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.

Histone Deacetylase Activity and Phytotoxic Effects Following Exposure of Duckweed (Lemna pausicostata L.) to Apicidin and HC-Toxin.

ABSTRACT The effects of two cyclic tetrapeptide fungal toxins, apicidin (from Fusarium spp.) and HC-toxin (from Cochliobolus carbonum), on duckweed (Lemna pausicostata L.) were examined. Both toxins inhibited histone deacetylase (HD) activity from duckweed plantlets; the effective concentration (EC(50)) for inhibition of HD was 5.6 and 1.1 muM for apicidin and HC-toxin, respectively. Approximately 65 and 85% of in vitro HD activity was inhibited by 50 muM apicidin or HC-toxin, respectively. Exposing duckweed for 72 h to apicidin or HC-toxin (25 or 50 muM) enhanced cellular leakage, impaired chlorophyll synthesis, and inhibited growth (cell division). At equivalent concentrations, the effects of HC-toxin were more pronounced than those of apicidin. In fronds, 72 h of exposure to 50 muM apicidin resulted in chloroplast deterioration indicated by loss of orientation and excess starch accumulation. In roots, a 72-h treatment with 50 muM apicidin resulted in the loss of the root cap and increased vacuolization and starch accumulation in plastids.

Isolation and characterization of glutathione S-transferase isozymes from sorghum.

Two glutathione S-transferase (GST) isozymes, A1/A1 and B1/B2, were purified from etiolated, O-1,3-dioxolan-2-yl-methyl-2,2,2, -trifluoro-4'-chloroacetophenone-oxime-treated sorghum (Sorghum bicolor L. Moench) shoots. GST A1/A1, a constitutively expressed homodimer, had a subunit molecular mass of 26 kD and an isoelectric point of 4.9. GST A1/A1 exhibited high activity with 1-chloro-2, 4, dinitrobenzene (CDNB) but low activity with the chloroacetanilide herbicide metolachlor. For GST A1/A1, the random, rapid-equilibrium bireactant kinetic model provided a good description of the kinetic data for the substrates CDNB and glutathione (GSH). GST B1/B2 was a heterodimer with subunit molecular masses of 26 kD (designated the B1 subunit) and 28 kD (designated the B2 subunit) and a native isoelectric point of 4.8. GST B1/B2 exhibited low activity with CDNB and high activity with metolachlor as the substrate. The kinetics of GST B1/B2 activity with GSH and metolachlor fit a model describing a multisite enzyme having two binding sites with different affinities for these substrates. Both GST A1/A1 and GST B1/B2 exhibited GSH-conjugating activity with ethacrynic acid and GSH peroxidase activity with cumene hydroperoxide, 9-hydroperoxy-trans-10, cis-12-octadecadienoic acid and 13-hydroperoxy-cis-9, trans-11-octadecadienoic acid. Both GST A1/A1 and GST B1/B2 are glycoproteins, as indicated by their binding of concanavalin A. Polyclonal antibodies raised against GST A1/A1 exhibited cross-reactivity with the B1 subunit of GST B1/B2. Comparisons of the N-terminal amino acid sequences of the GST A1, B1, and B2 subunits with other type I theta-GSTs indicated a high degree of homology with the maize GST I subunit and a sugarcane GST.