photoautotrophic growth of PSI- mutants

Kevin Redding Kevin.Redding at molbio.unige.ch
Tue Aug 13 09:20:56 EST 1996

This is in response to Keli Sato's message of Aug 1.  I wanted to post
something before we get fixed into textbooks the idea that plants can
grow photoautotrophically without PSI.  Although I think that the authors
of the recent Science Paper (Lee, Tevault, Owens, and Greenbaum; Science
273:364-367) have shown something fairly miraculous, I must say that it
is not at all clear whether PSI is really missing in the mutants that
they examined.  Elias Greenbaum and James Lee (of Oak Ridge) very
generously sent me samples of their photosynthetically-grown cultures of
B4 and F8 before publication of their article.  However, using an
antibody specific to the PsaA protein, I found significant levels of PsaA
protein in both.  (In the case of B4, a trans-splicing mutant of the psaA
mRNA, this should not be the case.)  It seems fairly clear that, at least
in the case of B4, some sort of reversion or suppression occurred along
the way.  Lee and Greenbaum have assured me that the strains had not
reverted before or during the time in which the published experiments
were performed, so they still stand.  However, this demonstrates the
potential problems in using nuclear mutants of as-yet unknown function
that result in lowered/no accumulation of the PSI complex.  I'm not yet
convinced that these mutants were not from the beginning "leaky" in that
they allow a small amount of PSI to accumulate.  The question seems to
be: are small amounts enough to explain the phototrophic growth?
Greenbaum and his colleagues would say no, that such small amounts (they
estimate an upper limit of 3-5% PSI in the mutants they examined) could
not explain the photoautotrophic growth or O2 evolution using the Z

The problem I have with this work is that I cannot repeat it with my
PSI-deficient mutants.  I have made defined deletions of the psaA and
psaB genes, which encode the major subunits of PSI.  As these deletions
remove more than 90% of the coding sequences, there is no way that they
can either be leaky or revert.  Under the same condtions in which F8 and
B4 grow phototrophically, my mutants do not.  I know that they do not
have secondary mutations that render them nonphotosynthetic, as they
instantly recover the ability to photosynthesize after reintroduction of
the wild-type gene.  In fact, even point mutants that accumulate very
small amounts of PSI (due to lowered assembly and/or stability) can allow
them to grow phototrophically as long as the light is not too strong
(PSI- mutants are very light-sensitive).  One hypothesis to explain this
is that there is something lacking in the strain background in which I
constructed these mutants, and this missing component is required for
"PSII-only" photosynthesis.  This would explain why some of the
PSI-deficient mutants from the Chlamy stock center are able to grow
photosynthetically and others are not.  Thus, there might be
heterogeneity in the "wild-type" Chlamy background.  Another hypothesis
is that growth/no growth is determined by presence/absence of small
amounts of PSI -- those that accumulate small amounts of PSI can continue
to grow under some conditions, while those that accumulate none cannot.
We are currently testing these hypotheses by a variety of experiments.  A
key one is to delete the genes for PsaA and/or PsaB in the same strains
that have been used by Lee et al.  The first hypothesis predicts that
this would make no difference, whereas the second predicts that this
would cause them to lose the ability to grow photosynthetically.  It
should be noted that even if the first hypothesis is disproved, this
would not necessarily mean that "PSII-only photosynthesis" does not
exist, as it could be that PSI is required at least in small amounts for
phototrophic growth, although not as the main conduit of electron flow
from PSII in certain cases.  In addition, I have sent my deletion mutants
to Oak Ridge to see if they perform similarly in terms of O2 and H2
evolution under the same conditions.  I think we should be able to
resolve these issues fairly rapidly.  So, the message I want to get
across is: WAIT! and keep an open mind.  We haven't heard the last of

Respectfully submitted,

Kevin Redding

Dept. Molecular Biology
University of Geneva
30 Quai Ernest-Ansermet
CH1211 Geneve
EMail: Kevin.Redding at molbio.unige.ch> CHLAMYDOMONAS/bionet.chlamydomonas
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Elizabeth Harris
chlamy at acpub.duke.edu

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