FIGURE 23.

5 A phototropic fluence-response curve for

Avena coleoptiles. First positive, first negative, and second positive curvatures are indicated.
There is a fundamental law of photochemistry,
called the Bunsen-Roscoe reciprocity law, which says
the product of a photochemical reaction is determined
by the total amount of energy presented, regardless of
fluence rate or presentation time. In other words, the
same result is obtained with either a brief exposure
to a high fluence rate or a longer exposure to a low
fluence rate. Numerous experiments have established
that the reciprocity law applies to first positive curvature but does not apply to first negative or second
positive curvatures. Second positive curvature is instead
very time-dependent. In other words, second positive
curvature is more dependent on presentation time than
on fluence rate. Failure of reciprocity for second positive curvature suggests the possibility that more than
one photoreceptor might be involved. However, action
spectra have been determined for both first and second
positive curvature and they are identical. Apparently
both first and second positive curvature are mediated by
the same photoreceptor and the complexities of second
positive curvature are due to subsequent events in the
signal transduction chain.
23.1.5 THE PHOTOTROPIC RESPONSE
IS ATTRIBUTED TO A LATERAL
REDISTRIBUTION OF
DIFFUSIBLE AUXIN
At the same time F. W. Went and his contemporaries
had chosen to study the influence of the apex on coleoptile elongation, parallel studies on the role of the root
apex were being conducted in Germany by N. Cholodny.
The result was independent proposals by Cholodny, in
1924, and Went, in 1926, that the apex was able to
influence cell elongation in the more basipetal extension
region of the organ. These ideas of these 2 investigators
were drawn together in the late 1920s in an attempt to
explain phototropism. The Cholodny-Went hypothesis states that unilateral illumination induces a lateral
redistribution of endogenous auxin near the apex of the
organ. This asymmetry in auxin distribution is maintained as the auxin is transported longitudinally toward
the base of the organ. The higher concentration of auxin
on the shaded side of the organ stimulates those cells
to elongate more than those on the lighted side. It is
this differential growth that causes curvature toward the
light source.
The experimental basis for the Cholodny-Went
hypothesis is derived largely from agar-diffusion experiments originally conducted by Went and described
earlier in Chapter18. In Went’s experiments, oat coleoptiles were first stimulated with unilateral light. The
coleoptile apices were then excised, split longitudinally,
and the two halves placed on agar blocks in order to collect the auxin that diffused out of the base. The amount
of auxin collected in the agar blocks was then assayed by
the Avena curvature test (Chapter 18). Went reported
that a significantly higher quantity of auxin was collected
from the shaded half of the coleoptile apex than from the
lighted half, indicating that unilateral lighting caused a
greater proportion of the auxin to be transported down
the shaded side of the coleoptile.
Doubts as to the validity of the Cholodny-Went
hypothesis arose from numerous unsuccessful attempts
to verify asymmetric auxin distribution by applying
14C-labeled IAA to tropically stimulated coleoptiles.
These problems, however, may be largely attributed to
poor experimental technique. It is now evident that a
large proportion of the radioactive auxin taken up by
the tissue in those experiments did not enter the auxin
transport stream. When care is taken to discount this
nondiffusible auxin, a clear differential in auxin transport
can be detected. For example, when maize coleoptile tips
were supplied with 14C-IAA, approximately 65 percent
of the radioactivity was recovered from the shaded side.
There was no significant asymmetry when subapical
sections were used, further evidence that the lateral
redistribution of auxin occurs at the very apex of the
coleoptile.
In the 1960s, the Cholodny-Went hypothesis was
systematically reevaluated by W. R. Briggs and his colleagues. Briggs repeated Went’s original split-tip
experiments but, unlike Went, he excised the tips and placed
them on agar blocks before presenting the phototropic
stimulus. The results (Figure 23.6) clearly demonstrate
that when the tip is partially split, leaving tissue continuity only at the very apex of the coleoptile, exposure
to unilateral light causes an increase in the amount of
diffusible auxin on the shaded side and a decrease on
the lighted side. The total amount of auxin recovered,
however, remains effectively constant. When lateral
diffusion of auxin is prevented throughout the entire
396 Chapter 23 / Tropisms and Nastic Movements: Orienting Plants in Space
DARK CONTROL
Intact tip
ILLUMINATED
Intact tip
ILLUMINATED
Partially split tip
ILLUMINATED
Totally split tip
A.
B.
C.
D.
25.6

