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 coleopteran insect western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte) is a devastating crop pest in North America and Europe. Although crop plants that produce Bacillus thuringiensis (Bt) proteins can limit insect infestation, some insect populations have evolved resistance to Bt proteins. Here we describe an insecticidal protein, designated IPD072Aa, that is isolated from Pseudomonas chlororaphis. Transgenic corn plants expressing IPD072Aa show protection from WCR insect injury under field conditions. IPD072Aa leaves several lepidopteran and hemipteran insect species unaffected but is effective in killing WCR larvae that are resistant to Bt proteins produced by currently available transgenic corn. IPD072Aa can be used to protect corn crops against WCRs.

Tobacco activase does not markedly facilitate the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) from non-Solanaceae species, including the green alga Chlamydomonas reinhardtii. To examine the basis of this specificity, we focused on two exposed residues in the large subunit of Rubisco that are unique to the Solanaceae proteins. By employing in vitro mutagenesis and chloroplast transformation, P89R and K356Q substitutions were separately made in the Chlamydomonas enzyme to change these residues to those present in tobacco. Both mutants were indistinguishable from the wild type when grown with minimal medium in the light and contained wild-type levels of holoenzyme. Purified Rubisco was assessed for facilitated activation by spinach and tobacco activase. Both wild-type and K356Q Rubisco were similar in that spinach activase was much more effective than tobacco activase. In contrast, P89R Rubisco was not activated by spinach activase but was well activated by tobacco activase. Thus, the relative specificities of the spinach and tobacco activases for Chlamydomonas Rubisco were switched by changing a single residue at position 89. This result provides evidence for a site on the Rubisco holoenzyme that interacts directly with Rubisco activase.

Xylulose 1,5-bisphosphate (XuBP) is synthesized from ribulose 1,5-bisphosphate (RuBP) at carbamylated catalytic sites on ribulose 1,5-bisphosphate carboxylase (Rubisco) with significant amounts of XuBP being formed at pH less than 8.0. XuBP has been separated by high performance liquid chromatography and identified by pulsed amperometry from compounds bound to 2Abbreviations: CAIP, 2-C-carboxy-i-arabinitol 1-phosphate; ACO2, activator CO2; CABP, 2-Ccarboxy-D-arabinitol 1,5-bisphosphate; HPAE-PAD, high performance anion exchange chromatography with pulsed amperometric detection; PGA, 3-phosphoglycerate; RuBP, ribulose 1,5-bisphosphate; tR, retention time; XuBP, xylulose 1,5-bisphosphate.scribed (1, 8-11, 18, 25) and the term "fallover" was used to describe this die-off phenomenon (9). During the preparation of this paper, Edmondson et al. (8-1 1)

Loss of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity during CO2 fixation, called fallover, occurred with or without loss of activator CO2 from catalytic sites depending on pH. At pH 7.5, but not at pH 8.5, the fraction of Rubisco sites that were carbamylated decreased during fallover. Inhibitors which formed during fallover were identified following NaBH4 reduction and separation of the products by high performance anion-exchange chromatography and pulsed amperometnc detection. They were xylulose 1,5-bisphosphate (XuBP) and 3-ketoarabinitol 1,5-bisphosphate. During fallover at pH 8.5, 3-ketoarabinitol-P2 was the only inhibitor binding to Rubisco and this binding was at carbamylated sites, although both inhibitors were made. At pH 7.5, both inhibitors were bound to catalytic sites of Rubisco with XuBP bound tightly to decarbamylated sites, whereas 3-ketoarabinitol-P2 bound to carbamylated sites. The pH during fallover also influenced the ratio of 3-ketoarabinitol-P2 to XuBP formed. When fallover occurred at pH 7.5, both the formation of XuBP and its binding affinity to decarbamylated Rubisco sites were increased compared with those at pH 8.5. 3-Ketoribitol-P2 was not found at either pH.