Cotton project

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Testing transgenic cotton plants
overexpressing antioxidant enzymes

Supported by USDA grant
#99-35100-7630
and grant from the Texas Advanced
Technology-Development program

ABSTRACT

On the Texas High Plains, where 20% of the nation's cotton is produced, morning temperatures are well below optimum for photosynthesis. Exposure to full sun light at these chilling temperatures results in the absorbtion of a great deal light energy in excess of that needed for photosynthetic CO₂ assimilation. The transgenic cotton plants with elevated activities of superoxide dismutase, ascorbate peroxidase, and gluthatione reductase were developed to improve the protection of photosynthetic apparatus to stress conditions. Using these genotypes our goals are 1) to elucidate the mechanism that affords protection to transgenic plants, 2) to determine the limitations to this approach to protecting the photosynthesis in cotton, 3) to determine the performance of these transgenic plants in the field under irrigated and dryland conditions. Our infestigations will identify the most sensitive sites of the photosynthetic apparatus of cotton to chilling/high light stress, and assess the interaction between other protective processes. Various aspects of photosynthetic function, as well as growth, cotton fiber yield and quality will be assessed from field-grown populations of transgenic and wild-type plants. These studies of the performance of transgenic cotton genotypes grown in the field in West Texas will provide the basis for a realistic assessment of the efficacy of this approach to improving crop performance.

Chilling temperatures limit the activity of Calvin cycle enzymes, thus reducing the utilization of absorbed light energy for CO₂ assimilation (Leegood 1995, Wise 1995). This diminished demand for absorbed light energy can render plants more susceptible to photoinhibition (Melis 1999). In fact, even relatively moderate photosynthetic photon flux densities (PPFDs) can induce photoinhibition at chilling temperatures.
The rate of oxygen photoreduction increases during chilling, increasing the production of the reactive oxygen species (ROS), superoxide (O₂^-), H₂O₂, and, potentially, the hydroxyl radical (Wise and Naylor 1987, Hodson and Raison 1992, Wise 1995, Prasad 1996). There is evidence that ROS may be produced at both PS II and PS I (Foyer and Harbinson 1994, Osmond and Grace 1995, Melis 1999). Because these highly reactive substances can damage proteins involved in photosynthetic electron transport and thylakoid membrane lipids (Halliwell and Gutteridge 1999), as well as deactivate certain Calvin cycle enzymes (Charles and Halliwell 1981), they are implicated in the mechanism of chilling-induced photoinhibition.
Although chloroplasts possess a system of antioxidant enzymes that can detoxify ROS before they damage cellular constituents, during chilling in the light, the rate of ROS formation may exceed the enzyme capacity to scavenge them (for reviews see Asada 1994, 1999, Foyer and Harbinson 1994, Foyer et al. 1994b, Logan et al. 1999). The enzymic system includes superoxide dismutase (SOD) and ascorbate peroxidase (APX), whose combined activities catalyze the conversion of O₂^- to H2O, while oxidizing ascorbate (Asada 1999). Two mechanisms to regenerate ascorbate involve reduced glutathione, either as a substrate for dehydroascorbate reductase (reviewed in Noctor and Foyer 1998) or perhaps via non-enzymatic reduction of oxidized ascorbate under the alkaline conditions present in the stroma during illumination (Winkler et al. 1994). Chloroplastic GR maintains the pool of reduced glutathione, using NADPH as a reductive substrate. The importance of antioxidant enzymes in the protection of the photosynthetic apparatus during photoinhibitory conditions, such as those that develop with chilling in the light, is indicated by the following observations: a) the activities of these enzymes generally increase during acclimation to chilling temperatures (Sch?er and Krause 1990, Prasad 1996, Hull et. al. 1997, Fryer et al. 1998, Logan et al. 1998), presumably to cope with increased ROS production; b) a correlation exists between resistance to chilling-induced photoinhibition and high antioxidant enzyme activities in comparative studies of chilling-sensitive versus chilling-resistant cultivars or species (Wise and Naylor 1987, Janke et al. 1991, Kocsy et al. 1996, Hodges et al. 1997); c) supplementing isolated thylakoid preparations with antioxidant enzymes reduces their susceptibility to photoinhibition (reviewed in Tyystj?vi et al. 1999).
These observations underlie attempts to increase plant resistance to chilling-induced photoinhibition via genetic manipulation of leaf antioxidant systems (for reviews see Foyer et al. 1994a, Allen 1995).

Identification of transgenic plants

To confirm that the SOD+ plants that we used had high levels of active MnSOD, the seedlings were screened by subjecting extracts prepared from fully expanded cotyledons to non-denaturating polyacrylamide gel electrophoresis according to Beauchamp and Fridovich (1971) as modified by van Camp et al. (1994). Representative gels are shown in Payton et al. (1997)

(See figure, left column - gel electrophoresis of extract from wild-type plants, right column - from transgenic plants.

