Chloroplast Studies Point to Crop Enhancements

With news about Dolly and embryonic stem cells the stuff of cocktail party conversation, cloning a transgenic sheep or cow seems like child's play. The recipe is simple: Insert a pet gene into the nucleus of a cultured cell, fuse it with an enucleated egg, and voilà--a cow with high-octane milk. But incorporating genes into nuclear chromosomes isn't the only road to fame and fortune. Animals and plants have other sources of genetic information--their respiratory mitochondria and photosynthetic chloroplasts. Both are partially autonomous, with loops of DNA containing important genes whose encoded proteins are made on the organelles' own protein synthesis machinery. The plant chloroplast houses about 120 genes--small potatoes compared to the nucleus. Though most have been identified, a few open reading frames of unknown function remain, says Jeffrey Palmer, professor of biology at Indiana University in Bloomington. In general, the genes govern two main functions: transcription and translation of the organelle's genetic information, and photosynthesis.1 The photosynthesis genes encode the large subunit of RuBP carboxylase (the chloroplast enzyme that binds carbon dioxide) and various membrane proteins. Modifying chloroplast genes, or even adding a few here and there, could improve photosynthesis or otherwise enhance crop yields. Recent papers show that scientists are on the threshold of such changes, and even a few steps across. Chloroplasts use two large protein/pigment photosystems inside their thylakoid membranes to generate the ATP, electrons, and protons needed to fix carbon dioxide.2 Light absorbed by chlorophyll molecules of photosystem I energizes electrons that are then used to make sugar. Those electrons are replaced by others derived from water when light excites photosystem II. A series of proteins and other carriers move electrons between the two photosystems. One of the first is a protein called D1, which is actually part of photosystem II. Despite its importance, D1 is a weak link since it's sensitive to herbicides including triazines. Jurg Frey, senior scientist at the Swiss Federal Research Station for Fruit-Growing, Viticulture and Horticulture in Wadenswil, found that a daisylike weed called groundsel has two different versions of the gene for D1, called psbA, that are marked by a single base substitution, or polymorphism--the codon GGT replaces AGT, thereby substituting the amino acid glycine for serine in the protein. That small change is enough to make D1 resistant to triazine.3 Frey used PCR to amplify a 277 base pair fragment of psbA spanning the polymorphism, and he identified susceptible and resistant forms by the way the fragment digests with restriction enzymes. Surprisingly, in plants from Europe and as far away as North America, different leaves and even different parts of the same leaf have chloroplasts with either version of the gene--something called heteroplasmy. Since every leaf cell can have 100 or more chloroplasts, each with many copies of its chromosome, the complexity is startling. Heteroplasmy was thought to disappear between generations as chloroplast types sort out. Says Palmer, "That it persists, and is found in widespread geographic lines, surprises me." On the other hand, Frey thinks heteroplasmy is more common: "The lack of polymorphism reported so far seems to be a technical artifact" of not doing enough sequencing. In any event, maintaining different forms of psbA enhances a plant's versatility and may speed the evolution of herbicide resistance. Adds Palmer, "It raises the specter that if you introduce changes into the chloroplast genome, they may persist." Plants tinker with the two photosystems to optimize photosynthesis in response to changing light conditions. For example, they move extra light-gathering power from one system to another by sticking phosphate onto a light-harvesting "antenna" complex.4 Now it seems they also turn photosystem genes on and off to balance the two. Using mustard plants, a team of researchers led by John Allen, professor of plant cell biology at the University of Lund in Sweden, reported that when light stimulates photosystem I to a greater extent than photosystem II, it enhances transcription of psbA, so more D1 is made.5 In other words, more D1 helps compensate for overstimulation of photosystem I. In contrast, psaAB, a gene-encoding part of photosystem I, elevates when light preferentially stimulates photosystem II. By mimicking these effects with inhibitors of electron flow, the researchers concluded that the redox state of a critical electron carrier called plastoquinone controls expression of the two genes, perhaps via a kinase enzyme that adds phosphate to