November, 2002

From: ISB News Report* November 2002
Crop Improvement through Alteration in the Photosynthetic Membrane
by Peter Horton

Photosynthesis is a pivotal process in plant growth—it is not only the primary process of energy conversion in the plant, but the machinery involved requires a large investment from external resources such as nitrogen, its function impacts upon water-use via stomatal conductance, and it is a main site for plant/environmental interactions. Therefore, for many different reasons, manipulation of photosynthesis is considered of central importance in efforts to increase crop yield.1 Firstly, the efficiency of conversion of intercepted radiation into biomass is a major yield parameter; many facets of the photosynthetic process that can give rise to inefficiency have been identified. Secondly, the photosynthetic apparatus is of prime importance in considerations of yield reduction by abiotic stress, common when crops are grown under sub-optimal conditions and detectable even under optimal conditions at light saturation. The latter topic is the subject of this article.

We have focused upon the photosynthetic membrane since it is not only the primary site of oxidative damage but also the main source of the reactive oxygen species (ROS) that cause widespread cell destruction under stress conditions (drought, extremes of temperature, nutrient deficiency). Our approach contrasts with that of many laboratories and consortia who focus on the plant cell processes that might confer resistance to abiotic stress by altering cell osmotic potential, expression of heat shock proteins, the levels of enzymes that scavenge ROS, and so on. We are interested in the processes in the photosynthetic membrane that are the origins of oxidative stress.

Our approach in turn leads to a consideration of the pigments in the membrane. The absorption of light by chlorophyll is the main origin of oxidative damage associated with stress. Photosynthesis has evolved into a process tuned to absorb and store light energy very efficiently. However, in many environmental conditions the level of sunlight is in excess of that which can be used in photosynthesis, and this excess light energy is potentially highly damaging to plants. Photosystem II (PSII), the starting point for photosynthetic electron transfer since it oxidizes water, is particularly susceptible to
damage. In fact, throughout the lives of all leaves, proteins damaged by oxidative stress in the PSII reaction center (the site of photochemical reactions) are continuously being replaced by new ones. Under stress, the repair process can be overloaded and the plant suffers photoinhibition. However, the problem is more widespread than just the PSII reaction center proteins—excess light energy increases the probability of forming ROS species in the pigment-protein complexes of the light harvesting system (where most of the chlorophyll is found). Furthermore, any damage to a constituent of the photosynthetic system increases the probability of further ROS production, a potentially lethal downward spiral of damage to protein, lipid, and nucleic acids, terminating in plant death.

It is therefore not surprising to learn that the photosynthetic membrane contains specific constituents and processes that are designed for "photoprotection."1,2 In recent years, enormous strides have been made in understanding the complex function of this membrane. High-resolution structural information is available for most of the main protein complexes, and new methods of image analysis are showing how these are assembled into giant macrostructures containing hundreds of proteins. One essential feature of the light harvesting system is its structural flexibility, which allows it to switch between two functional states—one for light harvesting, another to dissipate excess light energy harmlessly as heat. The latter, known as the state of feedback de-excitation, is readily measured non-invasively by chlorophyll fluorescence (nonphotochemical quenching or NPQ) and has been the subject of intensive study.

To dissipate excess light energy, two molecules in the membrane are needed. The first is a very hydrophobic membrane protein known as PsbS. When PsbS is absent from Arabidopsis plants, photoprotection is inhibited, and the plant becomes more susceptible to oxidative stress and suffers a large yield penalty in the field.3 Secondly, a particular type of carotenoid plays a crucial regulatory role, since it determines the rate of the switch between these two states.

Carotenoids are molecules widely known to protect against oxidative damage in all cells. In plants, the xanthophyll cycle (the reversible interconversion of two particular carotenoids, violaxanthin and zeaxanthin) has evolved to play this essential role in photoprotection. In fact, the xanthophyll cycle has two roles. Zeaxanthin accumulates under conditions of excess light because of the activation of violaxanthin de-epoxidase by the high transthylakoid pH gradient (DpH) generated under these conditions. In order for zeaxanthin to carry out its role in feedback de-excitation, it is bound to one or more of the proteins that constitute the macromolecular complexes of the chloroplast membrane. This binding, which has the characteristics of an allosteric regulator, is also dependent upon, or stimulated by, the increase in thylakoid DpH. Zeaxanthin is thought to bind to PsbS. In addition, zeaxanthin has a second protective role. Since zeaxanthin is hydrophobic, it is found mostly at the periphery of the light-harvesting complexes, and there it functions to prevent damage to the membrane lipids from ROS, lipid
peroxidation.

These elements of photoprotection, therefore, are promising targets for genetic engineering to enhance stress tolerance in crop plants.4 From studies in comparative ecology, it is known that the capacity for photoprotection is subject to genetic variation—plants inhabiting harsh environmental conditions tend to have a higher capacity for feedback de-excitation. We have focused on the manipulation of the
xanthophyll cycle, since the level of zeaxanthin is always higher in stressed plants, and took a metabolic engineering approach to increase the xanthophyll cycle pool size in Arabidopsis thaliana. Given the nutritional importance of carotenoids, several groups have manipulated carotenoid biosynthesis in plants. Although Arabidopsis mutants with an increased xanthophyll cycle pool size are available, they have large alterations in the content of other carotenoids and lesions in the biosynthesis of the plant growth regulator, abscisic acid.

