Paul C. Bethke

Nitric Oxide Acts as an Antioxidant and Delays Programmed Cell Death in Barley Aleurone Layers

Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of α-amylase. α-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective antioxidant in aleurone cells. The amounts of CAT and SOD, two enzymes that metabolize ROS, are greatly reduced in aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and SOD. We speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.

Suppression of the Vacuolar Invertase Gene Prevents Cold-Induced Sweetening in Potato

Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to prevent sprouting, minimize disease losses, and supply consumers and the processing industry with high-quality tubers throughout the year. Unfortunately, cold storage triggers an accumulation of reducing sugars in tubers. High-temperature processing of these tubers results in dark-colored, bitter-tasting products. Such products also have elevated amounts of acrylamide, a neurotoxin and potential carcinogen. We demonstrate that silencing the potato vacuolar acid invertase gene VInv prevents reducing sugar accumulation in cold-stored tubers. Potato chips processed from VInv silencing lines showed a 15-fold acrylamide reduction and were light in color even when tubers were stored at 4°C. Comparable, low levels of VInv gene expression were observed in cold-stored tubers from wild potato germplasm stocks that are resistant to cold-induced sweetening. Thus, both processing quality and acrylamide problems in potato can be controlled effectively by suppression of the VInv gene through biotechnology or targeted breeding.

Retrospective View of North American Potato (Solanum tuberosum L.) Breeding in the 20th and 21st Centuries

Cultivated potato (Solanum tuberosum L.), a vegetatively propagated autotetraploid, has been bred for distinct market classes, including fresh market, pigmented, and processing varieties. Breeding efforts have relied on phenotypic selection of populations developed from intra- and intermarket class crosses and introgressions of wild and cultivated Solanum relatives. To retrospectively explore the effects of potato breeding at the genome level, we used 8303 single-nucleotide polymorphism markers to genotype a 250-line diversity panel composed of wild species, genetic stocks, and cultivated potato lines with release dates ranging from 1857 to 2011. Population structure analysis revealed four subpopulations within the panel, with cultivated potato lines grouping together and separate from wild species and genetic stocks. With pairwise kinship estimates clear separation between potato market classes was observed. Modern breeding efforts have scarcely changed the percentage of heterozygous loci or the frequency of homozygous, single-dose, and duplex loci on a genome level, despite concerted efforts by breeders. In contrast, clear selection in less than 50 years of breeding was observed for alleles in biosynthetic pathways important for market class-specific traits such as pigmentation and carbohydrate composition. Although improvement and diversification for distinct market classes was observed through whole-genome analysis of historic and current potato lines, an increased rate of gain from selection will be required to meet growing global food demands and challenges due to climate change. Understanding the genetic basis of diversification and trait improvement will allow for more rapid genome-guided improvement of potato in future breeding efforts.

A gibberellin-regulated calcineurin B in rice localizes to the tonoplast and is implicated in vacuole function.

Many developmental and environmental signals are transduced through changes in intracellular calcium concentrations, yet only a few calcium-binding proteins have been identified in plants. Calcineurin B-like (CBL) proteins are calcium-binding proteins that are thought to function as plant signal transduction elements. RNA profiling using a rice (Oryza sativa cv Nipponbare) oligonucleotide microarray was used to monitor gene expression in de-embryonated rice grains. This analysis showed that a putative rice CBL gene responded to gibberellic acid, but not abscisic acid, treatment. The CBL gene family in rice contains at least 10 genes and these have extensive similarity to the CBLs of Arabidopsis (Arabidopsis thaliana). In yeast (Saccharomyces cerevisiae) two-hybrid assays, rice CBLs interact with the kinase partners of Arabidopsis CBLs. Only one rice CBL gene, OsCBL2, is up-regulated by GA in the aleurone layer. A homolog with 91% sequence identity to OsCBL2 was cloned from barley (Hordeum vulgare cv Himalaya), and designated HvCBL2. We examined the localization and function of OsCBL2 and HvCBL2 in rice and barley aleurone because changes in cytosolic calcium have been implicated in the response of the aleurone cell to GA. Green fluorescent protein translational fusions of OsCBL2 and OsCBL3 were localized to the tonoplast of aleurone cell protein storage vacuoles and OsCBL4-green fluorescent protein was localized to the plasma membrane. Data from experiments using antisense expression of OsCBL2 and HvCBL2 are consistent with a role for OsCBL2 in promoting vacuolation of barley aleurone cells following treatment with GA.

