Comparison of developmental gradients for growth, ATPase, and fusicoccin-binding activity in mung bean hypocotyls.

Abstract

A comparison has been made of the developmental gradients along a mung bean (Vigna radiata L.) hypocotyl of the growth rate, plasma membrane ATPase, and fusicoccin-binding protein (FCBP) activity to determine whether they are interrelated. The hook and four sequential 7.5 millimeter segments of the hypocotyl below the hook were cut. A plasma membrane-enriched fraction was isolated from each section by aqueous two-phase partitioning and assayed for vanadate-sensitive ATPase and FCBP activity. Each gradient had a distinctive and different pattem. Endogenous growth rate was maximal in the second section and much lower in the others. Vanadate-sensitive ATPase activity was maximal in the third section, but remained high in the older sections. Amounts of ATPase protein, shown by specific antibody binding, did not correlate with the amount of vanadate-sensitive ATPase activity in the three youngest sections. FCBP activity was almost absent in the first section, then increased to a maximum in the oldest sections. These data show that the growth rate is not determined by the ATPase activity, and that there are no fixed ratios between the ATPase and FCBP.As cells develop, they undergo changes in physiological potential and in their spectrum of proteins. The challenge is to relate changes in proteins to changes in physiological potential. Cells in a hypocotyl go through a period of elongation prior to maturation (7). This elongation is due, at least in part, to auxin-induced wall acidification, mediated by the PM2 ATPase (1 1). The first question addressed here is whether the peak in elongation in mung bean hypocotyls is paralleled by a peak of PM ATPase activity. Because hypocotyl development progresses from apex to base, with the youngest, meristematic cells contained in the hook, the growth rate and PM ATPase activity were compared in successive sections along the hypocotyl, starting with the hook region.Proton excretion via the PM ATPase is greatly enhanced in most higher plant cells by the fungal toxin FC (19,24 does not bind to the catalytic peptide of the ATPase, but to a separate FCBP (1,5,6,20). It has been suggested that the FCBP is a constitutive regulatory subunit of the ATPase (5, 10); if so, one would expect a constant ratio between ATPase and FCBP activities. The second question addressed here is the quantitative relationship between these two activities along the mung bean hypocotyl. Mung beans ( Vigna radiata L.) were selected because of the large body of information about other developmental gradients along its hypocotyl (7), and the relative ease of obtaining PM using aqueous twophase partitioning (21, 31). MATERIALS AND METHODS Plant MaterialMung bean (Vigna radiata L. cv Berken) seeds were obtained from Burpee Seed Co. Seeds were soaked with aeration overnight, then dark-grown at 25°C on soaked and drained coarse vermiculite. Three hundred milliliters ofa 1 mm CaSO4 solution were added to the vermiculite to prevent dampingoff symptoms. Hypocotyls were harvested after 5 d, when their lengths wer...

Isolation of plasma membranes from mung bean hypocotyl sections by two-phase partitioning: suitability of the technique for ageing tissues

“…Also, for mung beans, the absence of (or reduction in) K+-stimulation has been reported several times (Yoshidaetal. 1986, Kimuraet al 1988, Mitoetal, 1988, Basel and Cleland 1992, Tbis was confirmed by our own results (Tab, 1). The plasma membrane (U,) ATPase activity did not increase significantly in tbe presence of K*.…”

“…The addition of vanadate and calcium to the assay medium caused a 35 to 40% inhibition of Mg-*-dependent ATPase activity. The VO^-inhibition was relatively small compared to inhibition rates for mung bean plasma membrane ATPases reported by others (Yoshida et al 1986, Basel andCleland 1992). Although vanadate inhibition was expected to be enhanced by the addition of K* to the medium (Widell and Larsson 1990), in the present experiments the level of inhibition was not drastically affected: K+ induced a supplementary reduction of only 5 to 10% (data not shown).…”

A fusicoccin binding protein belongs to the family of 14-3-3 brain protein homologs.

“…(1) all organs and developmental stages of the plant express the FCBP (Meyer et al, 1993); (2) expression of the FCBP is under developmental control (Base1 and Cleland, 1992;Meyer et al, 1993); and (3) the structure and biochemical properties of the FCBP are highly conserved in different plant species Weiler et al, 1990;Oecking and Weiler, 1991).…”

“…Binding activity in the plasma membrane is cell-type specific (Meyer et al, 1993) To whom correspondence should be addressed. and is controlled by the developmental stage of the tissue (Basel and Cleland, 1992). Modulation of the receptor affinity for FC by binding of its own ligand (Basel et al, 1994) further accentuates the role of the FCBP in the physiology of plants.…”

Fusicoccin affects cortical microtubule orientation in the isolated epidermis of sunflower hypocotyls

“…In situ , there is a gradient of elongation rate along the peel, i.e., elongation of portion I is fastest, while that of portion III is slowest (data not shown), but no significant differences in the rate of fusicoccin‐induced strain along peels were observed. Basel & Cleland (1992) found that the rate of fusicoccin‐induced growth in segments cut from mung bean hypocotyls does not correlate with endogenous growth rate of intact hypocotyls, although some gradients of growth rate exist in both cases. They concluded that endogenous growth rate is not determined by the activity of plasma membrane H + ‐ATPases.…”