William J. Hurkman

Thioredoxin targets in plants: The first 30 years

The turn of the century welcomed major developments in redox biology. In plants, proteomics made possible the identification of proteins linked to thioredoxin (Trx), initially in chloroplasts and then other cell compartments. Two procedures, one based on thiol specific probes and the other on mutant Trx proteins, facilitated the labeling or isolation of potential Trx targets that were later identified with proteomic approaches. As a result, the number of targets in land plants increased 10-fold from fewer than 40 to more than 400. Additional targets have been identified in green algae and cyanobacteria, making a grand total of 500 in oxygenic photosynthetic organisms. Collectively these proteins have the potential to influence virtually every major process of the cell. A number of laboratories currently seek to confirm newly identified Trx targets by biochemical and genetic approaches. Almost certainly many new targets become redox active during oxidative stress, enabling the plant to cope with changing environments. Under these conditions, certain targets may be glutathionylated or nitrosylated such that reversion to the original reduced state is facilitated not only by Trx, but also, in some cases preferably, by glutaredoxin. When judging changes linked to Trx, it is prudent to recognize that effects transcend classical light/dark or oxidative regulation and fall in other arenas, in some cases yet to be defined. While future work will continue to give insight into functional details, it is clear that Trx plays a fundamental role in regulating diverse processes of the living cell.

Effect of temperature on expression of genes encoding enzymes for starch biosynthesis in developing wheat endosperm

The effect of high temperature on starch accumulation, starch granule populations, and expression of genes encoding key enzymes for starch biosynthesis was examined during grain development in wheat (Triticum aestivum L. cv. Butte 86). High temperature applied from anthesis to maturity reduced the duration of starch accumulation. Starch accumulation ceased approximately 6 days earlier for grain produced under a 37/17 8C (day/night) regimen and 21 days earlier under a 37/28 8C (day/night) regimen than for grain produced under a 24/17 8C (day/night) regimen. Compared to the 24/17 8C regimen, starch content was approximately 19% less for mature grain produced under the 37/17 8C regimen and 58% less under the 37/28 8C regimen. Based on relative volume, the smaller type B starch granules were the predominant class in mature grain produced under the 24/17 and 37/17 8C regimens, whereas the larger type A granules were predominant in grain produced under the 37/28 8C regimen. Under the 24/17 8C regimen, steady state transcript levels for ADP-glucose pyrophosphorylase, starch synthases I, II, and III, granule-bound starch synthase, and starch branching enzymes I and II were highest from 12/16 days post-anthesis (dpa). Under the 37/17 8C regimen, steady state levels of these transcripts followed the same temporal pattern, but were substantially lower. Under the 37/28 8C regimen, transcript levels peaked earlier, at 7 dpa. The high temperature regimens reduced the relativ el evels of transcripts for starch synthase more than the other starch biosynthetic enzymes. Published by Elsevier Science Ireland Ltd.

Thioredoxin-Linked Proteins Are Reduced during Germination of Medicago truncatula Seeds

Germination of cereals is accompanied by extensive change in the redox state of seed proteins. Proteins present in oxidized form in dry seeds are converted to the reduced state following imbibition. Thioredoxin (Trx) appears to play a role in this transition in cereals. It is not known, however, whether Trx-linked redox changes are restricted to cereals or whether they take place more broadly in germinating seeds. To gain information on this point, we have investigated a model legume, Medicago truncatula. Two complementary gel-based proteomic approaches were followed to identify Trx targets in seeds: Proteins were (1) labeled with a thiol-specific probe, monobromobimane (mBBr), following in vitro reduction by an NADP/Trx system, or (2) isolated on a mutant Trx affinity column. Altogether, 111 Trx-linked proteins were identified with few differences between axes and cotyledons. Fifty nine were new, 34 found previously in cereal or peanut seeds, and 18 in other plants or photosynthetic organisms. In parallel, the redox state of proteins assessed in germinating seeds using mBBr revealed that a substantial number of proteins that are oxidized or partly reduced in dry seeds became more reduced upon germination. The patterns were similar for proteins reduced in vivo during germination or in vitro by Trx. In contrast, glutathione and glutaredoxin were less effective as reductants in vitro. Overall, more than half of the potential targets identified with the mBBr labeling procedure were reduced during germination. The results provide evidence that Trx functions in the germination of seeds of dicotyledons as well as monocotyledons.

