For many years it was accepted that isopentenyl diphosphate, the common precursor of all isoprenoids, was synthesized through the well known acetate/mevalonate pathway. However, recent studies have shown that some bacteria, including Escherichia coli , use a mevalonate-independent pathway for the synthesis of isopentenyl diphosphate. The occurrence of this alternative pathway has also been reported in green algae and higher plants. The first reaction of this pathway consists of the condensation of (hydroxyethyl)thiamin derived from pyruvate with the C1 aldehyde group of d -glyceraldehyde 3-phosphate to yield d -1-deoxyxylulose 5-phosphate. In E. coli , d -1-deoxyxylulose 5-phosphate is also a precursor for the biosynthesis of thiamin and pyridoxol. Here we report the molecular cloning and characterization of a gene from E. coli , designated dxs, that encodes d -1-deoxyxylulose-5-phosphate synthase. The dxs gene was identified as part of an operon that also contains ispA , the gene that encodes farnesyl-diphosphate synthase. d -1-Deoxyxylulose-5-phosphate synthase belongs to a family of transketolase-like proteins that are highly conserved in evolution.

E1 enzymes facilitate conjugation of ubiquitin and ubiquitin‐like proteins through adenylation, thioester transfer within E1, and thioester transfer from E1 to E2 conjugating proteins. Structures of human heterodimeric Sae1/Sae2‐Mg·ATP and Sae1/Sae2‐SUMO‐1‐Mg·ATP complexes were determined at 2.2 and 2.75 Å resolution, respectively. Despite the presence of Mg·ATP, the Sae1/Sae2‐SUMO‐1‐Mg·ATP structure reveals a substrate complex insomuch as the SUMO C‐terminus remains unmodified within the adenylation site and 35 Å from the catalytic cysteine, suggesting that additional changes within the adenylation site may be required to facilitate chemistry prior to adenylation and thioester transfer. A mechanism for E2 recruitment to E1 is suggested by biochemical and genetic data, each of which supports a direct role for the E1 C‐terminal ubiquitin‐like domain for E2 recruitment during conjugation.

Summary: The genetic manipulation of both the mevalonic acid (MVA) and methylerythritol‐4‐phosphate (MEP) pathways, leading to the formation of isopentenyl diphosphate (IPP), has been achieved in tomato using 3‐hydroxymethylglutaryl CoA (hmgr‐1) and 1‐deoxy‐d‐xylulose‐5‐phosphate synthase (dxs) genes, respectively. Transgenic plants containing an additional hmgr‐1 from Arabidopsis thaliana, under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter, contained elevated phytosterols (up to 2.4‐fold), but IPP‐derived isoprenoids in the plastid were unaltered. Transgenic lines containing a bacterial dxs targeted to the plastid with the tomato dxs transit sequence resulted in an increased carotenoid content (1.6‐fold), which was inherited in the next generation. Phytoene and β‐carotene exhibited the greatest increases (2.4‐ and 2.2‐fold, respectively). Extra‐plastidic isoprenoids were unaffected in these lines. These data are discussed with respect to the regulation, compartmentalization and manipulation of isoprenoid biosynthetic pathways and their relevance to plant biotechnology.

Post-translational modification of proteins by small polypeptides, such as ubiquitin, has emerged as a common and important mechanism for regulating protein function. Small ubiquitin-like modifier (SUMO) is a small protein that is structurally related to but functionally different from ubiquitin. We report the identification and functional analysis of AtSUMO1, AtSUMO2, and AtSCE1a as components of the SUMO conjugation (sumoylation) pathway in Arabidopsis. In yeast-two hybrid assays, AtSUMO1/2 interacts specifically with a SUMO-conjugating enzyme but not with a ubiquitin-conjugating enzyme. AtSCE1a, the Arabidopsis SUMO-conjugating enzyme ortholog, conjugates SUMO to RanGAP in vitro. AtSUMO1/2 and AtSCE1a colocalize at the nucleus, and AtSUMO1/2 are conjugated to endogenous SUMO targets in vivo. Analysis of transgenic plants showed that overexpression of AtSUMO1/2 does not have any obvious effect in general plant development, but increased sumoylation levels attenuate abscisic acid (ABA)-mediated growth inhibition and amplify the induction of ABA- and stress-responsive genes such as RD29A. Reduction of AtSCE1a expression levels accentuates ABA-mediated growth inhibition. Our results suggest a role for SUMO in the modulation of the ABA signal transduction pathway.

The photoreceptor phytochrome (phy) A has a well-defined role in regulating gene expression in response to specific light signals. Here, we describe a new Arabidopsis mutant, laf1(long after far-red light1) that has an elongated hypocotyl specifically under far-red light. Gene expression studies showed that laf1 has reduced responsiveness to continuous far-red light but retains wild-type responses to other light wavelengths. As far-red light is only perceived by phyA, our results suggest that LAF1 is specifically involved in phyA signal transduction. Further analyses revealed thatlaf1 is affected in a subset of phyA-dependent responses and the phenotype is more severe at low far-red fluence rates. LAF1encodes a nuclear protein with strong homology with the R2R3–MYB family of DNA-binding proteins. Experiments using yeast cells identified a transactivation domain in the C-terminal portion of the protein. LAF1 is constitutively targeted to the nucleus by signals in its N-terminal portion, and the full-length protein accumulates in distinct nuclear speckles. This accumulation in speckles is abolished by a point mutation in a lysine residue (K258R), which might serve as a modification site by a small ubiquitin-like protein (SUMO).