We decided to overexpress the thylakoid membrane enzyme ß-carotene hydroxylase, which catalyses the conversion of ß-carotene to zeaxanthin, in order to specifically manipulate the xanthophyll cycle pool size. We transformed Arabidopsis with the chyB gene under the control of a constitutive promoter and established several stable lines derived from independent transformation events. These lines displayed up to 4-fold overexpression of ß-carotene hydroxylase. Overexpression of the chyB gene
specifically increased the xanthophyll cycle pool size two-fold, from 14% to nearly 30% of total carotenoid in low light grown plants, and from 22% to over 40% for moderate light plants. The latter value is significantly larger than any reported value for wild type Arabidopsis. The levels of other carotenoids were not perturbed, and so photosynthetic function was maintained at wild type levels and plant growth proceeded normally.

The extra xanthophyll was mostly associated with the photosystem II light-harvesting complexes (LHCII), which show a 3-fold increase in the amount of bound violaxanthin. The extra violaxanthin was also available for de-epoxidation when the plants were exposed to high light, so that the levels of zeaxanthin were at least twice as high in the sense chyB lines as in the wild type. We conclude that at least some of the extra xanthophyll is biologically active, allowing us to test whether an increased xanthophyll cycle pool will confer any improvement in stress tolerance.

Plants were grown for five weeks under low light conditions (100 mmol photons m-2 sec-1/20°C) and then were switched to stress conditions, 1000 mmol photons m-2 sec-1 and air temperature 40°C, for two weeks. One indicator of stress is the presence of the purple flavonoid anthocyanin, and sense chyB plants had significantly less anthocyanin than the wild type control (mean value 1.0 ± 0.47 and 1.6 ± 0.49). This difference in anthocyanin levels was obvious in the whole plants where the sense chyB lines were clearly greener and healthier, showing less leaf necrosis. An estimate was made of
general lipid peroxidation as determined by the amount of malondialdehyde (MDA), a secondary end product of the oxidation of polyunsaturated fatty acids, in wild type and sense chyB plants exposed to stress. The amount of lipid peroxidation differed between the wild type and sense chyB lines—the mean values were 9.2 ± 4.3 and 6.6 ± 3.9 respectively; the almost 30% drop in the amount of lipid peroxidation in the sense chyB lines was statistically significant.

The results show that increasing expression of the ß-carotene hydroxylase enzyme brings about an increase in the content of the xanthophyll cycle and zeaxanthin in the chloroplast membrane, and, most importantly, that this manipulation leads to an improved tolerance of high light and high temperature conditions. It is important to point out that these effects on anthocyanin production and lipid peroxidation were observed in plants exposed to stress conditions for two weeks; that is, it was a sustained property of the transformed plants. We conclude that genetic manipulation of a single enzyme in carotenoid metabolism can bring about a pronounced increase in the stress tolerance of
plants and represents a potentially powerful way forward in the production of stress-tolerant crops.

The exact mechanism by which the xanthophyll cycle provides increased stress tolerance remains undetermined. No increase in feedback de-excitation was found in these plants. Most likely, therefore, the increase in zeaxanthin provides direct protection against lipid peroxidation. Further work is needed to confirm this. The next phase in the research and development is to introduce this genetic manipulation into crop plants and to test its effect on yield in the field.

This research, showing a positive effect from the alteration of ß-carotene hydroxylase level, represents the first time that a change in any feature of the chloroplast thylakoid membrane has resulted in an enhancement of plant performance. Whilst there is widespread acknowledgement that the basic features of the photosynthetic process per se do not offer opportunities for genetic improvement, there is an increasing likelihood that manipulation of the secondary "regulatory" processes in the thylakoid membrane, associated with the large number of low abundance proteins and other molecules, such
as carotenoids, minor lipids and tocopherols, has considerable untapped potential.

References

1. Horton P, Murchie, EH, Ruban AV, and Ruban AV. 2001. Increasing rice photosynthesis by manipulation of the acclimation and adaptation to light. In Novartis Symposium No 236. Rice Biotechnology: improving yield, stress tolerance and grain quality, eds. J. Goode, D. Chadwick, Wiley and Sons Ltd, Cichester, England, 117-134.

2. Niyogi KK. 1999. Photoprotection revisited: genetic and molecular approaches. Annual Review of Plant Physiology and Plant Molecular Biology 50: 333-359.

3. Külheim C, Ågren J, and Jansson S. 2002. Rapid Regulation of Light Harvesting and Plant Fitness in the Field. Science 297: 91-93.

4. Davison P, Hunter CN, and Horton P. 2002. Overexpression of ß-carotene hydroxylase enhances stress tolerance in Arabidopsis. Nature 418: 203-206.

Peter Horton
Robert Hill Institute
Dept. of Molecular Biology and Biotechnology
University of Sheffield, United Kingdom
P.Horton@sheffield.ac.uk 

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