Photosynthetic Pigmentation—Variegations on a Theme

Plant pigmentation has been of major interest to horticulturists for millennia. The ancient Egyptians, for example, cultivated the madder plant (Rubia tinctorum) as a source of dye for the ornamentation of mummies, and tribal peoples living before the Common Era in northern Britain tattooed their skin with indigo dye, a fact recorded by Julius Caesar upon Roman colonization of Britain. The diligence with which Native Americans cultivated many different pigmented maize seeds can even be ascribed to an early systematic foray into plant genetics. During the present century, the chemical industry has largely obviated the exploitation of plant pigmentation, but basic scientists have pursued studies of plant coloring to the extent that the disciplines of plant physiology, biochemistry, development, and genetics have all flourished and, on occasion, converged. Such convergence of experimental disciplines and approaches is perhaps not surprising given the fundamental roles that pigments play in plant life. Certainly one of the most important functions to be effected by plant pigments is the capturing of light energy for photosynthesis. Chief players in this regard are the green chlorophylls, as well as the orange-red carotenoids. Both of these pigment types reside within chloroplasts, which in turn arise from colorless, non-differentiated proplastids. The appearance of green, photosynthetic tissue from dark-grown (etiolated) seedlings can thus provide an excellent system for addressing a wide range of cell biological themes. As an example, the assembly of the photosynthetic apparatus along with the performance of the carbon reduction cycle within the chloroplast requires the coordinated expression of genes residing in the nuclear and plastid genomes. Exactly how the cell orchestrates signaling pathways between cellular compartments is an experimental issue that continues to benefit from studies of the light-induced synthesis of photosynthetic pigments. Mutations that interfere with the proper greening of etiolated plant tissue upon exposure to light, therefore, may provide crucial insight into intracellular signaling events. Because albino plants suitable for developmental investigation can be hard to come by, however, a number of plant mutants that elaborate a variegated phenotype have been selected for study over the past few decades. A number of factors can trigger leaf variegation. It is clear, for example, that light and other variable environmental cues can differentially affect organ and tissue pigmentation: shaded branches may become chlorotic, illuminated branches may produce vibrantly colored foliage. Thus, in addition to mutations that would directly interfere with chloroplast development (e.g., because they affect photosystem assembly), mutations that potentiate plant responsiveness and sensitivity to environmental effects could also provoke variegated phenotypes. Furthermore, inasmuch as both nuclear and chloroplast DNA encode proteins and stable RNAs essential to chloroplast function (the vast majority of chloroplast proteins are in fact nuclear encoded), chloroplast genesis could be affected by the appropriate mutation of either genome. Indeed, variegated mutants of both categories exist, whereby chloroplasts segregate during leaf development to give clones of cells containing varying amounts of affected as opposed to normal (i.e., green) chloroplasts. In addition, mutations within the third source of genetic material in plant cells, the mitochondrion, have also been found to result in leaf variegation. Although the segregation of faulty mutant mitochondria, like that of mutant chloroplasts, represents a conceptually satisfying mechanism for variegation, the resulting mutant plants confront us with many questions and few answers. The nonchromosomal stripe (NCS) mutants of maize, for instance, as well as Arabidopsis mutations at the nuclear locus that, reasonably enough, was originally named chloroplast mutator (CHM), manifest maternally heritable mitochondrial DNA rearrangements that ultimately harm chloroplast structure and function (Roussell et al., 1991; Sakamoto et al., 1996). The biochemical and genetic interplay between mitochondrial and chloroplast compartments, although now recognized from such mutations as an intriguing dimension of plant cell biology, awaits further elucidation. Two papers in this issue of THE PLANT CELL report different investigative paths that have converged upon a variegated Arabidopsis mutant. On pages 43–55, Wu et al. update their efforts in investigating the communication pathways between nuclear and organellar genomes, and offer insight into the biochemical lesion that underlies the variegated phenotype of the Arabidopsis immutans (im) mutant. On pages 57–68, Carol et al. describe their success in further elucidating the biochemistry of plant pigment biosynthesis by isolating a variegated mutant of Arabidopsis that turns out to be an allele of IM. The experimental elegance displayed by each group will do much to further research into both the molecular biology and biochemistry of organelle development. The variegated patterning of im is highly intriguing in that increases in either temperature or light intensity increase the amount of white-sectored plant tissue (Rédei, 1963; Röbbelen, 1968). This is to say that the degree of green and white sectoring in any seedling arising from the self-pollination of im plants will reflect the lighting conditions that it was exposed to during early cotyledon development rather than the actual phenotype of the parent (Wetzel et al., 1994). Indeed, the name immutans was chosen to underscore the steadfast, “unchanging” nature of the mutation despite wide variability in phenotype color: even all-green im inflorescences, rather than representing