Certain Malvaceae Plants Have a Unique Accumulation of myo-Inositol 1,2,4,5,6-Pentakisphosphate

Abstract

Methods used to quantify inositol phosphates in seeds lack the sensitivity and specificity necessary to accurately detect the lower concentrations of these compounds contained in the leaves of many plants. In order to measure inositol hexakisphosphate (InsP₆) and inositol pentakisphosphate (InsP₅) levels in leaves of different plants, a method was developed to concentrate and pre-purify these compounds prior to analysis. Inositol phosphates were extracted from leaves with diluted HCl and concentrated on small anion exchange columns. Reversed-phase solid phase extraction cartridges were used to remove compounds that give peaks that sometimes interfere during HPLC. The method permitted the determination of InsP₆ and InsP₅ concentrations in leaves as low as 10 µM and 2 µM, respectively. Most plants analyzed contained a high ratio of InsP₆ to InsP₅. In contrast, certain members of the Malvaceae family, such as cotton (Gossypium) and some hibiscus (Hibiscus) species, had a preponderance of InsP₅. Radiolabeling of cotton seedlings also showed increased amounts of InsP₅ relative to InsP₆. Why some Malvaceae species exhibit a reversal of the typical ratios of these inositol phosphates is an intriguing question for future research.

Keywords: Malvaceae; cotton; hexakisphosphate; inositol phosphates; pentakisphosphate; phosphate; phytate; phytic acid.

Figure 1

Isocratic and gradient HPLC of inositol hexakisphosphate (InsP₆) standard and InsP₆ hydrolysate. Chromatographic conditions are described in the Experimental Section. Isocratic HPLC of InsP₆ standard (A); isocratic HPLC of InsP₆ hydrolysate (B); and gradient HPLC of InsP₆ hydrolysate (C); calibration curve to correct for recovery of InsP₆ from Ag1X8 anion exchange columns (D).

Figure 2

Isocratic and gradient HPLC of corn (Zea mays) leaves before and after solid phase extraction. Isocratic HPLC of concentrated corn leaf extract (A); isocratic HPLC of concentrated extract that did not bind to the solid phase extraction (SPE) cartridge (B); isocratic HPLC of concentrated extract that bound to the SPE cartridge and was eluted with 50% methanol (C); gradient HPLC of concentrated extract (D); and gradient HPLC of concentrated extract that bound to the SPE cartridge and was eluted with 50% methanol (E).

Figure 3

HPLC of cotton (Gossypium hirsutum) leaves. Isocratic HPLC of concentrated cotton leaf extract before (A) and after (B) passage through the SPE cartridge. Gradient HPLC of cotton leaf extract (C).

Figure 4

Overview of the analytical procedure for InsP₅ and InsP₆ in plant tissues.

Figure 5

HPLC of plant tissue labeled for four days with [2-³H(N)]myo-inositol. (A). Radiolabeled Arabidopsis wild-type seedlings were extracted, separated using anion exchange and quantified via an in-line radioisotope detector. InsP₅ elutes at Minutes 40–42 and InsP₆ at Minutes 46–48. This chromatogram is representative of seven independent seedling labeling experiments; (B). Extracts were prepared and fractionated by HPLC from radiolabeled G. hirsutum seedling shoot (red trace), root (blue) and young leaf from pre-flowering-stage plant (black). Root and shoot data were repeated two times (C). Scintillation counting of eluted fractions is shown in A and B. (Top) Arabidopsis seedling; (middle) G. hirsutum shoot (red), root (blue); (bottom) young leaf from pre-flowering-stage G. hirsutum.