Unraveling thioredoxin-linked metabolic processes of cereal starchy endosperm using proteomics

Application of a thiol‐specific probe, monobromobimane, with proteomics and enzyme assays led to the identification of 23 thioredoxin targets in the starchy endosperm of mature wheat seeds (Triticum aestivum cv. Butte), almost all containing at least two conserved cysteines. The identified targets, 12 not known to be thioredoxin‐linked, function in a spectrum of processes: metabolism (12 targets), protein storage (three), oxidative stress (three), protein degradation (two), protein assembly/folding (one) and unknown reactions (two). In addition to formulating metabolic pathways functional in the endosperm, the results suggest that thioredoxin acts in redox regulation throughout the life cycle of the seed.

Germin Gene Expression Is Induced in Wheat Leaves by Powdery Mildew Infection.

Germin gene expression is induced in wheat (Triticum aestivum L.) leaves by powdery mildew (Erysiphe graminis f. sp. tritici) infection. Germin is a protein marker for early cereal development and is an oxalate oxidase, an enzyme that catalyzes the conversion of oxalate to CO2 and H2O2. The induction of germin gene expression by powdery mildew infection is consistent with the importance of H2O2 to plant defense and identifies a mechanism for the elevation of H2O2 levels in wheat leaves. Germin mRNA levels increased 2 d after inoculation of seedlings with powdery mildew and continued to increase throughout an 8-d time course. The increase in accumulation of germin mRNA was accompanied by an increase in the germin oligomer, which reached maximal levels by d 6. An increase in oxalate oxidase activity paralleled germin oligomer accumulation. Germin gene expression was induced in a relatively resistant cultivar (Bobwhite) as well as in a susceptible cultivar (Cheyenne), suggesting that the induction of germin gene expression is an indicator of powdery mildew infection rather than cultivar resistance.

Effect of salt stress on plant gene expression: A review

Soil salinity is an important agricultural problem, particularly since the majority of crop plants have low salt tolerance. The identification of genes whose expression enables plants to adapt to or tolerate salt stress is essential for breeding programs, but little is known about the genetic mechanisms for salt tolerance. Recent research demonstrates that salt stress modulates the levels of a number of gene products. Although the detection of gene products that respons specifically to salt stress is a significant finding, they must be identified, functions assigned, and their relation to salt tolerance determined. This article focuses on a few of the salt-responsive proteins and mRNAs that have been discovered and the methods employed to identify and characterize them.

Comparative proteomic analysis of the effect of temperature and fertilizer on gliadin and glutenin accumulation in the developing endosperm and flour from Triticum aestivum L. cv. Butte 86

BackgroundFlour quality is largely determined by the gluten proteins, a complex mixture of proteins consisting of high molecular weight-glutenin subunits (HMW-GS), low molecular weight-glutenin subunits (LMW-GS), and α-, γ-, and ω-gliadins. Detailed proteomic analyses of the effects of fertilizer and high temperature on individual gliadin and glutenin protein levels are needed to determine how these environmental factors influence flour quality.ResultsWheat plants (Triticum aestivum L. cv. Butte 86) were grown in greenhouses under moderate and high temperature regimens with and without post-anthesis fertilizer. Quantitative two-dimensional gel electrophoresis was used to construct accumulation profiles in developing endosperm for the entire complement of gluten proteins identified previously by tandem mass spectrometry. Amounts of individual gliadins and glutenins were also determined in flour produced under each of the regimens. Under all environmental regimens, most HMW-GS, LMW-GS, γ- and ω-gliadins accumulated rapidly during early stages of grain development and leveled off during middle stages of development. A subset of LMW-GS showed a second distinct profile, accumulating throughout development, while α-gliadins showed a variety of accumulation profiles. In flour, fourteen distinct gluten proteins responded similarly to fertilizer, high temperature, and high temperature plus fertilizer. The majority of HMW-GS and ω-gliadins and some α-gliadins increased while two LMW-GS and a minor γ-gliadin decreased. Fertilizer did not influence gluten protein accumulation under high temperature conditions. Additionally, the effects of fertilizer and high temperature were not additive; very few changes were observed when plants that received fertilizer were subjected to high temperature.ConclusionsAlthough post-anthesis temperature and fertilizer have very different effects on grain development and yield, the two treatments elicit surprisingly similar effects on the accumulation of gluten proteins. The similarity of the responses to the different treatments is likely due to source-sink activities of nitrogen reserves in the wheat plant. Because each protein that showed a response in this study is linked to a gene sequence, the work sets the stage for transgenic studies that will better elucidate the roles of specific proteins in flour quality and in the response to the environment.

