Terence A. Walsh

Mutations in an Auxin Receptor Homolog AFB5 and in SGT1b Confer Resistance to Synthetic Picolinate Auxins and Not to 2,4-Dichlorophenoxyacetic Acid or Indole-3-Acetic Acid in Arabidopsis

Although a wide range of structurally diverse small molecules can act as auxins, it is unclear whether all of these compounds act via the same mechanisms that have been characterized for 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). To address this question, we used a novel member of the picolinate class of synthetic auxins that is structurally distinct from 2,4-D to screen for Arabidopsis (Arabidopsis thaliana) mutants that show chemically selective auxin resistance. We identified seven alleles at two distinct genetic loci that conferred significant resistance to picolinate auxins such as picloram, yet had minimal cross-resistance to 2,4-D or IAA. Double mutants had the same level and selectivity of resistance as single mutants. The sites of the mutations were identified by positional mapping as At4g11260 and At5g49980. At5g49980 is previously uncharacterized and encodes auxin signaling F-box protein 5, one of five homologs of TIR1 in the Arabidopsis genome. TIR1 is the recognition component of the Skp1-cullin-F-box complex associated with the ubiquitin-proteasome pathway involved in auxin signaling and has recently been shown to be a receptor for IAA and 2,4-D. At4g11260 encodes the tetratricopeptide protein SGT1b that has also been associated with Skp1-cullin-F-box-mediated ubiquitination in auxin signaling and other pathways. Complementation of mutant lines with their corresponding wild-type genes restored picolinate auxin sensitivity. These results show that chemical specificity in auxin signaling can be conferred by upstream components of the auxin response pathway. They also demonstrate the utility of genetic screens using structurally diverse chemistries to uncover novel pathway components.

Robust crop resistance to broadleaf and grass herbicides provided by aryloxyalkanoate dioxygenase transgenes

Engineered glyphosate resistance is the most widely adopted genetically modified trait in agriculture, gaining widespread acceptance by providing a simple robust weed control system. However, extensive and sustained use of glyphosate as a sole weed control mechanism has led to field selection for glyphosate-resistant weeds and has induced significant population shifts to weeds with inherent tolerance to glyphosate. Additional weed control mechanisms that can complement glyphosate-resistant crops are, therefore, urgently needed. 2,4-dichlorophenoxyacetic acid (2,4-D) is an effective low-cost, broad-spectrum herbicide that controls many of the weeds developing resistance to glyphosate. We investigated the substrate preferences of bacterial aryloxyalkanoate dioxygenase enzymes (AADs) that can effectively degrade 2,4-D and have found that some members of this class can act on other widely used herbicides in addition to their activity on 2,4-D. AAD-1 cleaves the aryloxyphenoxypropionate family of grass-active herbicides, and AAD-12 acts on pyridyloxyacetate auxin herbicides such as triclopyr and fluroxypyr. Maize plants transformed with an AAD-1 gene showed robust crop resistance to aryloxyphenoxypropionate herbicides over four generations and were also not injured by 2,4-D applications at any growth stage. Arabidopsis plants expressing AAD-12 were resistant to 2,4-D as well as triclopyr and fluroxypyr, and transgenic soybean plants expressing AAD-12 maintained field resistance to 2,4-D over five generations. These results show that single AAD transgenes can provide simultaneous resistance to a broad repertoire of agronomically important classes of herbicides, including 2,4-D, with utility in both monocot and dicot crops. These transgenes can help preserve the productivity and environmental benefits of herbicide-resistant crops.

Inhibition of Diabrotica larval growth by a multicystatin from potato tubers

Abstract Second instar western corn rootworm larvae have a higher proportion of total proteinase activity which is attributable to cysteine proteinase (92%) than southern corn rootworm larvae (75%). E-64, potato multicystatin (PMC) and tryptic fragments of PMC (T-PMC) are effective inhibitors of gut cysteine proteinase activity, in vitro. The presence of PMC in the diet causes a dose-dependent inhibition of growth in neonate southern corn rootworm and second instar southern corn rootworm and western corn rootworm. Neonate southern corn rootworm and second instar western rootworm have similar sensitivity to the inhibitor (50% inhibition at 25–43.8 μg/cm2), whereas second instar southern corn rootworm are about 5-fold less sensitive. In contrast to southern corn rootworm larvae, western corn rootworm growth is completely halted by PMC. Long-term exposure of southern corn rootworm larvae to PMC suggests that the larvae become less sensitive to the inhibitor during development. Hen egg cystatin (HEC) and T-PMC are unable to inhibit growth of either species but, in southern corn rootworm, co-feeding of potato carboxypeptidase inhibitor (PCI) with T-PMC causes growth inhibition. Direct measurements of gut cysteine proteinase activity after feeding of the inhibitors indicates that PMC and PCI+T-PMC cause significant inhibition of cysteine proteinase in the gut, whereas HEC, PCI and T-PMC do not. These observations indicate that multicystatins such as PMC may be effective cystatins for use in controlling larvae of Diabrotica species in transgenic plants.

