAS2 and TaLOBl in development of the lateral organs, transgenic plants were generated with ectopic expression of TaAS2 and TaLOBl under the 35S promoter in Arabidopsis.All of the 35S::TaAS2 transgenic plants were much more smaller than wild type plants. Cotyledons of transgenic plants were narrow and curled upward, toward the adaxial side. The 35S::TaAS2 plants formed narrow leaves that were curled upward, sometime twisted, and produced finger-like outgrowths on the abaxial leaf surface. There was no trichomes on the abaxial side of early leaves. Histological analysis of 35S::TaAS2 transgenic plants revealed abnormalities in th polarity of internal tissues in cotyledons and leaf blades. Cells that resembled palisade mesophyll were present on the abaxial side. That suggested a role for TaAS2 in the promotion of adaxial identity. The organization of vascular tissues in 35S.vTaAS2 transgenic plant was altered.Examing transcript level of the polarity genes in 35S::TaAS2 shoots showed that expressions of abaxial genes KAN1, FIL and YAB3 were reduced, whereas expression of adaxial gene PHB was increased. Detecting KNOX gene expression in the shoot of 35S::TaAS2 transformants showed reductions in BP, KNAT2 and KNAT6 transcipt levels.The similar phynotype of 35S::TaAS2 and 35S::AS2 in Arabidopsis, together with the resemble expression pattern of TaAS2 and AS2, suggested a fuctional conservation between these two genes.Ectopic expression of TaLOBl in Arabidopsis plants caused pleiotropic defects. Plants with severe phynotypes formed narrow cotyledons and leaves that were curled upward. In afew transformants, there were trichomes on the abaxial surface of the third rosette leaf. However, no polarity abnormality observed in internal tissues of the transgenic cotyledons and leaf blades, suggested that TaLOBl promoted adaxial identity partly.