a key protein. Plastoquinone also regulates movement of the antenna complex. Says Allen, "I think [phosphorylation] is very likely .... We have plans to look seriously into this." Allen's results also hint at why chloroplasts still have DNA millions of years after they descended from cyanobacterial endosymbionts. Most of their genes ended up in the nucleus, leaving behind a minimal set including those encoding photosystem proteins. Since the genes are bacterial in nature, the Swedish team believes they respond more rapidly to light than if they were located in the nucleus. That might be advantageous for quickly fine-tuning photosynthesis. But Palmer is skeptical: "It's hard to see how you could generalize it to all proteins encoded by the chloroplast ....There are a lot of theories out there for why genes were retained." Several years ago, researchers trained Arabidopsis plants to make a biodegradable plastic by inserting three bacterial genes into the nucleus.6 The encoded enzymes assembled in the cytoplasm but carried a transit sequence that transported them into the chloroplast where they were more effective. The same strategy was used earlier to get mutated bacterial EPSP synthase into chloroplasts.7 The potent herbicide glyphosate (RoundupTM) targets the plant version of this enzyme, but the bacterial protein is resistant. A better approach might be to modify chloroplast DNA directly. Building on a string of technical advances,8,9 a team led by Henry Daniell, now professor of molecular biology and microbiology at the University of Central Florida in Orlando, recently inserted a nuclear EPSP synthase gene from petunia into the chloroplast genome of tobacco.10 The gene quickly sorted to all chloroplasts in transformed plants--it became homoplasmic. Given the large number of chloroplasts (and therefore up to 10,000 gene copies per cell), overexpression made the plants resistant. Daniell's team expects even greater resistance with the bacterial EPSP synthase, which has genuine ecological implications. Since chloroplasts are maternally transmitted through the egg in many crops, targeting foreign genes to them reduces the chance they will spread to native or weedy relatives via pollen. The reliability of chloroplast transformation as a brake on transgene escape is hotly debated,11 but new evidence that chloroplast genes flow maternally in oilseed rape (Brassica napus), and hybridization with weedy relatives is rare,12 could give the procedure a boost. The toxic Bt proteins of Bacillus thuringiensis are darlings of plant biotechnology because they kill a variety of destructive caterpillars. Unfortunately, broad scale application creates another threat: eventual insect resistance. Expressing the toxins at higher levels by way of chloroplasts9,13 may be one way to forestall it. Being green may not be easy (apologies to Kermit), but it's definitely not boring. Barry A. Palevitz (palevitz@botany.dogwood.uga.edu) is a contributing editor for The Scientist. H.-W. Heldt, Plant Biochemistry and Molecular Biology, Oxford, United Kingdom, Oxford University Press, 1997. L. Taiz and E. Zeiger, Plant Physiology, Sunderland, Mass., Sinauer Associates, 1998. J. Frey, "Genetic flexibility of plant chloroplasts," Nature, 398:115-116, March 11, 1999. J.F. Allen, "How does protein phosphorylation regulate photosynthesis?" Trends in Biochemical Sciences, 17:12-17, 1992. T. Pfannschmidt et al., "Photosynthetic control of chloroplast gene expression," Nature, 397:625-8, Feb. 18, 1999. C. Nawrath et al., "Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation," Proceedings of the National Academy of Sciences (PNAS), 91:12760-4, 1994. G. della-Cioppa et al., "Targeting a herbicide-resistant enzyme from Escherichia coli to chloroplasts of higher plants," Bio/Technology, 5:579-84, 1987. J.E. Boynton et al., "Chloroplast transformation in Chlamydomonas with high velocity microprojectiles," Science, 240:1534-8, 1988. K.E. McBride et al., "Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco," Bio/Technology, 13:362-5, 1995. H. Daniell et al., "Containment of herbicide resistance through genetic engineering of the chloroplast genome," Nature Biotechnology, 16:345-8, April 1998. D. Scott, C.N. Stewart Jr., "Transgene escape and transplastomics," Nature Biotechnology, 17:330-1, April 1999. S.E. Scott, M.J. Wilkinson, "Low probability of chloroplast movement from oilseed rape (Brassica napus) into wild Brassica rapa," Nature Biotechnology, 17:390-2, April 1999. M. Kota et al., "Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance against susceptible and Bt-resistant insects," PNAS, 96:1840-5, March 2, 1999.

Chloroplast Studies Point to Crop Enhancements

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