709 SummaryEthanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize ( Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-β -1,4-glucanase from Acidothermus cellulolyticus , was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei , also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall.With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T 2 ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.

SummaryExpression of industrial enzymes in transgenic plants offers an alternative system to fungal fermentation for large-scale production. Very high levels of expression are required to make the enzymes cost-effective. We tested several parameters to determine the best method for achieving high levels of expression for a fungal laccase gene. Transgenic maize plants were generated using an Agrobacterium -mediated system. The molecular parameters that induced the highest expression were the maize embryo-preferred globulin 1 promoter and targeting of the protein to the cell wall. Two independent transgenic events that yielded multiple clonal plants were characterized in detail. Independent transgenic events 01 and 03 contained two or one copies of T-DNA, respectively. Plants derived from a single transgenic event varied in expression level, and the variation in expression levels was heritable. Within the seed, expression in these plants was primarily within the embryo, and was associated with seed browning and limited germination. High oil germplasm was used to increase germination, as well as to assist in increasing expression 20-fold in five generations through breeding and selection.

Phytochrome (phy) A mediates two distinct photobiological responses in plants: the very-low-fluence response (VLFR), which can be saturated by short pulses of very-low-fluence light, and the high-irradiance response (HIR), which requires prolonged irradiation with higher fluences of far-red light (FR). To investigate whether the VLFR and HIR involve different domains within the phyA molecule, transgenic tobacco (Nicotiana tabacum cv Xanthi) and Arabidopsis seedlings expressing full-length (FL) and various deletion mutants of oat (Avena sativa) phyA were examined for their light sensitivity. Although most mutants were either partially active or inactive, a strong differential effect was observed for the ⌬6-12 phyA mutant missing the serine-rich domain between amino acids 6 and 12. ⌬6-12 phyA was as active as FL phyA for the VLFR of hypocotyl growth and cotyledon unfolding in Arabidopsis, and was hyperactive in the VLFR of hypocotyl growth and cotyledon unfolding in tobacco, and the VLFR blocking subsequent greening under white light in Arabidopsis. In contrast, ⌬6-12 phyA showed a dominant-negative suppression of HIR in both species. In hypocotyl cells of Arabidopsis irradiated with FR phyA:green fluorescent protein (GFP) and ⌬6-12 phyA:GFP fusions localized to the nucleus and coalesced into foci. The proportion of nuclei with abundant foci was enhanced by continuous compared with hourly FR provided at equal total fluence in FL phyA:GFP, and by ⌬6-12 phyA mutation under hourly FR. We propose that the N-terminal serine-rich domain of phyA is involved in channeling downstream signaling via the VLFR or HIR pathways in different cellular contexts.Phytochromes (phy) comprise a family of photoreceptors that help adjust plant growth and development to the ambient light environment. These photoreceptors sense red light (R) and far-red light (FR) through photo-interconversion between two stable conformations, an R-absorbing Pr form and an FRabsorbing Pfr form. In seed plants such as Arabidopsis, as many as five phy isoforms are present (Mathews and Sharrock, 1997). One of the more influential is phyA, the most abundant isoform in darkgrown seedlings (Hirschfeld et al., 1998). phyA helps perceive (a) the brief light pulses that can promote seed germination (Botto et al., l996; Shinomura et al., 1996), (b) the difference between darkness and the FR-rich environment that initiates de-etiolation beneath dense canopies (Yanovsky et al., 1995), (c) the changes in irradiance associated with the presence of neighboring vegetation (Yanovsky et al., 1998), and (d) the duration of the photoperiod (Johnson et al., 1994).phyA can initiate two photobiologically distinct responses, the very-low-fluence responses (VLFRs) and the high-irradiance responses (HIRs). The VLFR can be achieved by short intermittent pulses of R or FR. For example, the VLFR that inhibits hypocotyl growth can be saturated in Arabidopsis by a 3-min pulse of FR every 2 h with the half-maximal effect requiring 0.1 mol m Ϫ2 s Ϫ1 of FR (Casal et al., 2000). In contra...