Determination of SOD, APX, and GR activities from whole-leaf extracts. Leaf discs were rapidly removed using a cork borer and immediately frozen in liquid nitrogen. Frozen leaf discs were ground to powder at liquid N₂ temperature using a mortar and pestle and then rapidly homogenized in 1 ml of the appropriate ice-cold extraction solution in a glass tissue grinder. Aliquots were taken before centrifugation for chlorophyll determination in 80% acetone according to Lichtenthaler (1987). The assays were initiated within 1.5 min after commencing the extraction with 25 ml of centifuged extract. The assay temperature was 25 ^oC. The SOD activity was measured by monitoring the inhibition of nitro blue tetrazolium (NBT) reduction at 560 nm. The tissue was homogenised in 50 mM KH₂PO₄ (pH 7.0). The reaction mixture contained 50 mM KH2PO4 (pH 7.0), 0.1 mM EDTA, 75 mM NBT, 2 mM riboflavin, and 13 mM methionine. The reduction of NBT preceded under 500 mmol photons m^-2s^-1 of white light for 8 min. One unit of SOD activity was defined as the amount that inhibits the reaction by 50% (Giannopolitis and Ries 1977).The extraction and assay solutions for APX and GR assays were as described by Sen Gupta et al. (1993). The activity of APX was determined by monitoring the H₂O₂-dependent oxidation of ascorbate at 290 nm. The activity of GR was measured spectrophotometrically by monitoring the oxidation of NADPH at 340 nm.

Field measurements of chlorophyll fluorescence
using potable fluorometer Hansatech FMS2

Field data were collected using portable fluorometer FMS2 (Hansatech Instruments Ltd., UK). During the measurements the leaves were kept at natural angle. The magnitudes of photon flux density (PFD) and temperature were monitored by means of sensors located on the measuring clip of the fluorometer. The experimental protocol described by Schreiber et al. (1986) and nomenclature of van Kooten and Snel (1990) were employed for fluorescence analysis. For some of the time-points the small areas of the leaf surface were darkened with special opaque clips after measurements of F, F_m', and F_o'. For those leaf parts the values of F_v/F_m ratio were determined without detachment after 1.5 h of dark incubation and were denoted as (F_v/F_m)_PI

Notes for lesson about studying PHOTOINHIBITION using chlorophyll fluorescence analysis

Published results

Kornyeyev D., Logan B.A., Allen R.D., Holaday A.S. (2005) Field-grown cotton plants with elevated activity of chloroplastic glutathione reductase exhibit no significant alteration of duirnal or seasonal patterns of excitation energy partitioning and CO2 fixation. Field Crops Research 94 (2-3) 165-175. (PDF file)

Kornyeyev D., Holaday A.S., Logan B.A.(2004) Minimization of the photon energy absorbed by 'closed' reaction centers of photosystem 2 as a photoprotective strategy in higher plants. Photosynthetica 42 (3) 377-386. (PDF file)

Kornyeyev D., Holaday A.S., Logan B.A. (2003) Predicting the extent of photosystem II photoinactivation using chlorophyll a fluorescence parameters measured during illumination. Plant & Cell Physiology 44 (10): 1064-1070. (PDF file)

Kornyeyev D., Logan B.A., Allen R.D., Holaday A.S. (2003) Effect of chloroplastic overproduction of ascorbate peroxidase on photosynthesis and photoprotection in cotton leaves subjected to low temperature photoinhibition. Plant Science 165 (5): 1033-1041. (PDF file)

Kornyeyev D., Logan B.A., Payton P.R., Allen R.D., Holaday A.S. (2003) Elevated chloroplastic glutathione reductase activities decrease chilling-induced photoinhibition by increasing rates of photochemistry, but not thermal energy dissipation, in transgenic cotton. Functional Plant Biology 30 (1): 101-110. (PDF file)

Logan B.A., Monterio G., Kornyeyev D., Payton P., Allen R.D., Holaday A.S. (2003) Transgenic overproduction of glutathione reductase does not protect cotton, Gossypium hirsutum (Malvaceae) , from photoinhibition during growth under chilling conditions. American Journal of Botany 90 (9): 1400-1403. (PDF file)

Kornyeyev D., Logan B.A., Holaday A.S. (2002) A chlorophyll fluorescence analysis of the allocation of radiant energy absorbed in photosystem 2 antennae of cotton leaves during exposure to chilling. Photosynthetica 40 (1): 77-84. (PDF file, 1.466 Mb)

Payton P., Webb R., Kornyeyev D., Allen R., Holaday A.S. (2001) Protecting cotton photosynthesis during moderate chilling at high light intensity by increasing chloroplastic antioxidant enzyme activity. Journal of Experimental Botany 52 (365): 2345-2354. (PDF file)

Kornyeyev D., Logan B.A., Payton P., Allen R.D., Holaday A.S. (2001) Enhanced photochemical light utilization and decreased chilling-induced photoinhibition of photosystem II in cotton overexpressing genes encoding chloroplast-targeted antioxidant enzymes. Physiologia Plantarum 113 (3): 323-331. (PDF file)