Isolation of Intact Protein Storage Vacuoles from Barley Aleurone (Identification of Aspartic and Cysteine Proteases)

Within the cereal aleurone reserve proteins are stored in specialized organelles, the protein storage vacuoles (PSV). We developed an aqueous method for the isolation of intact PSV. Barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts were gently lysed by passing them through a syringe needle. PSV were separated from cytoplasmic components by microfiltration and low-speed centrifugation. Isolated PSV appeared by light microscopy to be identical with those within barley aleurone protoplasts. Luminal contents were retained throughout the isolation procedure. We used isolated PSV to identify and characterize PSV-associated proteolytic activities. Isolated PSV contained cysteine proteases and aspartic proteases (APs). Gibberellic acid treatment of protoplasts increased cysteine protease activity. Protein blots probed with anti-H. vulgare aspartic proteinase (HvAP) indicated that one PSV-AP was HvAP. Immunocytochemical localization by electron microscopy confirmed the presence of HvAP within the lumen of PSV. We conclude that isolated barley aleurone PSV will be useful in further characterizing this organelle.

The Effects of Boiling and Leaching on the Content of Potassium and Other Minerals in Potatoes

The white potato (Solanum tuberosum L.) is a valuable source of potassium in the human diet. While most consumers benefit from high levels of potassium in potato tubers, individuals with compromised kidney function must minimize their potassium intake. This study was undertaken to determine the effects of leaching and boiling on levels of potassium and other minerals in potato tubers. Leaching alone did not significantly reduce levels of potassium or other minerals in tubers. Boiling tuber cubes and shredded tubers decreased potassium levels by 50% and 75%, respectively. Reductions in mineral amounts following boiling were observed for phosphorus, magnesium, sulfur, zinc, manganese, and iron. There was no difference between the leaching and boiling treatment and the boiling treatment. In addition, mineral levels in tubers of 6 North American potato cultivars are reported. Significant differences in mineral levels were detected among cultivars, but they were too small to be nutritionally important. Individuals wishing to maximize the mineral nutrition benefits of consuming potatoes should boil them whole or bake, roast, or microwave them. Those who must reduce potassium uptake should boil small pieces before consuming them.

Extensive Variation in Fried Chip Color and Tuber Composition in Cold-Stored Tubers of Wild Potato (Solanum) Germplasm

Cold-induced sweetening and browning in the Maillard reaction have driven extensive research in the areas of plant physiology, biochemistry, and food science in Solanum tuberosum because of its importance to the potato-processing industry. Prior research has not characterized wild Solanum relatives of potato for tuber composition and has not determined if relationships between tuber composition and chip color after cold storage in wild species are comparable to those found for cultivated potato. Extensive inter- and intraspecific variation for chip color and tuber composition were found in the wild Solanum species examined. Tuber sugar profiles suggested that invertase activity at low temperatures differed between and within species. Tuber fructose, glucose, and sucrose concentrations partially explained chip color variation in most accessions, but asparagine concentration and percent dry matter did not. Most wild species had reducing sugar concentrations and chip color scores after 2 degrees C storage that were less than those in S. tuberosum cultivar Snowden. Sugar profiles and relationships between specific sugars and chip color in Solanum pinnatisectum were unique among the species examined.

