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Project
IV. Signal transduction pathways and early responses to gravistimulation
in plants (Boss,
Allen,
Muday,
Davies,
Whetten,
and Tucker).
This research effort is one of five integrated projects that in their
totality comprise the NSCORT in Gravitational Biology. Project IV addresses
cellular and biochemical studies of signal transduction pathways in gravity
stimulated plants. We have used three model systems to study the signaling
pathways: 1) maize pulvini; 2) Arabidopsis roots; and 3) Physcomytrella
patens caulonema. The group has published the following new insights within
the past year:
1)
Maize and oat pulvini:
A) Rapid changes in pH are evident only in the amyloplast-containing bundle
sheath cells of the maize pulvinus and not in the parenchyma cells. There
was a decrease in the pH of the cytoplasm near the sides of the cells
gravi-stimulated pulvinus where changes in amyloplast orientation were
observed. In contrast, the pH of the cytoplasm at the base of the cell
where the amyloplast accumulated became more basic (Johannes et al. 2001).
B) Rapid changes occur in selective mRNA recruited to polysomes isolated
from the upper and lower sides of maize pulvini within minutes of gravi-stimulation
(described in Project III; Heilmann et al., 2001).
C) A differential increase in auxin occurs on the lower side of the pulvinus
after the commitment to differential growth, but prior to growth and the
increase in invertase (Long et al., 2002).
D) The actin cytoskeleton is present in root columella cells putting to
rest the controversy as to whether the gravi-sensing cells of the root
contain a functional cytoskeletal network. (Collings et al., 2001).
2)
Arabidopsis:
A) Reversible protein phosphorylation can regulate auxin transport. A
genetic and pharmacological approach was used to show that if protein
phosphatase 2A activity was reduced, basipetal auxin transport was increased
and there were defects in differential cell elongation. Reducing basipetal
auxin transport with the inhibitor, NPA, restored the normal gravitropic
response. Studies of gravitropism and lateral root growth indicated differential
regulation of acropetal and basipetal auxin transport (Rashotte et al
2000 and 2001; reviewed in Muday and DeLong, 2001; Muday 2001).
B) Flavonoids can function as negative regulators of auxin transport in
vivo. Mutants with reduced flavonoid biosynthesis had increased auxin
transport and the additional of flavonoids slowed down auxin transport.
(Peer et al., 2001; Brown et al., 2000).
C) Comparison of IAA and IBA transport. The transport of the two endogenous
auxins, indole 3-acetic acid (IAA) and indole 3- butyric acid (IBA) were
compared in several tissues of Arabidopsis in order to understand the
similarities and differences in the movement of these two auxins. Both
auxins are important in gravitropic bending, as mutations that reduce
the response to either auxin impair root gravitropic bending. IBA moves
in roots in similar directions and with similar rates, but uses different
transporters that are not sensitive to inhibition by IAA efflux inhibitors
(Rashotte et al. 2002)
D) Examination of gravity response in calreticulin transgenics. We are
currently analyzing the gravity response in plants that constitutively
overproduce the calcium binding domain (C-domain) of calreticulin. These
plants have a defect in root gravitropism when grown under limiting calcium
and this defect is not observed under conditions where calcium is abundant.
E.) A genetic approach was used to alter InsP3 levels in plants. To test
the hypothesis that a differential long term increase in InsP3 was necessary
for gravitropism, cells and plants were transformed with the human gene
encoding InsP 5-ptase which when expressed should metabolize InsP3 rapidly.
Biochemical characterization of the transformed cells grown in culture
has been published (Perera et al., 2002). We are currently characterizing
the transgenic Arabidopsis plants.
3)
Physcomytrella patens:
A) UV-A light induces calcium waves in Physcomytrella patens. In vivo
analysis of cytosolic calcium indicated that blue/UV-A light used for
the ratiometric measurements induced calcium waves and thus precluded
measurements of changes in calcium in response to gravity (Tucker, submitted).
B) Cryptochrome mutants of Physcomytrella patens can be used to distinguish
the role of calcium in blue and UV-A light responses. Light-insensitive
mutants have been obtained and are being used to study the role of calcium
in gravi-sensing (Tucker et al., in preparation).
C) Cytokinins affect the direction of the gravitropic response as well
as branching. Knockout mutants with selective deficiencies in auxin and
cytokinin biosynthesis as well as calcium-binding proteins are being selected
to delineated these aspects of the gravitropic response (Tucker et al.,
in preparation).