Biochemistry and molecular biology of binding proteins for plant growth regulators.
Plant growth regulators have a vital role in plant growth and development. The
cellular response to these regulators depends on the presence and the action of specific
receptors. The plant growth regulators and their receptors act together in complexes
which determine the final effects of the plant growth regulators. In the research
reported here, emphasis has been given to the regulation of the activity of the receptors
themselves. The regulation of the N-l-naphthylphthalamic acid (N~A) receptor
through phosphorylation and dephosphorylation and the regulation of the auxin
binding protein (ABP) through gene manipulation have been investigated.
NPA, an auxin transport inhibitor, was found to bind specifically to a crude membrane
preparation from sugar beet seedling leaf cell suspension cultures. The in vitro
binding was optimal at pH 4.5 and 4°C. Binding parameters for NP A binding were
determined by Scatchard analysis. The dissociation constant (Kd) and binding protein
concentration were found to be 1.71 x 10-7 mol dm-3 and 220 pmoles g-I membrane
protein respectively. It was found that the amount of specific 3H-NPA binding was
significantly increased by adding Mg2+ A TP to the binding assay solution; treatment of
membrane preparations with acid phosphatase, prior to the NP A binding assay,
resulted in lower specific binding. A TP activation and phosphatase inactivation were
culture stage dependent. Although a considerable effect could be detected when using
cells from day 8 (representing the linear phase), the same treatment did not alter the
binding if cells from day I (representing lag phase) or day 14 (representing the
stationary phase) were used. These observations have strongly highlighted the
possible involvement of a phosphorylation and dephosphorylation mechanism in vivo
in the regulation of the activity of the NP A receptor. High phosphatase activity was
found in the supernatant, but not in the membrane pellet, after 50,000 g centrifugation.
The presence of a membrane-bound auxin receptor, ABP, was demonstrated by
Scatchard analysis in sugar beet seedlings. The Kd value and the receptor
concentration were found to be 2.15 x 10-6 mol dm-3 and 68 pmoles g-I membrane
protein. The protein could be solubilised either with the detergent Triton X-I 00 or by
acetone-washing, with a recovery of about 40%. An acetone-solubilised ABP
preparation could be partially purified by DEAE-Sephacel ion exchange
chromatography, NAA-linked AH-Sepharose 4B affmity chromatography or Sephadex
G-200 gel filtration. The recovery after any of these chromatographic treatments was
very low so that successive chromatography for further purification was unsuccessful.
The low level of detectable binding after purification resulted mainly from the low
abundance of ABP in the plant material. Non-radioactive labelling and detection
techniques were used to show that an ABP-probe hybridized to sugar beet genomic
DNA during dot blotting.
The present study has indicated that receptor activity could be regulated by a
phosphorylation and dephosphorylation mechanism in plants. The investigation has
also suggested that the effect of plant growth regulators on plant development could
be regulated through the manipulation of the expression of their receptor genes.