The projects in the Grossman laboratory have always
been diverse, but they generally involve plant acclimation responses to light
and nutrient limitation. There are a number of new areas that have recently
been developed in the lab. Dr. Lorraine van Waasbergen, now an assistant
professor at the University of Texas at Arlington, has begun to define the webs
of regulatory circuits that link nutrient limitation and high-light responses.
Dr. van Waasbergen identified a sensor kinase, designated NblS, that acts as a
global regulator to control processes critical for altering the photosynthetic
activity of cyanobacteria in response to stress conditions.[1]
Among the processes controlled by NblS are light-dependent modulation of hliA gene expression, exchange of forms of D1 subunits
of photosystem II, and the biosynthesis and degradation of light-harvesting
phycobilisomes. In both bacteria and vascular plants, many genes that are regulated by
high-intensity light can be specifically controlled by blue/UV-A light; this is
a response that is also mediated by NblS. While acclimation of cyanobacteria to
both excess excitation energy and limiting nutrient levels requires NblS, this
regulator governs these processes through distinct signaling pathways that can
be differentially stimulated. The deduced polypeptide sequence of NblS revealed
the presence of a PAS domain, which has the potential to bind a redox carrier
such as a flavin. The association of NblS with a pigmented electron carrier could
allow for direct monitoring of both the light environment and the intracellular
redox status.
One
gene family controlled by NblS encodes the Hli polypeptides, which were first
shown to accumulate in cells during exposure to high light. The Hli proteins
show similarity to the light-harvesting polypeptides (Lhc) of vascular plants
and green algae; however, they have only a single transmembrane domain (Lhc
have three). There are four genes present on the cyanobacterial genome of Synechocystis PCC6803 encoding Hli polypeptides. Recently, Dr.
Qingfang He demonstrated that the levels of the specific Hli polypeptides
increase in response to high light, low temperature, and nutrient limitation.[2]
The kinetics of change in the steady-state levels of each of the Hli
polypeptides is specific, suggesting that the different members in the gene
family have distinct roles in the acclimation processes. To help determine the
function of the Hli polypeptides, the genes encoding these polypeptides were
inactivated both singly and in various combinations. Many strains defective in
specific Hli polypeptides could not cope with high light as effectively as
wild-type cells. Furthermore, a mutant in all four of the hli genes was high-light sensitive and seemed unable
to tolerate specific reactive oxygen species. The Hli polypeptides are the
evolutionary precursors of the Lhc polypeptides of vascular plants. They appear
to allow cyanobacteria to cope with high-light conditions and specifically the
production of reactive oxygen species. This suggests that the evolution of
systems to cope with reactive oxygen species evolved prior to light-harvesting
function. This is supported by the phenotype of an Arabidopsis mutant of lacking PsbS,
another evolutionary precursor of Lhc. PsbS is critical for dissipating excess
absorbed light energy as heat.[3]
While
NblS and Hli polypeptides are essential for the survival of cyanobacteria in
high light, other polypeptides help cyanobacteria acclimate to low-light
conditions. Such proteins include
those that function in the directional movement of cyanobacteria toward
low-intensity light. The unicellular cyanobacterium Synechocystis PCC6803 requires type IV pili in order to move
toward the light; pili are proteinaceous projections that cover the surface of
cyanobacterial cells. Dr. Devaki Bhaya demonstrated that there are different
pilus morphotypes with specific functions and that there are many proteins
required for the biogenesis and function of a pilus.[4]
We have also discovered a number of regulatory elements including a specific
photoreceptor that links the light cue to motor operation. The photoreceptor is
a hybrid protein that has one domain that is characteristic of a
chromophore-binding domain from vascular plant phytochromes and a second domain
that resembles the sensory transduction domain associated with chemotaxis
proteins of bacteria.
Interestingly, a mutant that is null for this photoreceptor still
responds to a unidirectional light source, but instead of moving toward the
light, the mutants move backward. This suggests that at least one additional
photoreceptor must be important for directional motility of cyanobacteria with
respect to light.
Other
alga that are highly responsive to light are the diatoms. Members of this
ubiquitous algal class are most noted for their highly patterned silica shells,
or frustules, and the amazing stability of their mitotic apparatus. We are
interested in many aspects of diatom biology and are working with Dr. Kirk Apt
at Martek Biosciences to tailor diatoms for efficient and profitable use in the
production of specific oils. Toward this end, we have thoroughly characterized
several genes from the diatom Phaeodactylum tricornutum that encode fucoxanthin-chlorophyll binding
light-harvesting polypeptides (FCPs).[5]
The promoters and terminators of these genes were fused to marker genes such as
sh ble, which encodes a protein
that confers Zeocin resistance to the alga, and reporter genes such as cat. These constructs were successfully used to
generate diatom transformation vectors. These vectors are being used to analyze
biological processes in diatoms and more specifically, to determine mechanisms
by which polypeptides are transported into plastids. However, they are also
becoming instrumental in engineering diatoms for increased production of
commercially valuable metabolites such as the C20:5 fatty acids. Transformation
technology has recently been employed to engineer a trophic conversion of P.
tricornutum from an obligate
photoautotroph to an organism that can grow heterotrophically in the dark. This
has allowed researchers at Martek to grow the organism in chemostats and obtain
cultures that are 20 times denser than cultures grown under photoautotrophic
conditions. This is not only the first time that this type of conversion has
been achieved using a photosynthetic eukaryote, but will also allow for the
more effective use of diatoms as commercial vehicles for the production of
valuable metabolites.
The
Grossman researchers are also taking a broader approach to probing gene
regulation with respect to light and nutrient conditions using high-density DNA
microarrays. They have already begun to define sets of genes that are
controlled by specific photoreceptors in cyanobacteria…but this story is
reserved for a future report.
[1] van Waasbergen, L., N. Dolganov, and A. R. Grossman, Environmental control of the photosynthetic apparatus depends on a PAS domain-bearing sensor protein, Proc. Natl. Acad. Sci., USA, submitted, 2000.
[2] He, Q. N., Dolhanov, and A. R. Grossman,
Expressions of the hli genes
and their role in high-light acclimation. J. Biol. Chem., in press, 2000.
[3] Li, Xiao-Ping, O. Björkman, C. Shih, A. R.
Grossman, M. Rosenquist, S. Jansson, and K. Niyogi, A chlorophyll-binding
protein necessary for energy dissipation but not photosynthetic light
harvesting, Nature 403,
391-395, 2000.
[4] Bhaya, D., D. Bryant, N. R. Bianco and A. R.
Grossman, Type IV pilus biogenesis and motility I the cyanobacterium Synechocystis
sp. Strain PCC6803, Molecular
Microbiology 37, 941-951, 2000.
[5] Zaslavskaia, L., J. C. Lippmeier, C. Shih, A. R.
Grossman and K. E. Apt, Trophic conversion of an obligate photoautotrophic alga
through metabolic engineering. in preparation, 2000.