Tuber Water and Pressure Potentials Decrease and Sucrose Contents Increase in Response to Moderate Drought and Heat Stress

Environmental stress during the growing season can reduce the quality of stored Solanum tuberosum (potato) tubers. Changes in greenhouse grown Russet Burbank tuber water potential and tuber composition following periods of moderate drought, heat, or drought and heat stress were characterized. Decreased leaf and tuber water potentials were observed at the end of stress treatments. Decreased tuber water potential caused dramatic declines in tuber pressure potential. Tuber sucrose content increased with the severity of stress. Changes in tuber glucose and malic acid contents were also observed. Stress during early tuber bulking had a larger effect on tuber sugars at the end of the stress period than stress during late bulking. Stem-end glucose concentration of tubers at harvest after vine senescence was greater in tubers from plants stressed at early tuber bulking compared to late bulking.ResumenEl agobio ambiental durante el ciclo de cultivo puede reducir la calidad de tubérculos de Solanum tuberosum (papa) almacenados. Se caracterizaron cambios en el potencial hídrico del tubérculo y su composición en Russet Burbank cultivada en invernadero, después de períodos de sequía moderada, calor, o sequía y agobio por calor. Se observó disminución en el potencial hídrico de la hoja y del tubérculo al final de los tratamientos de agobio. La disminución del potencial hídrico del tubérculo causó dramática disminución en el potencial de presión del tubérculo. Aumentó el contenido de sacarosa en el tubérculo con la severidad del agobio. También se observaron cambios en los contenidos de glucosa y ácido málico del tubérculo. El agobio al inicio de la tuberización tuvo un efecto mayor en azúcares del tubérculo al final del período de agobio que el agobio al final de la tuberización. La concentración de la glucosa en la base de los tubérculos a la cosecha, después de la senectud del follaje, fue mayor en tubérculos de plantas agobiadas al inicio de la tuberización comparada con agobio al final.

Acrylamide in Processed Potato Products

Trace amounts of acrylamide are present in many foods cooked at high temperatures. Acrylamide in processed potato products is formed from reducing sugars and asparagine and is a product of the Maillard reaction; this reaction typically occurs during frying and baking of food products. Processed potato products, including fries and chips, are relatively high in acrylamide compared with other foods and contribute substantially to dietary acrylamide. Acrylamide content in potato products is strongly affected by processing conditions, potato variety, field management, environmental conditions during tuber growth, and tuber storage conditions. Numerous approaches have been described that could potentially reduce the acrylamide content of potato products, but many influence finished product sensory attributes and may be difficult to implement. Health concerns related to acrylamide in food center on its role as a potential carcinogen. Research using feeding studies with rodent models and epidemiological studies with humans are ongoing and are likely to provide future guidance for acceptable amounts of acrylamide in food.ResumenSe presentan ligeras cantidades de acrilamida en muchos alimentos cocinados a altas temperaturas. La acrilamida en los productos procesados de papa se forma a partir de los azúcares reductores y asparagina y es un producto de la reacción de Maillard; esta reacción se presenta típicamente durante el freído y cocinado de productos alimenticios. Los productos procesados de la papa, incluyendo hojuelas y a la francesa, son relativamente altos en acrilamida comparados con otros alimentos y contribuyen substancialmente a la acrilamida de la dieta. Su contenido en los productos de papa esta fuertemente afectado por las condiciones del procesamiento, la variedad de papa, el manejo en el campo, condiciones ambientales durante el crecimiento del tubérculo y las condiciones de almacenamiento. Se han descrito numerosas estrategias que podrían potencialmente reducir el contenido de acrilamida de los productos de papa, pero muchas influencian los atributos sensoriales del producto final y pudieran ser difíciles de implementar. Las preocupaciones sobre la salud relacionadas con acrilamida en los alimentos se centran en su papel como un potencial carcinogénico. Esta en proceso la investigación usando estudios de alimentación con modelos de roedores y con estudios epidemiológicos con humanos y es probable que suministren guía a futuro para cantidades aceptables de acrilamida en alimentos.

Vacuolar invertase gene silencing in potato (Solanum tuberosum L.) improves processing quality by decreasing the frequency of sugar-end defects.

Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (VInv) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.