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Plant Physiol, December 2001, Vol. 127, pp. 1334-1338
SCIENTIFIC CORRESPONDENCE
When Simpler Is Better. Unicellular Green Algae for Discovering
New Genes and Functions in Carbohydrate Metabolism
Glenn R.
Hicks,*
Catherine M.
Hironaka,
David
Dauvillee,
Roel P.
Funke,
Christophe
D'Hulst,
Sabine
Waffenschmidt, and
Steven G.
Ball
Plant Genetics, Exelixis, Inc., 170 Harbor Way, P.O. Box 511, South
San Francisco, California 94083-0511 (G.R.H., C.M.H., R.F.);
Laboratoire de Chimie Biologique, Unite Mixte de Recherche du Centre
National de la Recherche Scientifique Number 111, Universite des
Sciences et Technologies de Lille Flandres-Artois, 59655 Villeneuve
d'Ascq cedex, France (D.D., C.D., S.G.B.); and Institut fur Biochemie,
Universitat zu Koln, Greinstr. 4, 50939 Koln, Germany D-50931
(S.W.)
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INTRODUCTION |
Of all algae, those in the
division chlorophyta (green algae) display the closest relationship to
the vascular plants. Chlorophytes harbor a chloroplast that is
considered to have originated from a single endosymbiotic event and
contain the same types of photosynthetic pigments as land plants. At
variance with other divisions of algae, starch is produced within
chlorophyte plastids and displays a structure very similar to that
described for vascular plants. Considerable variation is found in the
organization and composition of chlorophyte cell walls. Some are
characterized by the presence of a simple glycoprotein wall, whereas
others synthesize elaborate walls with a polysaccharide composition
like that of land plants. Algae often contain pulsatile vacuoles that
in some cases allow growth in the absence of a normal cell wall
structure, a useful feature for studying wall biology. Because of the
presence of plastids and plant-like cell walls, unicellular
chlorophytes may be considered true plant-like eukaryotic
microorganisms. Their microbial nature permits the use of extremely
powerful genetic techniques akin to those in yeast
(Saccharomyces cerevisiae) for the dissection of
plant pathways. In this scientific correspondence, we will emphasize
the potential of using unicellular chlorophytes to understand plant
pathways, including polysaccharide and cell wall metabolism. We hope to
encourage plant biologists to consider these species because of the
potential for rapid progress in understanding many basic plant pathways.
Among unicellular green algae,
Chlamydomonas reinhardtii is by far the most studied
system. Many recent reviews describing the speed and ease of C. reinhardtii genetics and molecular biology have appeared.
In fact, within this issue of Plant Physiology, several
detailed research articles depict the use of C. reinhardtii to study chloroplast biogenesis and cell motility. This alga grows rapidly in defined medium both in liquid and on agar, and its sexual
cycle can be as easily controlled as that of yeast. Single colonies
grow within 5 d and crosses can be analyzed in less than a month.
In addition, the nuclear genome can be efficiently transformed and gene
replacement via homologous recombination in the chloroplast genome is a
routine method. Unlike land plants, C. reinhardtii has the
remarkable ability to dispense with photosynthesis and to use acetate
as a carbon source; this has allowed the isolation of mutations in both
nuclear and plastid genes that affect all possible aspects of
chloroplast biogenesis. Similarly, the ease with which cell motility
mutants can be isolated has allowed a thorough description of the
structure, assembly, and function of flagella. These major achievements
have overshadowed many aspects of plant metabolism for which C. reinhardtii could be an extremely useful model system such as
commercially valuable pathways leading to carbohydrates, carotenoids,
lipids, and secondary products, as well as other essential pathways.
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UNRAVELING STARCH METABOLISM |
An excellent example of the power of unicellular algae is the use
of C. reinhardtii to understand starch metabolism, which is
resulting in the discovery of new functions even within enzymes that
are well characterized. Such knowledge can guide rational efforts to
manipulate starch composition for practical purposes (Slattery et al.,
2000 ). Starch is an extremely valuable polymer both nutritionally and
as an industrial raw material. It is stored in photosynthetically
active leaf chloroplasts as transient starch and in seeds or tubers of
economically important crops such as maize (Zea mays),
rice (Oryza sativa), and potatoes (Solanum
tuberosum) as storage starch. Starch is an insoluble crystalline
granule composed of two polysaccharide fractions. Amylopectin is the
predominant high-Mr polymer in storage
granules and contains an abundance of branched glucans having  -1,6
linkages. Amylose is a lesser component of the granule having
relatively few branch points (Ball et al., 1998; Buleon et al., 1998).
