Pioneer Hi-Bred International, DuPont Agriculture and Nutrition, Redwood City, California 94063Plant photosynthesis declines when the temperature exceeds its optimum range. Recent evidence indicates that the reduction in photosynthesis is linked to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) deactivation due to the inhibition of Rubisco activase (RCA) under moderately elevated temperatures. To test the hypothesis that thermostable RCA can improve photosynthesis under elevated temperatures, we used gene shuffling technology to generate several Arabidopsis thaliana RCA1 (short isoform) variants exhibiting improved thermostability. Wild-type RCA1 and selected thermostable RCA1 variants were introduced into an Arabidopsis RCA deletion (Drca) line. In a long-term growth test at either constant 268C or daily 4-h 308C exposure, the transgenic lines with the thermostable RCA1 variants exhibited higher photosynthetic rates, improved development patterns, higher biomass, and increased seed yields compared with the lines expressing wild-type RCA1 and a slight improvement compared with untransformed Arabidopsis plants. These results provide clear evidence that RCA is a major limiting factor in plant photosynthesis under moderately elevated temperatures and a potential target for genetic manipulation to improve crop plants productivity under heat stress conditions.

The herbicide glyphosate is effectively detoxified by N-acetylation. We screened a collection of microbial isolates and discovered enzymes exhibiting glyphosate N-acetyltransferase (GAT) activity. Kinetic properties of the discovered enzymes were insufficient to confer glyphosate tolerance to transgenic organisms. Eleven iterations of DNA shuffling improved enzyme efficiency by nearly four orders of magnitude from 0.87 mM-1 min-1 to 8320 mM-1 min-1. From the fifth iteration and beyond, GAT enzymes conferred increasing glyphosate tolerance to Escherichia coli, Arabidopsis, tobacco, and maize. Glyphosate acetylation provides an alternative strategy for supporting glyphosate use on crops.

5 The abbreviations used are: EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase; PEP, phosphoenolpyruvate; PDB, Protein Data Bank; S3P, shikimate-3phosphate; MESG, 2-amino-6-mercapto-7-methylpurine ribonucleoside. Optimized, glyphosate-desensitized plant EPSPSa Enzyme turnover (min Ϫ1 ) at 30 M PEP and S3P, 1 mM glyphosate (see "Results" for rational as a fitness parameter). b Calculated with K m for PEP, c lod; limit of detection calculated as Blank ϩ 3xSD of blank. The value is 1.5 min Ϫ1 . d Variant captured from the initial combinatorial library, having G101A plus three other mutations (E301S, E390G, V437R). e EPSPS from Agrobacterium sp. strain CP4, present in RoundUp Ready crops. f Variants of maize EPSPS having only the two mutations shown. g Variant having G101A, L97C, and V332A. ACA, G101A-L98C, and G101A-V332A were accessed commercially by gene synthesis. h D2c-A5 with Ala 101 changed back to glycine. Figure 3. Amino acid substitutions present in variants in the progressive optimization of maize EPSPS for activity in the presence of glyphosate.Numbers are the amino acid positions in the reference sequence (Fig. 2). Amino acids that differ from those in the native enzyme are highlighted. Optimized, glyphosate-desensitized plant EPSPS

N-Acetylation is a modification of glyphosate that could potentially be used in transgenic crops, given a suitable acetyltransferase. Weak enzymatic activity (k(cat) = 5 min(-1), K(M) = 1 mM) for N-acetylation of glyphosate was discovered in several strains of Bacillus licheniformis (Weigmann) Chester by screening a microbial collection with a mass spectrometric assay. The parental enzyme conferred no tolerance to glyphosate in any host when expressed as a transgene. Eleven iterations of DNA shuffling resulted in a 7000-fold improvement in catalytic efficiency (k(cat)/K(M)), sufficient for conferring robust tolerance to field rates of glyphosate in transgenic tobacco and maize. In terms of k(cat)/K(M), the native enzyme exhibited weak activity (4-450% of that with glyphosate) with seven of the common amino acids. Evolution of the enzyme towards an improved k(cat)/K(M) for glyphosate resulted in increased activity toward aspartate (40-fold improved k(cat)), but activity with serine and phosphoserine almost completely vanished. No activity was observed among a broad sampling of nucleotides and antibiotics. Improved catalysis with glyphosate coincided with increased thermal stability.