[show abstract][hide abstract] ABSTRACT: Microarray technology was used to identify the genes associated with disease defence responses in the model legume Medicago truncatula. Transcript profiles from M. truncatula cv. Jemalong genotype A17 leaves inoculated with Colletotrichum trifolii and Erysiphe pisi and roots infected with Phytophthora medicaginis were compared to identify the genes expressed in response to all three pathogens and genes unique to an interaction. The A17 genotype is resistant to C. trifolii and E. pisi, exhibiting a hypersensitive response after inoculation, and is moderately susceptible to P. medicaginis. Among the most strongly up-regulated genes in all three interactions were those encoding a hevein-like protein, thaumatin-like protein (TLP) and members of the pathogenesis response (PR)10 family. Transcripts of genes for enzymes in the phenylpropanoid pathway leading to the production of isoflavonoid phytoalexins increased dramatically in response to inoculation with the foliar pathogens. In P. medicaginis-inoculated roots, transcripts of genes in the phenylpropanoid pathway peaked at 5 days post-inoculation, when symptoms became visible. Transcript accumulation of three PR10 family members, a TLP and chalcone synthase (CHS) was assessed in M. truncatula genotype R108 plants. The R108 plants are resistant to C. trifolii and moderately susceptible to E. pisi and P. medicaginis. Transcript accumulation paralleled the stages of pathogen development. To evaluate the role of a TLP, a PR10 family member and CHS in disease resistance, transgenic R108 plants containing interfering RNA (RNAi) constructs were produced. Reduced expression of PR10 and TLP had no effect on the disease phenotype, whereas reduced expression of CHS resulted in increased susceptibility to necrotrophic pathogens.
[show abstract][hide abstract] ABSTRACT: Arabidopsis UDP-sugar pyrophosphorylase (AtUSP) is a broad substrate enzyme that synthesizes nucleotide sugars. The products of the AtUSP reaction can act as precursors for the synthesis of glycolipids, glycoproteins, and cell wall components including pectin and hemicellulose. AtUSP has no close homologs in Arabidopsis and its biological function has not been clearly defined. We identified two T-DNA insertional mutant lines for AtUSP, usp-1 and usp-2. No homozygous individuals were identified and progeny from plants heterozygous for usp-1 or usp-2 showed a 1:1 segregation ratio under selection. Despite decreased levels of both AtUSP transcript and USP activity (UDP-GlcA-->GlcA-1-P), heterozygous plants were indistinguishable from wild type at all stages of development. Reciprocal test crosses indicated the source of the segregation distortion was lack of transmission through the male gametophyte. Analysis of pollen tetrads from usp-1 in the quartet background revealed a 2:2 ratio of normal:collapsed pollen grains. The collapsed pollen grains were not viable as determined by Alexander's viability and DAPI staining, and pollen germination tests. The pollen phenotype of usp-1 was complemented by transformation of usp-1 with the AtUSP cDNA sequence. Surface and ultrastructural analyses of pollen from wild-type and usp mutants demonstrated that the mutation had no apparent effect on the outer wall (exine) but prevented the synthesis of the pectocellulosic inner wall (intine). Evidence presented here shows that AtUSP has a critical role in pollen development.
[show abstract][hide abstract] 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.
Plant Physiology and Biochemistry 04/2006; 44(4):171-80. · 2.78 Impact Factor
[show abstract][hide abstract] ABSTRACT: During soybean [Glycine max (L.) Merrill] embryo development, cell wall polysaccharides (CWPs) derived from UDP-glucuronic acid (UDP-GlcA) (uronic acids, arabinose, xylose) exhibited a linear increase during the period of 25-45 days after flowering (daf). At embryo maturity, CWPs derived from UDP-GlcA accounted for 39% of total CWPs. To ascertain the relative importance of the nucleotide sugar oxidation (NSO) and the myo-inositol oxidation (MIO) pathways to UDP-GlcA biosynthesis, UDP-glucose (UDP-Glc) dehydrogenase (UDP-Glc DH, EC 126.96.36.199) and UDP-glucuronic acid pyrophosphorylase (UDP-GlcA PPase, EC 188.8.131.52) activities, respectively, were measured in desalted extracts of developing embryos. UDP-Glc DH and UDP-GlcA PPase activities, expressed on a per seed basis, increased 3.5- and 3.9-fold, respectively, during the period of 25-45 daf. However, UDP-GlcA PPase activity was 35-50-fold greater than UDP-Glc DH activity. The soybean UDP-sugar pyrophosphorylase gene (USP1), a homolog of pea USP, and a candidate gene for UDP-GlcA PPase, was cloned and the recombinant enzyme characterized. Recombinant soybean USP1 (71 kDa) exhibited high activity with glucuronic acid 1-phosphate (GlcA-1-P), glucose 1-phosphate (Glc-1-P) and galactose 1-phosphate (Gal-1-P), but low activity with mannose 1-phosphate (Man-1-P), N-acetylglucosamine 1-phosphate and Glc-6-P. Determination of kinetic constants indicated that USP1 has a higher affinity for GlcA-1-P (Km= 0.14 ± 0.02 mM) than for Glc-1-P (Km= 0.23 ± 0.02 mM). Semiquantitative RT-PCR was used to measure transcript levels of the UDP-glucose DH (UGD) and USP gene families in developing soybean embryos. Transcript levels, normalized to the 18S rRNA controls, were greater for UGD than USP throughout embryo development. The possibility that USP serves as UDP-GlcA PPase, the terminal enzyme of the MIO pathway, is discussed.