Tehryung Kim

Subcellular Targeting of Methylmercury Lyase Enhances Its Specific Activity for Organic Mercury Detoxification in Plants

Methylmercury is an environmental pollutant that biomagnifies in the aquatic food chain with severe consequences for humans and other animals. In an effort to remove this toxin in situ, we have been engineering plants that express the bacterial mercury resistance enzymes organomercurial lyase MerB and mercuric ion reductase MerA. In vivo kinetics experiments suggest that the diffusion of hydrophobic organic mercury to MerB limits the rate of the coupled reaction with MerA (Bizily et al., 2000). To optimize reaction kinetics for organic mercury compounds, themerB gene was engineered to target MerB for accumulation in the endoplasmic reticulum and for secretion to the cell wall. Plants expressing the targeted MerB proteins and cytoplasmic MerA are highly resistant to organic mercury and degrade organic mercury at 10 to 70 times higher specific activity than plants with the cytoplasmically distributed wild-type MerB enzyme. MerB protein in endoplasmic reticulum-targeted plants appears to accumulate in large vesicular structures that can be visualized in immunolabeled plant cells. These results suggest that the toxic effects of organic mercury are focused in microenvironments of the secretory pathway, that these hydrophobic compartments provide more favorable reaction conditions for MerB activity, and that moderate increases in targeted MerB expression will lead to significant gains in detoxification. In summary, to maximize phytoremediation efficiency of hydrophobic pollutants in plants, it may be beneficial to target enzymes to specific subcellular environments.

Toward Detoxifying Mercury-Polluted Aquatic Sediments with Rice Genetically Engineered for Mercury Resistance

Mercury contamination of soil and water is a serious problem at many sites in the United States and throughout the world. Plant species expressing the bacterial mercuric reductase gene, merA, convert ionic mercury, Hg(II), from growth substrates to the less toxic metallic mercury, Hg(0). This activity confers mercury resistance to plants and removes mercury from the plant and substrates through volatilization. Our goal is to develop plants that intercept and remove Hg(II) from polluted aquatic systems before it can undergo bacterially mediated methylation to the neurotoxic methylmercury. Therefore, the merA gene under the control of a monocot promoter was introduced into Oryza sativa L. (rice) by particle gun bombardment. This is the first monocot and first wetland-adapted species to express the gene. The merA-expressing rice germinated and grew on semisolid growth medium spiked with sufficient Hg(II) to kill the nonengineered (wild-type) controls. To confirm that the resistance mechanism was the conversion of Hg(II) to Hg(0), seedlings of merA-expressing O. sativa were grown in Hg(II)-spiked liquid medium or water-saturated soil media and were shown to volatilize significantly more Hg(0) than wild-type counterparts. Further genetic manipulation could yield plants with increased efficiency to extract soil Hg(II) and volatilize it as Hg(0) or with the novel ability to directly convert methylmercury to Hg(0).

STUDIES ON THE EFFECT OF ZINC SUPPLY ON GROWTH AND NUTRIENT UPTAKE IN PECAN

ABSTRACT Zinc (Zn) deficiency is a nutrient disorder observed in pecan (Carya illinoinensis Wangenh. K. Koch) under field conditions, and can cause distorted leaf growth and severe rosetting of shoots. Conducting Zn studies with pecan in the field have been problematic because Zn nutrition is difficult to control. In this study, Zn nutrient disorders were induced in greenhouse-grown pecan seedlings using hydroponic culture. Zinc efficiency was compared in two pecan seedstocks, ‘Stuart’ and ‘Curtis’ by evaluating growth response, nutrient uptake, and leaf nutrient analysis. Zinc deficiency symptoms appeared in plants grown in the absence of Zn after six weeks. Deficiency symptoms were characterized by interveinal mottling, followed by interveinal chlorosis, interveinal necrosis, and marginal curling. Symptoms were confined to the youngest most distal three to five leaves. Differences in Zn efficiency between the two seedstock were observed. “Stuart” exhibited more severe deficiency ratings than “Curtis”. Zinc supply also had a differential effect on the foliar concentration and content of Zn and other nutrient. “Stuart” seedstocks grown under minus Zn vs. plus Zn conditions exhibited significantly higher foliar concentration of phosphorus (P), calcium (Ca), magnesium (Mg), and copper (Cu), while “Curtis” leaves contained significantly higher manganese (Mn) and lower sulfur (S). Results of this study concur with the observed frequency of Zn deficiencies of the cultivars in the field, i.e., “Stuart” shows Zn deficiency more frequently then “Curtis”. This study verifies that in pecan, there are genotypic differences in Zn efficiency, and that hydroponic culture can be utilized for screening and selection.

