Karen L. Klotz

Phytohormone control of the tobacco anionic peroxidase promoter

The tobacco anionic peroxidase gene encodes the predominant peroxidase isoenzyme in the aerial portions of tobacco. Three kb of the peroxidase promoter was joined to the coding region of theEscherichia coli β-glucuronidase gene (GUS), and transiently expressed in tobacco mesophyll protoplasts in the presence or absence of plant growth regulators. Benzyladenine, ethylene, and gibberellic acid did not affect peroxidase gene expression. Abscisic acid slightly inhibited expression at high concentrations. The auxins indole-3-acetic acid (IAA) and naphthaleneacetic acid strongly suppressed peroxidase expression. We observed half maximal suppression at 30 μM IAA. An antiauxin,p-chlorophenoxyisobutyric acid (PCIB), enhanced expression from the peroxidase promoter above that of untreated controls or restored activity when used in combination with IAA. Sequencing 3 kb of the peroxidase promoter revealed many potential regulatory elements based on sequence homology to previously characterized genes. This includes several consensus transcription factor binding sites found in auxin-regulated promoters. 5′ deletions of the peroxidase promoter/GUS fusion revealed several positive and negative regulatory elements. An upstream enhancer element was found between −3146 and −638 from the start of transcription. A strong silencer element was observed between −638 and −220. Removal of this silencer resulted in a truncated promoter (−220) with 100% activity of the full-length promoter (−3146). Inhibition by auxin was observed with all 5′ deletions.

Expression of the tobacco anionic peroxidase gene is tissue-specific and developmentally regulated

Transcriptionally regulated expression of tobacco anionic peroxidase was investigated with regard to tissue specificity and developmental regulation. Two tobacco species, Nicotiana sylvestris and Nicotiana tabacum cv. Xanthi, were stably transformed with a gene chimera composed of 3 kb of the tobacco anionic peroxidase promoter, the Escherichia coli β-glucuronidase (GUS) coding region and the nopaline synthase terminator. Gene expression was regulated spatially and developmentally in all organs, and generally increased with age and maturity of the plant, tissue or organ. In the aerial portions of the plant, GUS activity was strongly expressed in trichomes and epidermis at nearly all developmental stages. In later stages of development, activity was also detected in ground tissue and parenchyma cells associated with vascular tissues. Activity in roots was limited to cortical cells and vascular-associated parenchyma cells. In reproductive tissue, expression was observed in sepals and petals before anthesis, and in all floral organs after anthesis. Expression was never detected in vascular tissue and was poorly correlated with lignification except in the cells surrounding primary xylem and pericyclic fibers in N. sylvestris. These studies suggest that this peroxidase isoenzyme is only limitedly involved in lignification but may be important in plant defense, growth and development.

Postharvest Storage Losses Associated with Rhizomania in Sugar Beet

During storage of sugar beet, respiration and rots consume sucrose and produce invert sugar. Diseases that occur in the field can affect the magnitude of these losses. This research examines the storage of roots with rhizomania (caused by Beet necrotic yellow vein virus) and the effectiveness of rhizomania-resistant hybrids in reducing postharvest losses. Roots of susceptible hybrids from sites with rhizomania had respiration rates 30 days after harvest (DAH) that ranged from 0.68 to 2.79 mg of CO2 kg-1 h-1 higher than roots of the resistant hybrids. This difference ranged from 2.60 to 13.88 mg of CO2 kg-1 h-1 120 DAH. Roots of resistant hybrids from sites with rhizomania had 18 kg more sucrose per ton than roots from susceptible hybrids 30 DAH, with this difference increasing to 55 kg Mg-1 120 DAH. The invert sugar concentration of susceptible hybrids from sites with rhizomania ranged from 8.38 to 287 g per 100 g of sucrose higher than that for resistant hybrids 120 DAH. In contrast, differences between susceptible and resistant hybrids in respiration rate, sucrose loss, and invert sugar concentration in the absence of rhizomania were relatively small. Storage losses due to rhizomania can be minimized by planting resistant hybrids and processing roots from fields with rhizomania soon after harvest.

Respiration in postharvest sugarbeet roots is not limited by respiratory capacity or adenylates

Control of respiration has largely been studied with growing and/or photosynthetic tissues or organs, but has rarely been examined in harvested and stored plant products. As nongrowing, heterotrophic organs that are reliant on respiration to provide all of their metabolic needs, harvested plant products differ dramatically in their metabolism and respiratory needs from growing and photosynthetically active plant organs, and it cannot be assumed that the same mechanism controls respiration in both actively growing and harvested plant organs. To elucidate mechanisms of respiratory control for a harvested and stored plant product, sugarbeet (Beta vulgaris L.) root respiration was characterized with respect to respiratory capacity, adenylate levels and cellular energy status in roots whose respiration was altered by wounding or cold treatment (1 degrees C) and in response to potential effectors of respiration. Respiration rate was induced by wounding in roots stored at 10 degrees C and by cold temperature in roots stored at 1 degrees C for 11-13d. Alterations in respiration rate due to wounding or storage temperature were unrelated to changes in total respiratory capacity, the capacities of the cytochrome c oxidase (COX) or alternative oxidase (AOX) pathways, adenylate concentrations or cellular energy status, measured by the ATP:ADP ratio. In root tissue, respiration was induced by exogenous NADH indicating that respiratory capacity was capable of oxidizing additional electrons fed into the electron transport chain via an external NADH dehydrogenase. Respiration was not induced by addition of ADP or a respiratory uncoupler. These results suggest that respiration rate in stored sugarbeet roots is not limited by respiratory capacity, ADP availability or cellular energy status. Since respiration in plants can be regulated by substrate availability, respiratory capacity or energy status, it is likely that a substrate, other than ADP, limits respiration in stored sugarbeet roots.

Effects of Aphanomyces Root Rot on Carbohydrate Impurities and Sucrose Extractability in Postharvest Sugar Beet

Sugar beet (Beta vulgaris) roots with rot caused by Aphanomyces cochlioides often are incorporated into storage piles even though effects of disease on processing properties are unknown. Roots with Aphanomyces root rot were harvested from six fields over 2 years. For each field, roots with similar disease symptoms were combined and assigned a root rot index (RRI) value (0 to 100; 0, no rot symptoms; 100, all roots severely rotted). After 20 or 120 days storage at 4°C and 95% relative humidity, concentrations of the major carbohydrate impurities that accumulate during storage and sucrose extractability were determined. Root rot affected carbohydrate impurity concentrations and sucrose extractability in direct relation to disease severity symptoms. Generally, roots with active and severe infection (RRI ≥ 85) exhibited elevated glucose and fructose concentrations 20 and 120 days after harvest (DAH), elevated raffinose concentration 120 DAH, and reduced sucrose extractability 20 and 120 DAH. Roots with minor or moderate disease symptoms (RRI 20 to 69), or damaged roots with no signs of active infection, had similar carbohydrate impurity concentrations and sucrose extractability after 20 and 120 days storage. Processing properties declined when RRIs exceeded 43, as determined by regression analysis, or when storage duration increased from 20 to 120 days. Results indicate that both disease severity and anticipated duration of storage be considered before Aphanomyces-infected roots are incorporated into storage piles.