Storage starch has been studied extensively in higher plants
particularly in maize, rice, pea (Pisum sativum), and
potato, whereas mutants in leaf starch have been identified and studied
in Arabidopsis (for example, see Casper, 1994; Zeeman et al., 1998; Yu
et al., 2001). Such efforts have resulted in a relatively good
description of the major biosynthetic enzymes in the pathway. It is
unfortunate that this has also led to the impression among many
scientists that little remains to be understood. Quite to the contrary,
however, many aspects of starch metabolism remain poorly understood,
including granule nucleation and assembly, regulation of synthesis and
turnover, mechanisms of starch modification, and the contributions of
the many enzyme isoforms to starch composition and crystalline structure.
C. reinhardtii produces starch granules that are similar to
those in other plants morphologically as well as in composition and
fine structure (Buleon et al., 1997). An extremely valuable feature of
the alga is the ability to induce granule formation easily within
several days by simple nutrient limitation as opposed to flowering
plants where storage granule development requires seed set. Under
nitrogen-limited conditions, algal colonies can be scored directly for
starch composition by staining or biochemical methods. As mentioned, a
key feature of C. reinhardtii is its microbial nature, which
should permit large-scale screens that can be automated by the adoption
of existing colony picking and screening robots. The ability to rapidly
screen tens of thousands of colonies for mutants make algae an
excellent complement to research in crop species where such efforts
require significantly more time and labor.
To date, 12 loci have been identified genetically in C. reinhardtii that are involved in starch biosynthesis using as
mutagens UV, x-ray, and, in some cases, insertional disruption (Buleon et al., 1998). These loci define most of the components of the pathway
known in land plants and orthologs can be readily identified by
sequence comparisons, indicating the extremely high degree of
conservation within the plant kingdom. Mutations at these loci were
identified by a sensitive and straightforward iodine vapor screen for
altered starch structure (Delrue et al., 1992). The method results in a
variety of colors that are indicative of the length of the glucan
chains and starch structure, and mutants are easily identified compared
with wild-type cells that stain violet (Fig.
1A). In the examples
presented, mutations in AGPase, granule-bound starch synthase I
(GBSSI), and soluble starch synthase result in essentially no starch,
low amylose, and high amylose, respectively. As an illustration to the
general reader of the utility of C. reinhardtii in
dissecting starch metabolism, we performed a modest screen of 5,600 methyl methanesulfonic acid mutants. We initiated the screen to
establish methods that could be adapted for high-throughput screening
of a large mutant collection in C. reinhardtii and to search
for novel mutations using a chemical mutagen, which had not been tried
previously in the alga to dissect the starch pathway.
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Figure 1.
C. reinhardtii can be screened
efficiently for mutations in starch. A, Distinct mutants define the
starch pathway. Previously characterized mutants (Buleon at el, 1998)
illustrate the color-based detection of mutants for altered starch
(sta1, no color; sta2, red; sta3, olive; and cc1928 wild type, violet).
Left, The starch biosynthetic pathway is depicted with intermediates
indicated and addition of -linked Glc (n) to the
lengthening glucan chain (n + 1). Mutations resulting in
loss of enzyme function are indicated (X). Right, The affected enzymes
are shown along with their respective mutant loci in C. reinhardtii. From top to bottom the loci are: sta1-1, sta2-1,
sta2-2, sta3-1, sta3-2, and sta3-3, respectively. The sta2-1 mutation
results in less than 5% (w/w) total starch. B, Mutagenized
cells can be arrayed for efficient screening. Mutagenized cells are
grown on standard medium for 7 d then inoculated into microtiter
plates containing liquid medium and grown for an additional 5 d.
Once in microtiter plates, cells are easily arrayed on solid medium to
induce starch for screening. All steps indicated can be automated by
the use of robots to increase the throughput.