Characterization of a genomic sequence coding for potato multicystatin, an eight-domain cysteine proteinase inhibitor

A gene coding for potato multicystatin (PMC), the crystalline inhibitor of cysteine proteases which is found in tubers, was isolated and characterized. The deduced polypeptide product of this genomic sequence in 757 amino acids long and has a molecular mass of 86,778 Da. It consists exclusively of eight closely related domains, with 53–89% identity of residues. Each repeated unit is homologous to the cystatin superfamily of cysteine protease inhibitors. To date, no other member of this family has been found to contain so many inhibitor domains in one polypeptide. Eight introns are proposed in the 3.5 kb of genomic DNA coding for PMC, one in each cystatin unit. There is a family of 4 to 6 such large genes in potato, while in pea and maize the homologues are much smaller, and probably code for single-domain cystatins. PMC transcripts are abundant in tubers, but scarce in undamaged leaves or stems of field-grown potatoes. The tuber messages are derived from at least four genes (including the cloned example). The pattern of gene expression, as well as the properties of the protein, suggest that PMC has a role in the plants defense system.

Inhibition of Diabrotica larval growth by patatin, the lipid acyl hydrolase from potato tubers

Patatin, the nonspecific lipid acyl hydrolase from potato (Solanum tuberosum L.) tubers, dose-dependently inhibits the growth of southern corn rootworm (SCR) and western corn rootworm when fed to them on artificial diet. The 50% growth reduction levels are somewhat cultivar dependent, ranging from 60 to 150 [mu]g/g diet for neonate SCR larvae. A single patatin isoform also inhibits larval growth. Neonate SCR continuously exposed to patatin are halted in larval development. Treatment with di-isopropylfluorophosphate essentially eliminates patatins phospholipase, galactolipase, and acyl hydrolase activities. SCR growth inhibition is eliminated also, indicating that patatins serine hydrolase activity is responsible for the observed activities. Patatin-mediated phospholipolysis is highly pH and cultivar dependent, with specific activities up to 300-fold less at pH 5.5 than at pH 8.5. Esterase or phospholipase activities do not correlate with insect growth inhibition. Galactolipase activity, being cultivar and pH independent, correlates significantly with SCR growth inhibition. Insect-growth inhibition of patatin is significantly reduced with increased dietary cholesterol levels. In conclusion, patatin represents a new class of insect-control proteins with a novel mode of action possibly involving lipid metabolism.

Proteolysis of the 85-Kilodalton Crystalline Cysteine Proteinase Inhibitor from Potato Releases Functional Cystatin Domains

The protein crystals found in potato (Solanum tuberosum L.) tuber cells consist of a single 85-kD polypeptide. This polypeptide is an inhibitor of papain and other cysteine proteinases and is capable of binding several proteinase molecules simultaneously (P. Rodis, J.E. Hoff [1984] Plant Physiol 74: 907–911). We have characterized this unusual inhibitor in more detail. Titrations of papain activity with the potato papain inhibitor showed that there are eight papain binding sites per inhibitor molecule. The inhibition constant (Ki) value for papain inhibition was 0.1 nM. Treatment of the inhibitor with trypsin resulted in fragmentation of the 85-kD polypeptide into a 32-kD polypeptide and five 10-kD polypeptides. The 32-kD and 10-kD fragments all retained the ability to potently inhibit papain (Ki values against papain were 0.5 and 0.7 nM, respectively) and the molar stoichiometries of papain binding were 2 to 3:1 and 1:1, respectively. Other nonspecific proteinases such as chymotrypsin, subtilisin Carlsberg, thermolysin, and proteinase K also cleaved the 85-kD inhibitor polypeptide into functional 22-kD and several 10-kD fragments. The fragments obtained by digestion of the potato papain inhibitor with trypsin were purified by reverse-phase high-performance liquid chromatography, and the N-terminal amino acid sequence was obtained for each fragment. Comparison of these sequences showed that the fragments shared a high degree of homology but were not identical. The sequences were homologous to the N termini of members of the cystatin superfamily of cysteine proteinase inhibitors. Therefore, the inhibitor appears to comprise eight tandem cystatin domains linked by proteolytically sensitive junctions. We have called the inhibitor potato multicystatin (PMC). By immunoblot analysis and measurement of papain inhibitory activity, PMC was found at high levels in potato leaves (up to 0.6 mg/g fresh weight tissue), where it accumulated under conditions that induce the accumulation of other proteinase inhibitors linked to plant defense. PMC may have a similar defensive role, for example in protecting the plant from phytophagous insects that utilize cysteine proteinases for dietary protein digestion.