STUDIES ON EFFECTS OF NITROGEN FORM ON GROWTH, DEVELOPMENT, AND NUTRIENT UPTAKE IN PECAN

Leaf nutrient analysis coupled with fertilizer applications are routinely used in commercial pecan production. Nitrogen can be applied in a range of formulations. However, definitive studies to establish nitrogen (N) uptake preference in pecan are lacking, as are definitive studies on the effects of N form on plant biomass production. In this study, we evaluated the effects of N form on plant growth and nutrient uptake in pecan (Carya illinoinensis Wangenh. K. Koch) using hydroponic cultures. Plants were grown under three N ratios of ammonium:nitrate (25 : 75, 50 : 50, and 75 : 25). Results from this study indicate that in-_creasing ammonium nutrition inhibited seedling growth. Plants grown with the 75 : 25 ratio (ammonium:nitrate) exhibited sig-_nificantly lower biomass, decreased root/shoot ratio, and lower specific leaf weight than observed in the other treatments. The highest root growth was observed in the 50 : 50 ratio treatment (ammonium:nitrate). Levels of calcium (Ca), magnesium (Mg), and manganese (Mn) in leaves were higher in plants grown under 25 : 75 (ammonium:nitrate) than in 75 : 25. Total N uptake on a dry weight basis was highest in the 75 : 25 ammonium:nitrate treatment. Plants exhibited preferential uptake of ammonium nitrogen under all nitrogen regimes. Ammonium nitrogen is generally applied in pecan orchard practices. This data suggest that further studies evaluating the effects of nitrogen form are warranted to determine if similar detrimental effects on pecan growth occur in the field. Such studies would be useful for optimizing current fertilization practices.

A Quantitative and Histological Comparison of GUS Expression With Different Promoter Constructs Used In Microprojectile Bombardment of Peanut Leaf Tissue

Summaryβ-glucuronidase (GUS) expression driven with different promoter constructs was quantitatively and histologically compared in peanut leaf tissue following microprojectile bombardment. X-Gluc staining patterns varied with the construct used. Tissues bombarded with the pAC2GUS construct had larger foci and a greater percentage of area staining blue. pEmuGN exhibited the greatest numbers of blue spots compared to pAC2GUS and pTRA140. Histological evaluations of blue staining foci showed a diffusion gradient of blue precipitate from a central, prominently-staining cell outward to as many as seven cell layers. The intensity of X-gluc product in centrally-staining cells varied. Gold microprojectile particles were usually located within the three surface cell layers. Depending on the plasmid construct, 72–90% of the centrally-staining cells had at least one gold particle. However, the presence of GUS expression did not appear to require a microprojectile within the nucleus, which was observed in 37% or fewer of the centrally-staining cells. With the pAC2GUS construct, staining patterns varied with location within leaflets and had an “edge effect,” i.e., blue spots were frequently larger at the margin versus central regions. This enhanced activity could be anticipated with an actin promoter in the more mitotically active marginal leaf cells. Total GUS activity as determined by fluorometric analyses was correlated with the percentage of X-gluc stained area. The pAC2GUS construct exhibited the highest total GUS activity among the three constructs.

