INDUCING, OVERCOMING, EVOLVING, STRESS TOLERANCES

Jonathan Gressel, Ziva Amsellem, Barry Cohen, Noa Cohen, Efrat Halaf, Dafna Michaeli, Ilana Rogachev, Bin Ye, Jian Zhang

Tel. 972-8-9343481 Fax. 972-8-9344181, e-mail: jonathan.gressel@weizmann.ac.il

Objectives of Research:

Understanding, inducing and overcoming oxidative stress tolerance:- Oxidative stress is caused by adverse environmental conditions (transient drought), by environmental pollutants (ozone, sulfur dioxide) and xenobiotics (herbicides such as paraquat, and many others). We have been studying the mechanisms of tolerance in a paraquat-resistant mutant of Conyza bonariensis where a single dominant gene confers resistance. A series of oxidant detoxifying enzymes was elevated. As the first two enzymes contain copper, we have been testing specially synthesized chelators of copper designed to traverse plant cuticles, (in conjunction with A. Warshawsky, Organic Chemistry, WIS), to ascertain whether they could (a) inhibit these enzymes; (b) abolish tolerance; and (c) act as synergists to allow the use of less oxidant generating herbicides.

As the enzymes conferring oxidant tolerance might confer transient drought tolerance, we are (a) correlating their transcript levels in drought sensitive and drought-tolerant wheat varieties; (b) transforming wheat with constructs that should cause overexpression of these enzymes in wheat to ascertain whether transient drought tolerance is achieved (with E. Galun and G. Galili, this department).

Overcoming stress caused by weeds with biotech-derived herbicide-resistant crops: Weeds can devastate crop production and we are dealing with two cases where there are no selective herbicides to decimate the weeds. The parasitic flowering Orobanche spp. and Striga spp. attach to plant roots and suck the crops dry. We have pioneered the use of biotech-derived (tissue culture isolated mutations or bearing transgenes) herbicide-resistant crops. We are now continuing this effort by introducing such genes into crops of interest (with D. Aviv, this department, and D. Joel, Newe Yaar Research Center). We are also developing novel seed treatment technologies to lower the amount of herbicide needed, and preclude the need of spraying.

Many grass weeds of wheat have evolved resistance to all the herbicides presently utilizable in wheat, probably due to elevated levels of cytochrome P450 oxidases. The only solution is to introduce alien genes to confer novel herbicide resistances in wheat. Such genes could also "second" as new selectable markers (with E. Galun and G. Galili, this department).

Preventing/delaying the evolution of herbicide resistance in weeds:There is a need to keep the more environmentally friendly, cost-effective herbicides in use. We have modeled various ways to delay herbicide resistance, using population genetics and dynamics models that factor in use patterns, as well as various parameters relating to the properties of the herbicides. These models have been developed in conjunction with leading ecologists, population dynamicists, and applied mathematicians (see "Recent Findings").

Overcoming stress protection responses that preclude efficient biocontrol of weeds: The use of weed-specific pathogens has been touted as a "natural" (i.e. non chemical) way of preventing damage by weeds. The problem is that huge inocula are needed to overcome the natural stress tolerance systems activated by the weeds. We are studying two of these systems to ascertain how to overcome the weeds responses: (1) callose, a sticky polysaccharide is often produced as a response to fungal attack; (2) we wish to ascertain what are the defenses used by parasitic weeds to two specific pathogens. In the case of callose we are synthesizing analogs of chemicals known to inhibit callose synthase in vitro (in conjunction with A. Warshawsky, Organic Chemistry, WIS). We are measuring how these synergize a Colletotrichum that attacks a pernicious weed, Abutilon.

Recent findings:

Oxidant stress: There is a variability in the levels of oxidative stress tolerance, and the enzymes in the paraquat-resistant Conyza (Ye and Gressel 1994). Transcripts of the genes coding for these enzymes are also elevated. Elevated levels of three enzymes not previously reported as conferring oxidant tolerance in this species: dehydroascorbate reductase, mono-dehydroascorbate reductase and glutathione peroxidase were correlated with resistance. We developed methodology to directly detect the latter enzyme in plants (Ye and Gressel in preparation). Increased transcript levels for glutathione peroxidase were found (in conjunction with G. Ben Hayim, D. Holland and J. Eshat, Volcani Centre). We developed a highly sensitive sequential double staining technique to measure glutathione reductase activity on gels.

In addition to the elevated constitutive levels of these enzymes, we found that the tolerant Conyza biotype could be induced to higher yet levels of resistance by giving low levels of oxidant stress.

Herbicide-resistant crops to overcome stress by weeds: Model crops bearing target-site herbicide resistance genes could be used to control parasitic weeds (Joel et al, 1995, Gressel, Ransom and Hassan, 1996). In Kenya, seed treatments of imidazolinone-resistant maize with herbicides could triple yields while devastating the parasites (Abayo et al. 1996). Parasite control was achieved with resistant crop seeds treated with glyphosate, a foliar herbicide. Transgenic potato and tomato bearing two different genes for herbicide resistance genes have been generated, and are being tested.

Wheat transformed with a dehalogenase that degrades dalapon has been regenerated on levels of dalapon toxic to normal wheat, and is just forming seed. Southern blots suggest that the gene has been integrated onto wheat chromosomes.

Modeling resistance management strategies The use of mixtures of herbicides was proposed as a means to preclude or delay the evolution of resistance. We compared the present mixtures to criteria that we delineated for such mixtures to work, we found them wanting (Wrubel and Gressel, 1994).

The strategies suggested to prevent monogenically-inherited resistance promote the evolution of polygenically-inherited resistance and vice versa, a Catch 22. We modeled techniques of using different pesticide rates in different seasons to delay both types of resistance (Gressel, 1995; Gressel, Gardner and Mangel, 1996, Gardner, Gressel and Mangel, submitted).

The herbicide used for the seed treatments of herbicide-resistant maize is known to have resistance quickly evolve to it. Modeling has shown that resistance will evolve slowly when the crop is hand harvested, and we delineated an integrated management strategy that could delay resistance problems for over 15 seasons (Gressel, Segel and Ransom, 1996).

Overcoming the stress responses to biocontrol agents: We have some preliminary evidence that the callose synthase suppressing chemicals enhance infectivity (Michaeli and Gressel, prelim. observations). We have isolated two different Fusaria spp. that specifically attack the parasitic Orobanche spp. on vegetables, without affecting the crops (Amsellem et al 1996). Mixing the two organisms gives an enhanced infection, with less inoculum, probably because one organism penetrates cells and grows through cells, the other to pathogenizes between the cells.