Metabolic Engineering of Seeds Can Achieve Levels of ω-7 Fatty Acids Comparable with the Highest Levels Found in Natural Plant Sources

Plant oils containing ω-7 fatty acids (FAs; palmitoleic 16:1Δ9 and cis-vaccenic 18:1Δ11) have potential as sustainable feedstocks for producing industrially important octene via metathesis chemistry. Engineering plants to produce seeds that accumulate high levels of any unusual FA has been an elusive goal. We achieved high levels of ω-7 FA accumulation by systematic metabolic engineering of Arabidopsis (Arabidopsis thaliana). A plastidial 16:0-ACP desaturase has been engineered to convert 16:0 to 16:1Δ9 with specificity >100-fold than that of naturally occurring paralogs, such as that from cats claw vine (Doxantha unguis-cati). Expressing this engineered enzyme (Com25) in seeds increased ω-7 FA accumulation from <2% to 14%. Reducing competition for 16:0-ACP by down-regulating the β-ketoacyl-ACP synthase II 16:0 elongase further increased accumulation of ω-7 FA to 56%. The level of 16:0 exiting the plastid without desaturation also increased to 21%. Coexpression of a pair of fungal 16:0 desaturases in the cytosol reduced the 16:0 level to 11% and increased ω-7 FA to as much as 71%, equivalent to levels found in Doxantha seeds.

Maize Ribosome-Inactivating Protein (b-32) (Homologs in Related Species, Effects on Maize Ribosomes, and Modulation of Activity by Pro-Peptide Deletions)

The ribosome-inactivating protein (RIP) from maize (Zea mays L.) is unusual in that it is produced in the endosperm as an inactive pro-form, also known as b-32, which can be converted by limited proteolysis to a two-chain active form, [alpha][beta] RIP. Immunological analysis of seed extracts from a variety of species related to maize showed that pro/[alpha][beta] forms of RIP are not unique to maize but are also found in other members of the Panicoideae, including Tripsacum and sorghum. Ribosomes isolated from maize were quite resistant to both purified pro- and [alpha][beta] maize RIPs, whereas they were highly susceptible to the RIP from pokeweed. This suggests that the production of an inactive pro-RIP is not a mechanism to protect the plants own ribosomes from deleterious action of the [alpha][beta] RIP. RIP derivatives with various pro-segments removed were expressed at high levels in Escherichia coli. Measurement of their activity before and after treatment with subtilisin Carlsberg clearly identified the 25-amino acid intradomain insertion, rather than the N- or C-terminal extensions, as the major element responsible for suppression of enzymatic activity. A RIP with all three processed regions deleted had activity close to that of the native [alpha][beta] form.

Chemical genetic identification of glutamine phosphoribosylpyrophosphate amidotransferase as the target for a novel bleaching herbicide in arabidopsis

A novel phenyltriazole acetic acid compound (DAS734) produced bleaching of new growth on a variety of dicotyledonous weeds and was a potent inhibitor of Arabidopsis (Arabidopsis thaliana) seedling growth. The phytotoxic effects of DAS734 on Arabidopsis were completely alleviated by addition of adenine to the growth media. A screen of ethylmethanesulfonate-mutagenized Arabidopsis seedlings recovered seven lines with resistance levels to DAS734 ranging from 5- to 125-fold. Genetic tests determined that all the resistance mutations were dominant and allelic. One mutation was mapped to an interval on chromosome 4 containing At4g34740, which encodes an isoform of glutamine phosphoribosylamidotransferase (AtGPRAT2), the first enzyme of the purine biosynthetic pathway. Sequencing of At4g34740 from the resistant lines showed that all seven contained mutations producing changes in the encoded polypeptide sequence. Two lines with the highest level of resistance (125-fold) contained the mutation R264K. The wild-type and mutant AtGPRAT2 enzymes were cloned and functionally overexpressed in Escherichia coli. Assays of the recombinant enzyme showed that DAS734 was a potent, slow-binding inhibitor of the wild-type enzyme (I50 approximately 0.2 μm), whereas the mutant enzyme R264K was not significantly inhibited by 200 μm DAS734. Another GPRAT isoform in Arabidopsis, AtGPRAT3, was also inhibited by DAS734. This combination of chemical, genetic, and biochemical evidence indicates that the phytotoxicity of DAS734 arises from direct inhibition of GPRAT and establishes its utility as a new and specific chemical genetic probe of plant purine biosynthesis. The effects of this novel GPRAT inhibitor are compared to the phenotypes of known AtGPRAT genetic mutants.

Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid

Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving.