Effect of processing on the recovery of recombinant beta-glucuronidase (rGUS) from transgenic canola

This study addresses the processing of transgenic canola seed for production of recombinant proteins by using beta-glucuronidase (rGUS) as a model protein. The major processing steps that were investigated included dry and wet grinding of the seed, solvent extraction of canola oil, and protein extraction. rGUS in canola seed was stable for at least 2 weeks of incubation at 38 degrees C and for more than 5 months at 10 degrees C. At 70 degrees C, the residual activity changed inversely to the initial moisture content of the seed. The comparison of wet and dry processing revealed no significant differences in protein recovery. rGUS was stable during the defatting of transgenic canola flakes with hexane at 66 degrees C, whereas 2-propanol extraction at the same temperature reduced the extractable enzyme activity by almost 50%. The particle size of the ground seed was important for the extraction efficiency. A faster extraction and greater protein yield was achieved by extracting particles with an average diameter equal to or smaller than 255 microm. More than 80% rGUS was extracted in one stage with sodium phosphate buffer of pH 7.5.     

Processing of transgenic corn seed and its effect on the recovery of recombinant beta-glucuronidase

The tools of plant biotechnology that have been developed to improve agronomic traits are now being applied to generate recombinant protein products for the food, feed, and pharmaceutical industry. This study addresses several processing and protein recovery issues that are relevant to utilizing transgenic corn as a protein production system. The gus gene coding for beta-glucuronidase (rGUS) was stably integrated and expressed over four generations. The accumulation level of rGUS reached 0.4% of total extractable protein. Within the kernel, rGUS was preferentially accumulated in the germ even though a constitutive ubiquitin promoter was used to direct gus expression. Fourth-generation transgenic seed was used to investigate the effect of seed processing on the activity and the recovery of rGUS. Transgenic seed containing rGUS could be stored at an ambient temperature for up to two weeks and for at least three months at 10 degrees C without a significant loss of enzyme activity. rGUS exposed to dry heat was more stable in ground than in whole kernels. The enzyme stability was correlated with the moisture loss of the samples during the heating. Transgenic seed was dry-milled, fractionated, and hexane extracted to produce full-fat and defatted germ fractions. The results of the aqueous extraction of rGUS from ground kernels, full-fat germ, and defatted-germ samples revealed that approximately 10 times more rGUS per gram of solids could be extracted from the ground full-fat germ and defatted-germ than from the kernel samples. The extraction of corn oil from ground germ with hot hexane (60 degrees C) did not affect the extractable rGUS activity. rGUS was purified from ground kernels and full-fat germ extracts by ion exchange, hydrophobic interaction, and size exclusion chromatography. Similar purity and yield of rGUS were obtained from both extracts. Biochemical properties of rGUS purified from transgenic corn seed were similar to those of E. coli GUS.     

A seed coat-specific promoter for canola

The canola industry generates more than $11 billion of yearly income to the Canadian economy. One problem of meal quality is the dark polyphenolic pigments that accumulate in the seed coat. Seed coat-specific promoters are a pre-requisite to regulate the genes involved in seed coat development and metabolism. The β-glucuronidase (GUS) reporter gene was used to test an Arabidopsis promoter in developing and mature seeds of canola (Brassica napus). The promoter tested is the regulatory region of the laccase gene (AtLAC15) from Arabidopsis thaliana. The AtLAC15 promoter::GUS construct was inserted into canola double haploid line DH12075 using Agrobacterium-mediated transformation. Southern blot analysis using a 536 bp GUS probe showed variation among the transformed plants in the T-DNA copy numbers and the position of the insertion in their genomes. Histochemical assay of the GUS enzyme in different tissues (roots, leaves, stem, pollen grains, flowers, siliques, embryos and seed coats) showed ascending GUS activity only in the seed coat from 10 days after pollination (DAP) to the fully mature stage (35 DAP). GUS stain was observed in the mucilage cell layer, in the outer integument layer of the seed coat but not in the inner integument. The AtLAC15 promoter exhibited a specificity and expression level that is useful as a seed coat-specific promoter for canola.     

Two seed coat-specific promoters are functionally conserved between <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Brassica napus</Emphasis>

Canola is one of the most important cash crops in Canada, and a national project named “Designing Oilseeds for Tomorrow’s Market” was undertaken to improve seed meal quality of this strategically important crop. As a part of this project, our group is focusing on identifying seed coat-specific promoters for canola (Brassica napus). These promoters will be used to genetically modify canola seed coat to reduce or eliminate anti-nutritional components from the meal. The Arabidopsis thaliana BAN promoter (AtBANpro) and δVPE promoter (AtδVPEpro) were isolated and fused to GUS reporter gene to generate transgenic canola plants. These plants were analyzed by GUS staining and microtome sectioning which showed that both promoters are seed coat-specific in canola: AtBANpro in inner seed coat layer and AtδVPEpro in outer seed coat layer. Therefore, the two Arabidopsis promoters can be used to modify genes in seed coat of canola for further improving its seed qualities.     

Distribution of intact and core membrane lipids of archaeal glycerol dialkyl glycerol tetraethers among size-fractionated particulate organic matter in hood canal, puget sound

There is great interest in the membrane lipids of archaea (glycerol dialkyl glycerol tetraethers [GDGTs]) as tracers of archaeal biomass because of their utility as paleoproxies and because of the biogeochemical importance of archaea. While core GDGTs (formed by hydrolysis of polar head groups of intact GDGTs after cell death) are appropriate for paleostudies, they have also been used to trace archaeal populations. Also, despite the small size (0.2 by 0.7 μm) of cultivated marine archaea, 0.7-μm glass-fiber filters (GFFs) are typically used to collect GDGTs from natural waters. We quantified both core and intact GDGTs in free-living (0.2- to 0.7-μm), suspended (0.7- to 60-μm), and aggregate (>60-μm) particle size fractions in Puget Sound (Washington State). On average, the free-living fraction contained 36% of total GDGTs, 90% of which were intact. The intermediate-size fraction contained 62% of GDGTs, and 29% of these were intact. The aggregate fraction contained 2% of the total GDGT pool, and 29% of these were intact. Our results demonstrate that intact GDGTs are largely in the free-living fraction. Because only intact GDGTs are present in living cells, protocols that target this size fraction and analyze the intact GDGT pool are necessary to track living populations in marine waters. Core GDGT enrichment in larger-size fractions indicates that archaeal biomass may quickly become attached or entrained in particles once the archaea are dead or dying. While the concentrations of the two pools were generally not correlated, the similar sizes of the core and intact GDGT pools suggest that core GDGTs are removed from the water column on timescales similar to those of cell replication, on timescales of days to weeks.     

Wheat, canola and grain legume access to soil phosphorus fractions differs in soils with contrasting phosphorus dynamics

Despite the high phosphorus (P) mobilizing capacity of many legumes, recent studies have found that, at least in calcareous soils, wheat is also able to access insoluble P fractions through yet unknown mechanism(s). We hypothesized that insoluble P fractions may be more available to non-legume plants in alkaline soils due to increased dissolution of the dominant calcium(Ca)-P pool into depleted labile P pools, whereas non-legumes may have limited access to insoluble P fractions in iron(Fe)- and aluminium(Al)-P dominated acid soils. Four crop species (faba bean, chickpea, wheat and canola) were grown on two acid and one alkaline soil under glasshouse conditions to examine rhizosphere processes and soil P fractions accessed. While all species generally depleted the H₂O-soluble inorganic P (water Pi) pool in all soils, there was no net depletion of the labile NaHCO₃-extractable inorganic P fraction (NaHCO₃ Pi) by any species in any soil. The NaOH-extractable P fraction (NaOH Pi) in the alkaline soil was the only non-labile Pi fraction depleted by all crops (particularly canola), possibly due to increases in rhizosphere pH. Chickpea mobilized the insoluble HCl Pi and residual P fractions; however, rhizosphere pH and carboxylate exudation could not fully explain all of the observed Pi depletion in each soil. All organic P fractions appeared highly recalcitrant, with the exception of some depletion of the NaHCO₃ Po fraction by faba bean in the acid soils. Chickpea and faba bean did not show a higher capacity than wheat or canola to mobilize insoluble P pools across all soil types, and the availability of various P fractions to legume and non-legume crops differed in soils with contrasting P dynamics.     

Revaluation of the role of cholesterol in stabilizing rafts implicated in T cell receptor signaling

T lymphocytes contain two kinetic pools of cholesterol extractable with methyl-beta-cyclodextrin (m-beta-CD): a fast pool (31.5%, t1/2=17 s) and a slow pool (68.5%, t1/2=15 min). Purification of detergent-resistant membranes (DRMs) shows that the fast pool corresponds to buoyant cholesterol. Cholesterol extraction of the fast pool (i.e. cholesterol from rafts) still allows the buoyancy of signaling proteins and their phosphorylation under CD3 stimulation. Cholesterol depletion of the slow pool (i.e. cholesterol from membranes other than rafts) is accompanied by the extraction of the whole raft followed by the inhibition of CD3-induced tyrosine-phosphorylations. Cholesterol oxidase (COase) allows a specific oxidation of raft cholesterol into cholestenone. Cholestenone leaves the DRMs and accumulates as Triton X-100-soluble material. Specific cholesterol-rich raft disruption by COase does not inhibit the activation of either Jurkat cells or T CD4+ lymphocytes. Our study challenges the real role of cholesterol-rich rafts in CD3/TCR signaling and suggests that a cholesterol-poor subtype of rafts is involved in signal transmission via the TCR.     

Labile Phosphorus in Soils of Forest Fallows and Primary Forest in the Bragantina Region, Brazil

We used the Hedley sequential extraction procedure to measure nine different organic inorganic soil phosphorus fractions in forest soil of the Bragantina region of Para, Brazil. We compared the labile fractions (resin‐extractable P + HCO3‐extractable inorganic and organic P) in Oxisols from three secondary forests (10, 20, and 40 years old) and a primary forest. These stands were located in an area that has supported shifting agriculture for approximately a century. After agricultural use, total P and labile P in soils of young secondary forests are diminished compared to the amounts presents in the primary forest soil. Within each stand, organic carbon content was a good predictor of labile organic and inorganic P, consistent with the large body of research indicating that mineralization of organic matter is important to plant nutrition in tropical ecosystems. During the reorganization of P pools during forest development, the pool of labile organic P (HCO3‐extractable) diminishes more than the other labile fractions, suggesting that it is directly or indirectly an important source of P for the regrowing forest vegetation. Across the four age classes of forest, the soil reservoir of labile P was equal to or greater than the total amount of P in the vegetation. If labile P measured by this method adequately represents P available to plants in the short term (as suggested by the current consensus), we would conclude that plant‐available P is reasonable abundant, and that the effects of agriculture on available P pools are detectable but not sufficient to compromise forest regrowth in this area.     

A seed coat outer integument-specific promoter for Brassica napus

In search for seed coat-specific promoters for canola (Brassica napus), transgenic plants carrying a 2,121 bp fragment of Arabidopsis thaliana At4g12960 promoter (AtGILTpro) fused to the uidA reporter gene (GUS) were generated. Out of 7 independent events in transgenic canola plants raised, 2 exhibited GUS activity exclusively in the outer integument of the seed coat. GUS activity in other tissues was also observed in the remaining five transformants. Therefore, the AtGILT promoter can be used as a canola seed coat outer integument-specific promoter after the generation and selection of desired transformants from several transgenic lines.     

Heterogeneous Cell Density and Genetic Structure of Bacterial Pools Associated with Various Soil Microenvironments as Determined by Enumeration and DNA Fingerprinting Approach (RISA)

Abstract The cell density and the genetic structure of bacterial subcommunities (further named pools) present in the various microenvironments of a silt loam soil were investigated. The microenvironments were isolated first using a procedure of soil washes that separated bacteria located outside aggregates (outer part) from those located inside aggregates (inner part). A nondestructive physical fractionation was then applied to the inner part in order to separate bacteria located inside stable aggregates of different size (size fractions, i.e., two macroaggregate fractions, two microaggregate fractions, and the dispersible day fraction). Bacterial densities measured by acridine orange direct counts (AODC) and viable heterotrophic (VH) cell enumerations showed the heterogeneous quantitative distribution of cells in soil. Bacteria were preferentially located in the inner part with 87.6% and 95.4% of the whole AODC and VH bacteria, respectively, and in the microaggregate and dispersible clay fractions of this part with more than 70% and 80% of the whole AODC and VH bacteria, respectively. The rRNA intergenic spacer analysis (RISA) was used to study the genetic structure of the bacterial pools. Different fingerprints and consequently different genetic structures were observed between the unfractionated soil and the microenvironments, and also among the various microenvironments, giving evidence that some populations were specific to a given location in addition to the common populations of all the microenvironments. Cluster and multivariate analysis of RISA profiles showed the weak contribution of the pools located in the macroaggregate fractions to the whole soil community structure, as well as the clear distinction between the pool associated to the macroaggregate fractions and the pools associated to the microaggregate ones. Furthermore, these statistical analyses allowed us to ascertain the influence of the clay and organic matter content of microenvironments on the genetic structure relatedness between pools. Received: 15 December 1999; Accepted: 5 April 2000; Online Publication: 19 May 2000     

Solid-phase distribution of heavy metals as affected by single reagent extraction in dredged sediment derived surface soils

ABSTRACT

EDTA is useful to assess mobile metal pools in polluted soils and sediments. There is a need to enhance our understanding of the significance of metal fractions released. The impact of single reagent extraction with 0.05 mol L^?1 EDTA on the solid phase distribution of trace metals in surface soils sampled from confined dredged sediment disposal sites was investigated. Not extracted and EDTA extracted soils were subjected to sequential extraction to fractionate the total contents into: (1) easily exchangeable and carbonate bound fraction; (2) reducible fraction; (3) oxidisable fraction; and (4) residual fraction. With EDTA, significant portions of metals associated with the acid extractable and reducible fractions were released. The oxidisable and residual fractions remained unaffected for most of the investigated metals except for the organic matter associated metals (Cu and Pb). A decrease in the residual fraction after EDTA-extraction for Cu and Pb was attributed to artifacts of the sequential extraction procedure.     

Genetic engineering strategies for purification of recombinant proteins from canola by anion exchange chromatography: an example of beta-glucuronidase

The elution behavior of native canola proteins from different anion-exchange resins was determined. The elution profiles showed the potential for simplified recovery of acidic recombinant proteins from canola. When Q-sepharose fast flow was used, there were three optimal salt elution points at which a recombinant protein would have minimal contamination with native proteins. The feasibility of exploiting this advantage was examined for recovery of the acidic protein beta-glucuronidase (GUS/GUSD0 from the Escherichia coli gene) along with three polyaspartate fusions to the wild-type GUS. The fusions contained 5 (GUSD5), 10 (GUSD10), or 15 (GUSD15) aspartic acids fused to the C-terminus and were chosen to extend the elution time. The three fusions and the wild-type enzyme were produced in E. coli, purified, and added to canola extracts before chromatography. The equivalence of this spiking experiment to that of extracting a recombinant protein from transgenic canola was determined in a control experiment using transgenic canola expressing the wild-type enzyme. Behavior in the transgenic and spiked experiments was equivalent. GUSD0 eluted at the earliest optimal elution point; the addition of polyaspartate tails resulted in longer retention times and better selective recovery. If one assumes binding through a single fusion (the protein is a tetramer), there is a nearly linear shift in elution within the salt gradient of 17 mM per added charge up to 10, with a reduced increment from 10 to 15. The fusions and their enzymatic activity proved very stable in the canola extracts through 7 days in cold storage, providing flexibility in process scheduling.     

Internalization of nerve growth factor by PC12 cells. A description of cellular pools

Binding and internalization of nerve growth factor (NGF) by responsive cells is a complex process. We have incubated rat pheochromocytoma cells (PC12) with 125I-NGF at 37 degrees C and measured the association of ligand after removal of subsets of bound ligand by different methods. Chase with unlabeled NGF at either 4 or 37 degrees C, acid stripping, nonionic detergent stability, and combinations of these protocols were utilized. These variations of the binding assay were able to distinguish ligand bound to fast versus slow cell surface receptors, NGF bound to slow receptors at the cell surface versus cell interior, and soluble ligand versus cytoskeletally attached NGF. Quantitative and temporal relations among five cellular pools were defined. Experiments with the inhibitors chloroquine, cytochalasin B, and colchicine defined pools of NGF in terms of the route through the cell from the plasma membrane to the lysosome. Chloroquine caused accumulation of NGF only in the pool that was not associated with the cytoskeleton, implicating the involvement of this pool in supplying ligand to the lysosome. Results with cytochalasin B and colchicine suggest that both microfilaments and microtubules are involved in pathways leading to NGF degradation. A semiquantitative model for the movement of NGF through the cell is presented based on these observations.     

A plasma membrane pool of cholesteryl esters that may mediate the selective uptake of cholesteryl esters from high-density lipoproteins

Selective uptake of high-density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL particles occurs by a nonendocytotic pathway that requires no specific apolipoprotein and results in the net delivery of cholesteryl esters to cells. Here we examine a reversibly cell-associated pool of cholesteryl ester tracer and its relationship to selective uptake. A fraction of cholesteryl ester tracer selectively taken up from HDL by rat primary or mouse Y1-BS1 adrenocortical cells was chased from the cells by subsequent incubation with unlabeled HDL. This pool of cholesteryl ester tracer was distinct from that irreversibly internalized, and in excess of that accounted for by dissociation of labeled HDL particles bound to the cell surface. In response to various metabolic effectors, cholesteryl ester tracer in this reversibly cell-associated pool of Y1-BS1 cells correlated linearly with irreversible selective uptake. Both reversibly and irreversibly cell-associated pools of cholesteryl ester tracer displayed similar saturation kinetics for uptake from HDL, and both pools correlated inversely with cell-free cholesterol levels. Cholesteryl ester tracer in the reversible pool was shown to serve as a precursor for irreversible selective uptake. A pool with properties similar to the reversibly cell-associated pool was identified in plasma membrane fractions; enough tracer was incorporated into this pool to account for the reversibly cell-associated pool of intact cells. The data suggest that a pool of cholesteryl esters in the plasma membrane is involved in selective uptake at a step prior to irreversible internalization.     

Efflux of cholesterol from different cellular pools

Free cholesterol is very efficiently removed from cells by 2-hydroxypropyl-beta-cyclodextrins. The efflux of cholesterol occurs from two distinct kinetic pools: the half-times (t(1/2)) for the two pools in CHO-K1 cells are 15 +/- 5 s and 21 +/- 6 min and they represent 25% +/- 5% and 75% +/- 5% of the readily exchangeable cell cholesterol, respectively. In this study we have determined that the fast pool and the majority of the slow kinetic pool for cholesterol efflux are apparently present in the plasma membrane. Numerous agents that inhibit intracellular cholesterol trafficking are unable to affect either the size or the t(1/2) for efflux of either kinetic pool. In contrast, treatment of the cells with N-ethylmaleimide (NEM), exogenous lipases such as sphingomyelinase and phospholipase C, calcium ionophore A23187, or heat resulted in the dramatic increase in the size of the fast kinetic pool of cholesterol. These changes in the kinetics of cholesterol efflux are not specific to the nature of the extracellular acceptor indicating that they are a consequence of changes in the cell plasma membrane. The above treatments disrupt the normal organization of the lipids in the plasma membrane via either hydrolysis or randomization. The phosphatidylcholine and sphingomyelin present in the plasma membrane are critical for maintaining the two kinetic pools of cholesterol; any alteration in the amount or the location of these phospholipids results in an enhancement of efflux by redistributing cholesterol into the fast kinetic pool.     

Calcium compartments and fluxes are affected by the src gene product of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus

Total cellular calcium content (determined by atomic absorption spectrometry) of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus decreases with cell density, but is found not significantly different at permissive and at non-permissive temperature. Kinetic analysis of 45Ca efflux from preloaded cells exhibits three separable pools of exchangeable calcium. The ratio of pool size of the fast-exchanging Ca-compartment (bound to cell surface) to pool size of the intermediate Ca-compartment (cytoplasmic) was found to decrease from 2.5 to 1.3 upon shift from non-permissive to permissive temperature. The slowly exchanging Ca-pool (presumably mitochondrial) did not change significantly upon temperature shift. These and further data demonstrate a close correlation between distribution of cellular Ca among different cellular compartments and characteristics of cellular proliferation, both attributable to the function(s) of a single oncogene.     