Surface-Associated Proteins of Wheat Starch Granules : Suitability of Wheat Starch for Celiac Patients

Wheat starch is used to make baked products for celiac patients in several European countries but is avoided in the United States because of uncertainty about the amounts of associated grain storage (gluten) proteins. People with celiac disease (CD) must avoid wheat, rye, and barley proteins and products that contain them. These proteins are capable of initiating damage to the absorptive lining of the small intestine in CD patients, apparently as a consequence of undesirable interactions with the innate and adaptive immune systems. In this study, starch surface-associated proteins were extracted from four commercial wheat starches, fractionated by high-performance liquid chromatography and gel electrophoresis, and identified by tandem mass spectrometry analysis. More than 150 proteins were identified, many of which (for example, histones, purothionins, and glutenins) had not been recognized previously as starch-associated. The commercial starches were analyzed by the R-5 enzyme-linked immunosorbent assay method to estimate the amount of harmful gluten protein present. One of these starches had a low gluten content of 7 ppm and actually fell within the range proposed as a new Codex Alimentarius Standard for naturally gluten-free foods (maximum 20 ppm). This low level of gluten indicates that the starch should be especially suitable for use by celiac patients, although wheat starches with levels up to 100 ppm are deemed safe in the proposed Codex standards.

Starch-branching enzymesSbe1 andSbe2 from wheat (Triticum aestivum cv. Cheyenne): Molecular characterization, development expression, and homoeologue assignment by differential PCR

Starch is the main component of the wheat kernel, and wheat flour is used for hundreds of food and nonfood products. We are exploring ways to improve wheat quality and to develop new uses for wheat based on altered starch characteristics. To understand the molecular basis for variations in the physical and chemical properties of starch, we examined transcripts for starch biosynthetic enzymes. cDNAs encoding 2 isoforms of starch-branching enzyme (Sbe1, Sbe2) were isolated from wheat endosperm. The longestSbe1 andSbe2 cDNAs were 2797 and 2975 bp, respectively, and they shared extensive identity withSbe sequences reported for wheat and other species. With orthologue-specific primer pairs, homoeologue assignments to chromosome 7 were made forSbe1#19 (TRIae: Sbe1A.1) andSbe1#9 (TRIae:Sbe1D.1) using the wheat cv. Chinese Spring nullisomic-tetrasomic-7 lines. This strategy may prove useful for future mapping of expressed sequence tag (EST) data. TheSbe cDNAs and a granule-bound starch synthase cDNA (GbssI) (from an EST sequencing project) were used to examine the steady-state RNA levels during development of the wheat. Steady-state levels ofSbe2 mRNA were detectable 5 d postanthesis (DPA) and reached a maximum at 10 DPA. Steady-state levels ofSbe1 andGbssI began to rise at 10 DPA and peaked at 15 DPA. Levels of all messages declined rapidly at 20–25 DPA. Reported here is the first analysis of transcripts for these enzymes in the same RNA pools and demonstration that expression patterns are unique and developmentally regulated.

The need for agriculture phenotyping: “Moving from genotype to phenotype”

UNLABELLED Increase in the world population has called for the increased demand for agricultural productivity. Traditional methods to augment crop and animal production are facing exacerbating pressures in keeping up with population growth. This challenge has in turn led to the transformational change in the use of biotechnology tools to meet increased productivity for both plant and animal systems. Although many challenges exist, the use of proteomic techniques to understand agricultural problems is steadily increasing. This review discusses the impact of genomics, proteomics, metabolomics and phenotypes on plant, animal and bacterial systems to achieve global food security and safety and we highlight examples of intra and extra mural research work that is currently being done to increase agricultural productivity. BIOLOGICAL SIGNIFICANCE This review focuses on the global demand for increased agricultural productivity arising from population growth and how we can address this challenge using biotechnology. With a population well above seven billion humans, in a very unbalanced nutritional state (20% overweight, 20% risking starvation) drastic measures have to be taken at the political, infrastructure and scientific levels. While we cannot influence politics, it is our duty as scientists to see what can be done to feed humanity. Hence we highlight the transformational change in the use of biotechnology tools over traditional methods to increase agricultural productivity (plant and animal). Specifically, this review deals at length on how a three-pronged attack, namely combined genomics, proteomics and metabolomics, can help to ensure global food security and safety. This article is part of a Special Issue entitled: Translational Plant Proteomics.