Vol. 16—1962 - NorthEastern Weed Science Society

30
One approach would appear to be by the use of'labeled auxins. satya
we now have techniques available for homogenizing,tnd differentially centrifuging
out the intact organolles of.a cell" theoretJ:cally at least we could
feed a labeled auxin to a plant eeJ.l and after spjlDning out. eacib. component
of the cell we could count each .fraction to see 'Nha'e theradioactivityi'S'
localized. I ,shall return to /fame experiments on this subject later. This
technique is tricky in that the homogenization procedure may" in fact" cause
the removal of labeled auxin frama particular organelle or, conversely" may
artifactually cause its adsorpt1onon to an organelle to which it is not
normally attached. '
Perhaps a better technique would be histochemic4l autoradiography.,
This technique has been of great utility for the m~clil&1" cytologist,S±noe
it has permitted him to localize nucleic acids in the cell with great precision.
Unfortunately the plant.growth hormone is present in the cell in,
such low concentrations that this technique cannot ~e.employed. In 'factI
do not know of anY work along these lines which hasi1yielded useful data4,:or
the plant physiologist.
I would like now to describe some experiments performed several years
ago in collaboration with Dr. Ravingar Kaur and later with Drs. Satishand
Nirmala Maheshwari. We decided to 'at!:llck the probJ,em of the .localization.a!
auxin action in the cell by feeding C 4 carboxyl-labelled 2,,4-D to th61iJ:OWing
cells of the pea plant and then atter hamoget4zat.ion and, differential
centrifugation of the cell, counting each of the f,ractione. Since both ,
the fractionation scheme and the basic results have. alreacl,y been pUblishecl."
I will summarize them only briefly here. After the ,first homogenization
we found that the cell walls and unbroken tissue fr~ents, which deposited
at lowest speeds, contained a, si¢ficant number of counts. These however.,
were readily removed by regrinding ,and rewashing.' We concluded that, in
reality, the wall did not contain f:$.rmly bound 2,4-i:l. Other particulate ,
fractions, such as the chloroplasts, mitochondria and ~crosomcs containe~
smaller activity, which was very readily removed by gentlo washing and
recentrifugation. In fact, the results of many such experiments convinced
us that the great bulk of the labeled 2,4-D fed to tbe plant cell does not
attach to any particulate fraction but remainsint~final centrifugal
supernatant'fluid after homogeni~ation and centr1~uistion. This means
that it is either in the vacuole cr' dn the clear Ils'\;ructureless ll fraction,'
of the cytoplasm. ' '
In order to see whether the 2,4-D 10calized1n.this way might poss1l:>ly
be attached to a macromolecule, such as protein, weatt~pted both dialysi,
experiments and coagulation of proteins. The dialysis experiments told us
that,the labelled 2,4-Dpassed readily out of the bag, ~lthough not SO
rapidly as similar material not 'in contact With pro~~in. All in all, ,the l
data indicated no firm binding ot aUXin to protein..~he precipit.:ation
experilnents, also, showed some small activity onthe,;coagulatedproteins."
but whether this occurs in vivo or in vitro is hard to tell. ,What intore"ted
us most was the finding that auxin'markedlY depresse,r:i t~e heat coagula'billty
of the proteins in this phase. In fact in certaih~eriments the coagulability
is so altered that there is no protein precipitate at all in the
experimental (auxin treated) series, but/the control (auxin free) series
4~