Seasonal fluctuations in nutrients and carbohydrates in pecan leaves and stems

Summary An integrated study of changes in carbohydrates and nutrients in pecan was conducted and related to developmental changes during a growing season. Perennial woody plants exhibit marked seasonal changes in nutrient content, carbohydrate metabolism, and organ development. Information on nutrient changes may provide insight into seasonal demands and requirements and be useful in developing fertilisation regimes for this crop. Leaf and stem tissues collected at full bloom, leaf maturation, full cotyledon expansion, fruit maturation, and leaf abscission were analysed for starch, specific soluble sugars, and 11 other macro- and micro- nutrients. During leaf expansion, foliar levels of fructose and glucose declined from 12 mg g–1 DW to 5 mg g–1 DW and from 7 mg g–1 DW to 3 mg g–1 DW, respectively. After fruit maturation, stems exhibited an increase in sucrose, from 15 mg g–1 DW to 37 mg g–1 DW, and a marked decrease in starch, from 59 mg g–1 DW to 8 mg g–1 DW. Seasonal variations in elemental content indicated developmental stage-related nutrient movement. During leaf development, all nutrients accumulated in leaves, with more than a 70% increase in N, K, Ca, Mg, S, B and Fe content. During fruit development, the foliar content of many nutrients decreased, suggesting fruit are a strong sink for essential elements, as well as carbohydrates. However, nutrient changes differed with the stage of fruit development. For example, foliar concentrations of Mg and Zn decreased by 23% and 24%, respectively during cotyledon formation; while P, K, S, B, Cu and Mn levels decreased during reserve accumulation by 4, 15, 7, 12, 8 and 10%, respectively. During the period leading-up to leaf abscission, leaf nutrient resorption was observed with decreased contents of N (6%), P (14%), K (7%), Ca (7%), Mg (10%) and S (3%). Pecan leaf expansion and fruit development are critical periods, when nutrient movement can be appreciable. Maintaining adequate fertility levels during developmental periods of high nutrient demand may be important to optimise tree function. Management practices that promote late-season leaf retention would prevent nutrient losses associated with premature defoliation.

Multigene Strategies for Engineering the Phytoremediation of Mercury and Arsenic

Hundreds of millions of people worldwide suffer the consequences their environment, which make them suitable to process related toxic field-adapted plant species that can extract mercury and arsenic from soil or water, process them in high concentrations, and prevent them Z. Xu et al. (eds.), Biotechnology and Sustainable Agriculture 2006 and Beyond, 49–60. of being exposed to toxic levels of mercury and arsenic. Our labophytoremediation of elemental pollutants relies on plants to extract ratory is focused on developing simple genetic engineering stratethem aboveground for later harvest. Plants possess many natural systems to uptake and manage 15 essential elemental nutrients from gies for the phytoremediation of these two toxic elements. The engineering. The long-term goal of our work is to develop vigorous, toxicants from soil and water, manage their toxicity, and concentrate elemental pollutants. These properties may be enhanced via genetic properties, such as using photosynthetic energy and pervasive root 50 R.B. Meagher et al. from entering the food-chain. Our initial research efforts have focused on enhancing several of these properties including: 1) increasing plant tolerance to mercury and arsenic; 2) transforming these toxic elements into the chemical species that promote their shortand longdistance transport from roots to shoots; 3) transforming these toxic elements to the best chemical species for storage in leaves, stems, and vascular tissues above ground; 4) enhancing plant chemical ‘sinks’ that can trap these toxicants aboveground; and 5) enhancing transporters for uptake and storage. It is likely that the next decade can cleanup mercury and arsenic and restore contaminated land and water resources. Introduction Many dangerous, elemental pollutants are present at unacceptably high levels in the environment because of industrial, defense, agricultural, and municipal processes, and they are adversely affecting the health of millions of people worldwide (Nriagu, 1994; Wolz et al., 2003). Elemental pollutants include heavy metals, metalloids, and radionuclides such as mercury, lead, cadmium, arsenic, technetium, tritium, and deuterium. Unlike organic pollutants that can be degraded to harmless small molecules, elemental pollutants are immutable by all biochemical reactions, making them particularly difficult to remediate (Meagher, 2000; Kramer and Chardonnens, 2001). The phytoremediation of some elemental pollution may require genetically engineering several plant properties to enhance plant extraction, tolerance, and hyperaccumulation of these toxicants. Research on engineered phytoremediation is still in its infancy, and a better understanding of natural hyperaccumulators will be a tremendous aid in our understanding of which genes and which cellular and organismal processes can be most effectively manipulated (Baker, 2000; Wang, 2002; Kerkeb and Kramer, 2003). The genes of soil bacteria, also have much to contribute to phytoremediation schemes, because they have evolved many properties that direct the aggressive transformation or management of elemental pollutants (Meagher et al., 2000). Another focal point for research on phytoremediating will see unprecedented progress in developing specialized plants that