SummaryPhotoconversion of the plant photoreceptor phytochrome A (phyA) from its inactive Pr form to its biologically active Pfr form initiates its rapid proteolysis. Previous kinetic and biochemical studies implicated a role for the ubiquitin/26S proteasome pathway in this breakdown and suggested that multiple domains within the chromoprotein are involved. To further resolve the essential residues, we constructed a series of mutant PHY genes in vitro and analyzed the Pfr-specific degradation of the resulting photoreceptors expressed in transgenic tobacco. One important site is within the C-terminal half of the polypeptide as its removal stabilizes oat phyA as Pfr. Within this half is a set of conserved lysines that are potentially required for ubiquitin attachment. Substitution of these lysines did not prevent ubiquitination or breakdown of Pfr, suggesting either that they are not the attachment sites or that other lysines can be used in their absence. A small domain just proximal to the C-terminus is essential for the form-dependent breakdown of the holoprotein. Removal of just six amino acids in this domain generated a chromoprotein that was not rapidly degraded as Pfr. Using chimeric photoreceptors generated from potato PHYA and PHYB, we found that the N-terminal half of phyA is also required for Pfr-specific breakdown. Only those chimeras containing the N-terminal sequences from phyA were ubiquitinated and rapidly degraded as Pfr. Taken together, our data demonstrate that, whereas an intact C-terminal

A cerulenin insensitive 3-ketoacyl-acyl carrier protein synthase has been assayed in extracts of spinach (Spinacia oleracea) leaf. The enzyme was active in the 40 to 80% ammonium sulfate precipitate of whole leaf homogenates and catalyzed the synthesis of acetoacetyl-acyl carrier protein. This condensation reaction was five-fold faster than acetyl-CoA:acyl carrier protein transacylase, and the initial rates of acyl-acyl carrier protein synthesis were independent of the presence of cerulenin. In the presence of fatty acid synthase cofactors and 100 micromolar cerulenin, the principal fatty acid product of de novo synthesis was butyric and hexanoic acids. Using conformationally sensitive native polyacrylamide gel electrophoresis for separation, malonyl-, acetyl-, butyryl-, hexanoyl, and long chain acyl-acyl carrier proteins could be detected by immunoblotting and autoradiography. In the presence of 100 micromolar cerulenin, the accumulation of butyryland hexanoyl-acyl carrier protein was observed, with no detectable long chain acyl-acyl carrier proteins or fatty acids being produced. In the absence of cerulenin, the long chain acyl-acyl carrier proteins also accumulated.biosynthesis to cerulenin can be attributed entirely to the inhibition of 3-ketoacyl-ACP synthase I.Similarly, the 3-ketoacyl-ACP synthases found in Escherichia coli, are characteristically cerulenin sensitive (5). Recently, a third condensing enzyme has been reported in E. coli. which has been named acetoacetyl-ACP synthase (6). This 3-ketoacyl-ACP synthase is distinctly different from the other 3-ketoacyl-ACP synthases in several important respects. It is cerulenin insensitive, is specific for very short chain acylACPs, and it prefers acetyl-CoA over acetyl-ACP (7). As a consequence of this last property, it would appear that this enzyme can bypass the much slower acetyl transacylase step and as a result, the acetyl transacylase would not be rate limiting in fatty acid biosynthesis.In light of these findings in the E. coli system, we have reexamined the spinach system and report here the presence ofa third 3-ketoacyl-ACP synthase which is completely insensitive to cerulenin, active only with short chain acyl thioesters, and appears to prefer acetyl-CoA to acetyl-ACP. MATERIALS AND METHODS MaterialsIt has been known for decades that fatty acid biosynthesis involves extending an acyl-ACP' stepwise by two carbons. In plants and bacteria, this synthesis is catalyzed by a type II fatty acid synthase, a readily dissociable group of enzymes which can be individually isolated and studied (1). One of the initial reactions leading to chain elongation is catalyzed by the enzyme 3-ketoacyl-ACP synthase, frequently referred to simply as the 'condensing enzyme.' The spinach fatty acid synthase was initially separated into its individual components by Shimakata and Stumpf (13,14). In that study, they found two 3-ketoacyl-ACP synthases, designated I and II. 3-Ketoacyl-ACP synthase I was responsible for the majority of the condensations, using acyl-ACPs r...