State of oncomarker protein B23/nucleophosmin in HeLa cells

Western blot after SDS-PAGE for protein separation showed two immunoreactive bands corresponding to monomers (38–40 kDa) and oligomers (210–230 kDa) of nucleophosmin in HeLa cell lysates. Decreasing the buffer ionic strength during the incubation of cells and nuclei destabilized these oligomers. We also showed the existence of two B23/nucleophosmin pools in nuclei of HeLa cells with different sensitivity to hypotonic buffer treatment: one extractable from the nucleus and the other non-extractable and tightly bound to the nucleus. A detailed structural analysis of the extractable B23 pool was carried out: two closely related nucleophosmin isoforms (B23.1 and B23.2) were identified as a result of analysis of C-terminal amino acid sequences using carboxypeptidase hydrolysis; the N-termini of both isoforms are blocked by an acetyl group. As a result of sequencing of the deacetylated proteins, it has been established that the N-terminal amino acid sequence of nucleophosmin in these preparations is truncated by nine amino acid residues and the acetylated residue is Ser. The truncated monomer of nucleophosmin (represented only by the extractable part of the protein) on addition of magnesium ions to low ionic strength buffer or increase in buffer ionic strength was shown to form oligomers with molecular weights (210–230 kDa) similar to those revealed in the total cell lysate. It should be noted that the set of oligomers in this case differs from the one in total cell lysate. Our strategy of characterization of B23 forms for HeLa cells can be applied for other tumor cells.     

Enhanced channelling of sulphate through a rapidly exchangeable sulphate pool in response to stimulated glycosaminoglycan synthesis in pancreatic epithelial cells.

The ability of cells to decorate glycosaminoglycans (GAGs) with sulphate in highly specific patterns is important to extracellular matrix biogenesis and placing appropriate glycosulphated ligands on the cell surface. We have examined sulphate metabolism in two pancreatic duct epithelial cell lines - PANC-1 and CFPAC-1 (derived from a cystic fibrosis patient) with a view to understanding how pancreatic cells utilise intracellular sulphate. [35S]Sulphate uptake was rapid and reached near steady state levels within 10 min. However, the intracellular specific activity of [35S]sulphate for PANC-1 and CFPAC-1 reached only 35 and 10%, respectively, of the medium specific activity at 10 min. Therefore, sulphate appears to reside within two compartments; a rapidly exchangeable sulphate pool (RESP) and a slowly exchangeable sulphate pool (SESP). Reducing chloride in the medium, increased the specific activity of [35S]sulphate within cells and increased the size of the inorganic sulphate pool, suggesting that the RESP was enlarged. Sulphate pools were not different in size between the two cell lines in physiological NaCl. Increasing the size of the sulphate pool had no effect on [35S]sulphate:[3H]glucosamine ratios incorporated into glycosaminoglycans (GAGs); however, stimulating the synthesis of GAGs with 4-methylumbelliferyl-beta-d-xyloside, stably elevated [35S]:[3H] ratios. This was due to higher [35S]sulphate incorporation. [35S]Cysteine contributed less than 0.1% of the cells' sulphate requirements. We conclude that in the face of elevated demand for sulphate, pancreatic cells appear to channel a greater proportion through the RESP.     

Considerations for the recovery of recombinant proteins from plants

The past 5 years have seen the commercialization of two recombinant protein products from transgenic plants, and many recombinant therapeutic proteins produced in plants are currently undergoing development. The emergence of plants as an alternative production host has brought new challenges and opportunities to downstream processing efforts. Plant hosts contain a unique set of matrix contaminants (proteins, oils, phenolic compounds, etc.) that must be removed during purification of the target protein. Furthermore, plant solids, which require early removal after extraction, are generally in higher concentration, wider in size range, and denser than traditional bacterial and mammalian cell culture debris. At the same time, there remains the desire to incorporate highly selective and integrative separation technologies (those capable of performing multiple tasks) during the purification process from plant material. The general plant processing and purification scheme consists of isolation of the plant tissue containing the recombinant protein, fractionation of the tissue along with particle size reduction, extraction of the target protein into an aqueous medium, clarification of the crude extract, and finally purification of the product. Each of these areas will be discussed here, focusing on what has been learned and where potential concerns remain. We also present details of how the choice of plant host, along with location within the plant for targeting the recombinant protein, can play an important role in the ultimate ease of recovery and the emergence of regulations governing plant hosts. Major emphasis is placed on three crops, canola, corn, and soy, with brief discussions of tobacco and rice.