3
1
0
6.
.
2
7

2
2
2
3.
.
0
3

25.8
h
h
h
FIGURE 23.6 Phototropic stimulation establishes an
asymmetric distribution of diffusible auxin in excised Zea
mays coleoptile apices. (A, B) Intact control apices. A was
maintained in darkness and B was provided light unilaterally from the right. (C) Tips were partially split, leaving
tissue continuity only at the very apex. A microscope
cover slip was inserted to provide a barrier to lateral
diffusion. The tips were then presented with unilateral
light from the right. (D) Tips were totally split and the
diffusion barrier passed through the apex before being
presented with unilateral light from the right. Numbers
indicate the amount of auxin collected in the agar blocks
over a 3-hour period, based on degrees of curvature in
the Avena curvature bioassay. Values are for auxin collected from 3 tips (A, B) or 6 half-tips (C, D). (Data from
Briggs, W. R. 1963. Plant Physiology 38:237.)
length of the tip, no such asymmetric auxin distribution
is observed. These results clearly support the principal tenet of the Cholodny-Went hypothesis, namely,
that unilateral light induces a preferential migration of
auxin down the shaded side of the coleoptile. Their
experiments also confirmed that auxin production in
coleoptiles of Zea mays is confined to the apical 1 to 2 mm
and that lateral redistribution during phototropic stimulation probably occurs within the most apical one-half
mm.
Compelling support for the Cholodny-Went theory has been provided by a more recent study of auxin
redistribution in Brassica oleraceae hypocotyls. The free
IAA concentration found on the shaded side of the
hypocotyl was found to be at least 20 percent higher
than on the lighted side following phototropic stimulation. Moreover, the differential auxin concentration was
accompanied by a several-fold increase in the expression of auxin-regulated genes on the shaded side of the
hypocotyls, including two members of the α-expansin
family of genes that are necessary for cell wall extension
(see Chapter 17). Finally, both the auxin differential and
the differential in auxin-regulated gene expression could
be detected before there was any noticeable curvature of
the hypocotyls.
23.1.6 PHOTOTROPISM AND RELATED
RESPONSES ARE REGULATED BY
A FAMILY OF BLUE-SENSITIVE
FLAVOPROTEINS
Two lines of study led to the discovery of the photoreceptor for phototropism, now called phototropin. In
the late 1980s, it was reported that blue light stimulated
the phosphorylation of a 120 kDa plasma membrane
protein localized in the actively growing regions of etiolated pea seedlings. This is the same region that is most
responsive to the phototropic stimulus. After extensive
biochemical and physiological characterization, the protein was found to be a kinase that autophosphorylates in
blue light. There was also a strong suggestion that this
kinase was the photoreceptor for phototropism.
A short time later, a mutant characterized by a
failure to respond to the phototropic stimulus (nonphototropic hypocotyl 1, or nph1) was isolated from
Arabidopsis. Plants carrying the nph1 mutant not only
failed to exhibit phototropism, but coincidentally lacked
the 120 kDa membrane protein. When the NPH1 gene
was cloned, it was found, as expected, to encode the
120 kDa protein. The NPH1 holoprotein was subsequently renamed phototropin 1 (phot1) because of its
functional role in phototropism.
Phototropin 1 is a flavoprotein with two flavin
mononucleotide (FMN) chromophores (Figure 23.7)