Characterization and expression of Arabidopsis UDP-sugar pyrophosphorylase.

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.

Soil nitrogen availability, plant luxury consumption, and herbivory by white-tailed deer.

N is often cited as a limiting factor for sapling growth in northeastern USA forests. However, under conditions of elevated soil N, seedlings and saplings of some tree species exhibit luxury consumption of N, leading to elevated tissue N concentration. While this pool of plant N may have benefits for saplings if light levels change, it may also increase the risk of herbivory by white-tailed deer (Odocoileus virginianus L.). We conducted a field fertilization experiment to test the hypothesis that saplings increase stem tissue N when soil N availability is elevated. We fertilized saplings of nine tree species under closed and open canopies. Two of the nine species, yellow birch (Betula alleghaniensis) and black cherry (Prunus serotina), had significant increases in radial growth when fertilized under high light. Six of the nine species showed increased stem N concentration under either low light or high light conditions. Under low light conditions, black cherry and sugar maple (Acer saccharum) had significantly higher concentrations of stem N when fertilized. Regardless of fertilization treatment, yellow birch had significantly greater stem N in saplings under low light conditions when compared with saplings found in high light conditions. Under high light conditions, fertilization resulted in increased stem N in saplings of white ash (Fraxinus americana) and eastern hemlock (Tsuga canadensis). There was a trend for saplings of red maple (Acer rubrum) and white ash to show elevated stem N concentration when fertilized under low light. Red oak (Quercus rubra), American beech (Fagus grandifolia) and white pine (Pinus strobus) did not show any evidence of luxury consumption. We compared browse frequency of fertilized and unfertilized saplings within similar forest types. Browse frequency was consistently higher on fertilized saplings. When averaged across all species, however, the difference between treatments was not significant. In contrast, we found significantly higher browse rates for fertilized saplings that had been previously identified as luxury N consumers. Our results indicate that increased soil N availability has positive effects on at least some of the species under high light, but, has potentially negative indirect effects on a larger group of species under both high and low light due to a higher risk of herbivory.

Characterization of the AMP-binding protein gene family in Arabidopsis thaliana: will the real acyl-CoA synthetases please stand up?

One of the most prominent and important topics in modern agricultural biotechnology is the manipulation of oilseed triacylglycerol composition. Towards this goal, we have sought to identify and characterize acyl-CoA synthetases (ACSs), which play an important role in both de novo synthesis and modification of existing lipids. We have identified and cloned 20 different genes that bear strong sequence homology to known ACSs from other organisms. Through sequence comparisons and functional characterization, we have identified several members of this group that encode ACSs, while the other genes fall into the broader category of genes for AMP-binding proteins (AMPBPs). Distinguishing ACSs from AMPBPs will simplify our efforts to understand the role of ACS in triacylglycerol metabolism.

Characterization of an acyl-CoA synthetase from Arabidopsis thaliana.

One of the major goals of modern plant biotechnology is to manipulate lipid metabolism in oilseed crops to produce new and improved edible and industrial vegetable oils. Lipids constitute the structural components of cellular membranes and act as sources of energy for the germinating seed and are therefore essential to plant cell function. Both de novo synthesis and modification of existing lipids are dependent on the activity of acyl-CoA synthetases (ACSs). To date, ACSs have been recalcitrant to traditional methods of purification due to their association with membranes. In our laboratory, several isoforms of ACSs have been identified in Arabidopsis thaliana. Reverse genetics allowed us to identify a mutant containing a transfer DNA-interrupted ACS gene. Results will be presented that describe the isolation and characterization of this mutant. The elucidation of the specific roles of ACSs will lead to a greater understanding of plant lipid metabolism.

The CaMV-35S promoter is sensitive to shortened photoperiod in transgenic tobacco.

The CaMV-35S promoter is one the most widely used promoters in transgenic plant research because it exhibits a high level of transcriptional activity in a variety of plant tissues. Here, the CaMV-35S promoter fused to the GUS gene was used as a model for constitutive expression in transgenic Nicotiana tabacum (cv 'Xanthi') leaves. The transgenic plants were placed under a shortened photoperiod to determine if GUS expression changed; measurements were made using fluorometry and ribonuclease protection assays. When the plants were moved from a 16 : 8-h photoperiod to a 8 : 16-h photoperiod, an increase in both specific GUS activity and gus RNA levels was observed, and these levels decreased upon returning to the 16 : 8-h photoperiod. These results indicate that photoperiod plays an important role in the regulation of the CaMV-35S promoter. Studies involving a comparison of this promoter to others should be limited to constant photoperiod conditions.

Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation.

The effects of timentin on shoot regeneration of tobacco (Nicotiana tabaccum) and Siberian elm (Ulmus pumila L.) and its use for the suppression of Agrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were determined. Timentin is a mixture of ticarcillin and clavulanic acid, and at concentrations of 200-500 mg/l with ratios of ticarcillin:clavulanic acid of 50:1 and 100:1, it had little effect on shoot regeneration of tobacco or Siberian elm. Timentin was as effective in suppressing A. tumefaciens as carbenicillin and cefatoxime at concentrations commonly used in transformation. The disarmed A. tumefaciens strain LBA4404 in infected tobacco leaf tissues was visually undetectable after three subcultures on medium with 500 mg/l of timentin and 250 mg/l carbenicillin. Timentin was stable in solid agar medium and remained effective for at least 70 days, but was unstable when stored as a mixed stock solution or as separate ticarcillin and clavulanic acid stock solutions at -20°C or -80°C freezer for 4 weeks. Timentin may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.