reversions, can be used to produce predominantly white progeny (Rédei, 1975). For close to thirty years after im was first described, the nature of the genetic lesion remained unknown. Because cytoplasmic RNase activities were recognized to be elevated in early studies (Rédei, 1967), the suggestion was long propagated that a disturbance in RNA metabolism might account for defective chloroplast development (Koncz and Rédei, 1994). Rodermel's group has made important strides in characterizing im (Wetzel et al., 1994; Wu et al., 1999). In addition to establishing definitively that the mutation is not maternally inherited, these investigators also found that the observed increase in RNase activity relative to wild-type tissue is a secondary effect not related to the genetic constitution of the variegated mutant. Through the use of electron and fluorescence microscopy, the same authors also established that cells from the white sectors of im plants are in fact heteroplastidic, containing primarily defective, but occasionally also normal, plastids. The cells of green sectors, however, appear phenotypically to be wild type. Biochemical characterization of the im plants extended to chlorophyll and carotenoid content (Wetzel et al., 1994), which confirmed the earlier determinations of reduced pigment production. Perhaps most significant of all was the group's finding that the white tissue of the variegated plants accumulates phytoene, the precursor of ζ-carotene and thus of all the carotenoids. In this way, the chlorotic nature of im would appear to result directly from a lesion in pigment biosynthesis, and the obvious hypothesis to explore is that phytoene desaturase (PDS), the enzyme that converts phytoene into ζ-carotene, is defective in im. After all, PDS is encoded within the nuclear genome (chromosome 4, where IM had also been previously mapped [Koornneef, 1987]) and is active in the chloroplast, the site of carotenoid biosynthesis. PDS is also the site of action of several herbicides, such as Norflurazon, that block carotenoid synthesis and cause the concomitant photobleaching of leaves (Sandmann and Albrecht, 1990). Levels of PDS protein and mRNA within white im sectors, however, prove to be normal, and appropriate crosses of im placed the IM locus approximately 13 cM from pds. Although PDS has been studied in a variety of species (reviewed in Sandmann, 1994), many of the mechanistic details that characterize the conversion of phytoene to carotene have yet to be elucidated. In fact, it was their interest in elucidating the intricacies of carotenoid biosynthesis and in identifying ways in which such intricacies might affect chloroplast development that led Carol et al. to initiate their mutagenesis experiments. Working across the Atlantic from Wu et al., Carol et al. crossed a line harboring a transposable element on chromosome 4 with a corresponding line carrying an Activator (Ac) tranposase so as to promote mutational rounds of Ac excision and reinsertion within the nuclear genome (Carol et al., 1999). Albino progeny grown in vitro were screened for the ability to produce variegated offspring as a consequence of a stable (non-reverting) mutation, and were confirmed upon further crossing to be allelic with im. Because transposon insertions can be directly probed, Carol et al. were able to clone the gene that had been mutated so as to yield the variegated phenotype. In addition, Wu et al. performed a sequence walk of chromosome 4 in order to characterize the im mutants that they had been studying. The combined efforts of both groups thereby resulted in the incontrovertible identification of IM, the wild-type allele of im. And the identity of the IM protein? Not homologous to PDS, nor to any other known chloroplast protein for that matter, the predicted 40.5-kD translation product of IM in fact shares homology in all searched databases solely to the alternative oxidase (AOX) of plant mitochondria. AOX provides plants with an alternative pathway for the shuttling of electrons from reduced ubiquinone (ubiquinol) of the mitochondrial respiratory chain (for a review see Siedow and Umbach, 1995) and is encoded by a nuclear multigene family (Saisho, et al., 1997). It is not clear what a “mitochondrial” terminal oxidase is doing in chloroplasts. Significantly, however, plastoquinone of the photorespiratory chain has been identified as a necessary cofactor in carotenoid production, thereby indicating that an oxidoreductase must be involved in the oxidation of phytoene as carried out by PDS (Norris et al., 1995). The IM protein, by the way, tends towards greater homology to AOX in its C-terminal sequence, whereas its N terminus manifests features of a chloroplast transit signal. Carol et al. in fact show that the IM product is indeed translocated into chloroplasts where it associates with the thylakoid membrane. Based on the conclusion that the IM protein acts as an oxidase to support phytoene turnover in carotenoid biosynthesis, both research groups offer a model to explain the elaboration of the light-induced white sectors seen in im. Carotenoids, in addition to being light-harvesting accessory molecules, protect tissues from photooxidative damage (Siefermann-Harms, 1987). The obstruction in im of phytoene turnover into ζ-carotene thus deprives tissues of photoprotection, just as Norflurazon does by inhibiting PDS activity. In this way, the differential exposure of developing im seedling tissues to light provides a mechanism to explain the production of the light-sensitive variegated phenotype that can range from near-albinism to full verdancy. In view of the intriguing data that have become available this month, it remains to be seen whether further study of the im mutant will provide additional clues into intracellular communication or the interorganellar exchange of macromolecular materials.