ISPMB Update for Stomataphiles

kearns at MIT.EDU kearns at MIT.EDU
Tue Jul 5 19:05:36 EST 1994

Hi Everyone,

Here's the update you've been waiting for. There were about 3000 people at
the ISPMB meeting and over 2000 posters, so I may have missed something, but 
hopefully Thomas Altmann or Nick Provart can fill in any gaps I leave.

Let's start with Nick's work. He presented a poster describing a lambda zap II
epidermal cDNA library from Solanum tuberosum leaves. This library is 50 to
100 fold enriched for guard cells compared to whole leaf. Guard cells are
approximately 1.5% of the total leaf. Sucrose transporter was highly expressed
as was malate dehydrogenase and starch biosynthesis enzymes. Out of 48 clones
picked randomly, there were potato virus ESTs, but no G-proteins. In addition
to this useful library, Nick also has a guard cell specific promoter from the
guard cell potassium channel, KST 1.

Also from the Willmitzer lab, a poster was presented describing the cloning of
KST 1. KAT1, a potassium channel clone derived from yeast via complementation,
was used as a probe on the epidermal library above. A clone with 81% similarity
to KAT1 was obtained and named KST 1. KST 1 in Xenopus looks like a K+ channel.
It appears to be in stomata in in situ hybridizations.

And again from the Willmitzer lab, Thomas Altmann gave a nice talk in which
he described mutants derived from his AC/DS system. At the end of this talk he
mentioned that he had gotten ems mutants with 4 fold more densely spaced 
stomata and with abnormal stomata. They were visualized by GUS staining plants
which were carrying GUS behind a stomata specific promoter and then mutagenized
with ems. I suspect that the promoter was KST 1. Am I correct? This was a 
clever screen for stomatal pattern mutants.

G-proteins! Hong Ma has two beta subunits. One is from maize tassel and named
ZGB1. The other is from Arabidopsis and named AGB1. They have 76% predicted
similarity to one another and 41% to mammalian beta subunits. They are 
expressed in several organs: root, leaf, and flower.

Cho from Korea found an alpha subunit of a G-protein in rice. From a southern,
it looks like two genes. 

Jones and Hooley showed that gibberellic acid turns on two RNAs, 1.2 and 1.7 kb
which hybridize to a 700bp PCRed probe of a G-protein alpha subunit in Avena.

Calcium! Kendrick of Japan presented a poster describing Ca2+, heptanedioic
acid, hexadiecanoic acid, p-cumaric, ferulic, and cinnamic acids in the 
cotyledon bodies of Pharbitis nil. Are cotyledon bodies the same ae glyoxisomes
? Is this another complication to the calcium stores story? 

Meulen and Wang showed that Rab 16 induction by 5microM ABA is blocked by Ca2+
channel blockers, 0.5mM Cd2+ and La3+ in barley aleurone.

COS cells and you! Kammerloher described an interesting system for finding 
plasma membrane proteins. This could work with the epidermal library, too.
In a talk on the research, Schaffner cited Brian Seed's work at Mass General 
Hospital. Essentially, one introduces cDNAs into COS cells and they replicate
the plasmids to high copy number and fold hydrophobic membrane proteins. Using
this protocol, they were able to find plasma membrane intrinsic proteins which
may be H20 channels. At the end of this talk, Jullian Schroeder mentioned that
gammaTIP is tonoplast specific H2O channel from Arabidopsis. Wow!

Jullian Schroeder gave an interesting talk about tonoplast channels and 
attempts to find a cascade similar to the channel cascade in the plasma 
membrane. Ca2+ comes into the cell or is released from internal stores in 
response to ABA. Some day this will all make sense, but what is pretty clear
about ABA is that K+ comes out of the vacuole. I assume that it is still true
that K+ enters the guard cells through plasma membrane channels, too, in 
response to ABA, and that the tonoplast cascade is happening at the same time
as the plasma membrane cascade, like a double waterfall. Anyway, 5microM Ca2+
in the cytosol turns on a highly selective K+ channel in the tonoplast. This
channel is much more selective than the plasma membrane channel and is of
high abundance. This was done using a whole vacuole patch technique. 5 microM
Ca2+ activated the K+ channel which brought the membrane potential to 0mV, 
activating an ion channel selective for Ca2+ with Keq=25mV, not selective for
K+. There we have it, a positive feedback cascade in the vacuole! This was
done in red beet. K+ channels were also found in patched root hair cells and
may be used in nutrition. Thermodynamics with pH v. mV imply that one H+
is exchanged for one K+, as expected. These K+ channels are expressed in the 
cortex of the root. There is a small gene family with possibily one member
boardering the vascular tissue. 

ATPase! Using reverse PCR with degenerate primers on a template of Vicia faba
guard cell protoplast mRNA, Larry Smart obtained two ATPase genes, VHA1 and
VHA2. From Southern analysis, there may be as many as ten genes in the ATPase
family. During in situ hybridization, VHA1 lights up the whole guard cell, but
so does the control transcript, actin. VHA2 lights up splotches on the guard 
cell, which could be chloroplasts to my eye, but again since the actin 
control is heavily expressed in the guard cell, I agree with Larry's 
assessment that these are not guard cell specific clones. On northerns, they 
are detected in all tissues: leaf, shoot, root, and flower.

I learned of many more things which are also not stomatal specific, so I will
not relate them here. However, I do want to thank the organizers of this
year's ISPMB meeting for a wonderful time and to point out that Amsterdam is
a really friendly and fun city to visit. Oh, and, of course, as of this
message, Holland is still in the World Cup!

Oranje! Ellen Kearns 

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