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Cells were mutagenized and grown in the
light, then inoculated into microtiter plates (Fig. 1B). The cells were
arrayed onto plates containing standard medium (stock plate) or onto
medium without nitrogen to induce starch formation then stained by
iodine vapor. Starch prepared from induced cultures of the mutants was examined for structure and relative amylose and amylopectin (Libessart et al., 1995). The spectral properties of the mutants, another indication of altered structure, were also measured. Several mutants displayed an apparent increase in amylose content; whereas other mutants could not be associated with known defects based upon starch
analysis and other approaches such as isozyme assays, indicating the
possibility of novel mutations. More informative in the short term are
mutations within characterized genes, which provide comparative results. Therefore, we focused attention upon mutants defective in the
well-characterized enzyme GBSSI, which is essential for the synthesis
of amylose. Waxy starch from maize, a commercially valuable
starch, is the result of natural mutations in GBSSI leading to reduced
function and a high relative content of amylopectin due to a decrease
in amylose biosynthesis. Three mutants were defective in GBSSI as
indicated by the characteristic loss of amylose, altered spectral
properties of the amylopectin fraction (Delrue et al., 1992), and
isozyme analysis. It is interesting that one of the mutants retains
granule-associated GBSSI protein, and subsequent mapping suggests the
presence of at least one mutation within the protein structure that was
not associated previously with loss of function. Detailed
characterization is in progress, but here is an excellent example where
an algal system may lead to the discovery of new regions necessary for function.
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NEW TOOLS |
Algae such as C. reinhardtii offer many advantages, yet
have lacked the coordinated development of resources aimed at
sequencing the genome or producing genetic tools that are useful to the
scientific community, such as large numbers of single nucleotide
polymorphisms and insertion and expression tagged lines as are
available in Arabidospsis. This is now changing with the development of
genome-wide single nucleotide polymorphisms (Vysotskaia et al., 2001).
To speed the discovery of novel components and functions in the starch pathway, we have constructed a collection of 50,000 insertion lines in
C. reinhardtii using a vector that contains a gene essential for Arg biosynthesis as the selectable marker for insertion. The collection is in a genetic background containing the sta2-1
mutation in the GBSSI gene (and Arg auxotrophy). Because the
sta2-1 allele causes the cells to stain red by iodine vapor,
it will be possible to detect a broader range of mutations, such as
those selectively defective for amylopectin synthesis, that cannot be
detected easily in a wild-type background. Because disrupted genes will
usually be linked to the selection marker, rapid cloning of affected
genes should be possible. The results may aid in the discovery of
useful genes that can be engineered into crops directly or provide
valuable information about metabolism that can help focus our efforts
in crop species. Although the collection was designed with starch metabolism in mind, many unrelated screens are possible for lipids or
other valuable metabolites.
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UNDERSTANDING THE CELL WALL |
The plant cell wall is a complex and dynamic mosaic of three
coextensive and interactive networks of cellulose/xyloglucan, pectin,
and proteins (Showalter, 1993). Although significant inroads have been
made recently in understanding the molecular details of cell wall
synthesis (for example, see Faik et al., 2000), much of this research
has focused on proteins in the Hyp-rich glycoprotein (HRGP)
superfamily. In the last decade, numerous HRGPs have been characterized
at both the protein and DNA level. As a consequence, a lot is known
about the structure and regulation of these proteins, but the precise
function of any particular HRGP remains unclear. An alternative
approach is to develop a model system for studying wall assembly using
C. reinhardtii, whose walls are composed almost exclusively
of HRGPs. In the walls that surround vegetative cells and gametes
(Goodenough et al., 1986; Snell and Adair, 1990), the HRGPs are
arranged in two major layers: an inner layer (W2) whose constituent
HRGPs are insoluble and a contiguous outer crystalline layer (W6) of
HRGPs that are soluble but salt extractable. The assembly of the
vegetative wall can be induced by treating cells with a cell wall lytic
enzyme produced by gametes (Claes, 1971; Kinoshita et al., 1992).
Within several hours, the protoplasts secrete a new and insoluble wall.
Analysis of soluble enzyme activity combined with studies in which
inhibitors that interfere with cross-linking are applied reveal that a
peroxidase and a transglutaminase are involved in wall regeneration
(Waffenschmidt et al., 1993, 1999). Because C. reinhardtii
is haploid, mutations will produce a phenotype directly. Thus,
mutagenesis and screening for defects in wall regeneration provide
information about essential structural proteins or cross-linking
enzymes as well as perception or signaling in response to stresses such
as wall rupture. In contrast to vascular plants, most C. reinhardtii mutants defective in wall regeneration will remain
viable due to the presence of pulsatile vacuoles, which obviates the
need for tedious selection of conditional phenotypes. In fact, cell
wall-defective colonies of C. reinhardtii can be distinguished easily by their mucoid morphology. Although C. reinhardtii is useful for understanding HRGPs, its wall lacks
several components of vascular plants, including cellulose, xyloglucan,
and pectin. However, other chlorophyte cell walls do possess elaborate
vascular plant-like organization. The desmidiales are unicellular
organisms with highly ornate walls organized in two
symmetrical semicells. They synthesize cellulose through hexagonal
arrays of rosettes and their walls contain pectins and arabinogalactan
proteins. Several heterothallic species of desmidiales are available
and conjugation has been mastered in the laboratory. Thus, desmidiales are a potentially useful system to study plant cell wall morphogenesis, although much work remains to develop them as genetic and molecular models equivalent in utility to C. reinhardtii.
