Characterization and expression of Arabidopsis UDP-sugar pyrophosphorylase

Abstract

At5g52560, a homolog of pea ( Pisum sativum) UDP-sugar pyrophosphorylase ( PsUSP) was functionally annotated by expression in Escherichia coli and subsequent characterization of substrate specificity and kinetic properties. Arabidopsis contains a single USP gene ( AtUSP) and evaluation of gene databases suggests that USP is unique to plants. The 69 kDa AtUSP gene product exhibited high activity with Glc-1-P, GlcA-1-P and Gal-1-P, but low activity with GlcNAc-1-P, Fuc-1-P, Man-1-P, inositol-1-P or Glc-6-P. AtUSP was activated by magnesium and preferred UTP as co-substrate. Apparent K m values for GlcA-1-P, Glc-1-P and UTP were 0.13 mM, 0.42 mM and 0.14 mM, respectively. In the reverse direction (pyrophosphorolysis), the apparent K m values for UDP-GlcA, UDP-Glc and pyrophosphate were 0.56 mM, 0.72 mM and 0.15 mM, respectively. USP enzyme activity (UDP-GlcA → GlcA-1-P) was detected in Arabidopsis tissues with highest activity found in the inflorescence. As determined by semi-quantitative RT-PCR, AtUSP transcript is widely expressed with high levels detected in the inflorescence. To evaluate tissue-specific expression of AtUSP, histochemical GUS staining of plants transformed with AtUSPprom:GUS constructs was performed. In 7-day-old seedlings, GUS staining was detected in cotyledons, trichomes and vascular tissues of the primary root. In the inflorescence of older plants, high levels of GUS staining were detected in cauline leaves, the epidermis of the stem and in pollen. In silico analysis of AtUSP expression in developing pollen indicates that transcript levels increase as development proceeds from the uninucleate to the tricellular stage. The results suggest that AtUSP plays an important role in pollen development in Arabidopsis.