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NEW MODELS |
An important and perhaps overlooked aspect of unicellular green
algae is the potential for reduced functional gene redundancy, which is
displayed by higher plant genomes. Whereas C. reinhardtii displays a genome complexity approaching that of Arabidopsis, some
recently characterized microalgae may have genomes simpler than that
even of yeast. Ostreococcus tauri, a unicellular
chlorophyte belonging to the prasinophyceae, defines the smallest
eucaryote known to date (0.8 µm in diameter) (Courties et al., 1994;
Chrétiennot-Dinet et al., 1995). It also has one of the smallest
genomes (10.2 Mbp; Courties et al., 1998). Yet O. tauri, recently identified as a picophytoplanktonic
organism, displays all major features of chlorophytes and other plant
cells. Picophytoplanktonic organisms were discovered only 2 decades
ago, when it was realized that cell counts based on chlorophyll
measurements from the surface of the open seas did not agree with the
cell counts performed by classical techniques. It was then discovered
that the seas contain tiny planktonic cells in abundance. Among these
picophytoplanktonic organisms (between 0.3-3 µm in diameter),
phycologists found a great diversity of picoeukaryotes. Although axenic
cultures of O. tauri are not yet available, other
picochlorophytes appear to grow well both on defined solid media and in
liquid cultures. Small size seems to offer a selective advantage
to oceanic planktonic species. To achieve such a small size,
other picochlorophytes will likely have simplified genomes with reduced
nDNA content. It is unlikely that O. tauri and its
picoeukaryotic cousins, with roughly one-tenth the nDNA content of
Arabidopsis, could maintain a level of "functional" redundancy
equivalent to that of C. reinhardtii or Arabidopsis. This is
particularly true given the need to maintain chloroplast, mitochondrion, and other major aspects of the eukaryotic way of life.
It is more likely that these organisms will have streamlined pathways
and will have reduced functional gene redundancy to core functions.
Thus, a green Escherichia coli might very well be lurking out in the open seas, an organism that could turn out to greatly simplify functional studies of vascular plant pathways.
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A PROMISING FUTURE |
We are just beginning to see the true value of algal species
for the elucidation of important pathways in plants. Classical discoveries in chloroplast and flagella function in C. reinhardtii were only the beginning. Recent results in
understanding carbohydrate metabolism indicate that chlorophytes like
C. reinhardtii provide the efficiency and speed necessary to
permit us to saturate mutationally basic pathways such as starch or
cell wall synthesis more fully. This will lead to a new understanding
of these and other important pathways. It will be essential ultimately
to understand such pathways in vascular plants if we are to reap the
practical benefits from our research. However, algae can serve as
powerful microbial models for the rapid identification of novel genes
and functional domains much as yeast has elucidated many core functions
that are essential in animals. In fact, examples of the use of
microbial genes for crop improvement are well known. To move forward
requires a concerted effort to fully develop algae such as C. reinhardtii in terms of genome sequence and tools to further
increase the speed of large genetic screens. We are developing tools
such as insertion collections, and as we approach the completion of
genome sequencing for a number of higher plant species, the opportunity
to expand our list to include unicelluar plants will present itself. As we have discussed, the identification of the picoeukaryotic algae such
as O. tauri highlights the potential to develop models that avoid the functional gene redundancy found in vascular plants. Given
the indications that their genomes will be quite small, sequencing
should be relatively straightforward compared to the efforts applied in
Arabidopsis and rice. The comparison of simple unicellular plant
genomes to those in multicellular plants will in itself be quite illuminating.
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ACKNOWLEDGMENTS |
The authors are very grateful to Drs. Claude Courties and
Hervé Moreau (Observatoire Oceanologique de Banyuls, Laboratoire Arago, Banyuls-sur-mer, France) for their generous sharing of O. tauri data prior to publication. We also wish to
thank Jacqueline McLaughlin (Exelixis, South San Francisco, CA)
for excellent graphic arts.
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FOOTNOTES |
Received September 7, 2001; returned for revision September 14, 2001; accepted September 20, 2001.
*
Corresponding author; e-mail ghicks{at}exelixis.com; fax
650-837-8122.
www.plantphysiol.org/cgi/doi/10.1104/pp.010821.
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TOP
INTRODUCTION
UNRAVELING STARCH METABOLISM