Myo-inositol hexakisphosphate, isolated from female gametophyte tissue of loblolly pine, inhibits growth of early-stage somatic embryos

Myo-inositol hexakisphosphate, isolated from femalegametophyte tissue of loblolly pine, inhibits growth ofearly-stage somatic embryos

Di Wu, M. Cameron Sullards, Charlie D. Oldham, Les Gelbaum, Jacob Lucrezi, Gerald S. Pullman and

Sheldon W. May

School of Chemistry and Biochemistry, Petit Institute for Bioengineering and Biosciences, School of Biology, and Institute of Paper Science and Technology, Georgia Institute of Technology,

Atlanta, Georgia 30332, USA

Author for correspondence:Sheldon W. MayTel: +1 404 894 4052

Email: [email protected]

Received: 16 May 2011

Accepted: 4 September 2011

New Phytologist (2012) 193: 313–326doi: 10.1111/j.1469-8137.2011.03928.x

Key words: cellular proliferation, loblollypine, myo-inositol hexakisphosphate, Pinus

taeda, somatic embryogenesis.

Summary

• Myo-inositol hexakisphosphate (InsP6), abundant in animals and plants, is well known for

its anticancer activity. However, many aspects of InsP6 function in plants remain undefined.

We now report the first evidence that InsP6 can inhibit cellular proliferation in plants under

growth conditions where phosphorus is not limited.

• A highly anionic molecule inhibitory to early-stage somatic embryo growth of loblolly pine

(LP) was purified chromatographically from late-stage LP female gametophytes (FGs), and

then characterized structurally using mass spectrometry (MS) and nuclear magnetic resonance

(NMR) analyses.

• Exact mass and mass spectrometry-mass spectrometry (MS-MS) fragmentation identified

the bioactive molecule as an inositol hexakisphosphate. It was then identified as the myo-

isomer (i.e. InsP6) on the basis of 1H-, 31P- and 13C-NMR, 1H-1H correlation spectroscopy

(COSY), 1H-31P heteronuclear single quantum correlation (HSQC) and 1H-13C HSQC. Topical

application of InsP6 to early-stage somatic embryos indeed inhibits embryonic growth.

• Recently evidence has begun to emerge that InsP6 may also play a regulatory role in plant

cells. We anticipate that our findings will help to stimulate additional investigations aimed at

elucidating the roles of inositol phosphates in cellular growth and development in plants.

Introduction

Myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP6), also calledphytic acid, is myo-inositol with phosphate groups attached toeach of its six carbons. myo-Inositol is a member of the B vita-mins, that play a central role in growth and development (Abelet al., 2001). InsP6 is ubiquitous in animal and plant cells; it isthe major form of phosphorus in seeds and also accumulates inother plant tissues and organs, including pollen, roots, tubers andturions (Cosgrove, 1980; Sasakawa et al., 1995; Raboy, 1997;Loewus & Murthy, 2000; Abel et al., 2001). It can representfrom 1% to several per cent of a typical seed’s DW, c. 75 ± 10%of a seed’s total phosphorus, and normally > 90% of a matureseed’s total, acid-extractable inositol phosphates (Raboy, 1997).In mammalian cells, total cellular InsP6 concentration rangesfrom 10 to 100 lM, depending on cell type and developmentalstage (Szwergold et al., 1987; Sasakawa et al., 1995; Shears,2001).

Myo-inositol-1,2,3,4,5,6-hexakisphosphate was historicallyconsidered as an antinutrient in that it can decrease the bioavail-ability of essential dietary minerals and proteins in monogastricanimals (chickens, swine, humans) as a result of its ability to bind

them (Urbano et al., 2000; Palacios et al., 2007). Monogastricanimals are unable to digest InsP6; their excreted InsP6 is trans-ferred to surface waters via rain, drainage, surface runoff andwind erosion. Excreted phosphorus contributes to cyanobacterialblooms, hypoxia, and death of aquatic animals (Brinch-Pedersenet al., 2002; Turner et al., 2002; Vats et al., 2005).

In the late 1980s, InsP6 was shown to possess striking anti-cancer action. InsP6 inhibited the growth of all tested cell lines ina dose- and time-dependent manner (Shamsuddin & Vucenik,2005). Reduction of the elevated rate of cell proliferation to normalvalues has been observed both in intact animals (Wattenberg,1995; Challa et al., 1997; Gupta et al., 2003) and in cultures ofhuman malignant cells (Shamsuddin et al., 1992; Sakamotoet al., 1993; Shamsuddin & Yang, 1995; Ferry et al., 2002;Singh et al., 2003). In terms of plants, InsP6 has been investi-gated for its effect on plant growth as a source of phosphorusunder conditions where phosphorus is limited (Hayes et al.,2000; Richardson et al., 2007). However, reduction of prolifera-tion in plant cells by InsP6, under conditions where phosphorusis not limited, has not been reported to date.

Loblolly pine (LP, Pinus taeda L.), a species of the pine family,constitutes the primary commercial species in the southern forests

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of the US covering 13.4 million ha. Somatic embryogenesis (SE)is a vegetative propagation system currently used to reproducewhole LP and other plants from suspension cultures (Jain et al.,1995; Pullman & Johnson, 2002; Pullman et al., 2003a). Well-controlled SE cannot only maintain the desirable genetic compo-sition of the progeny, but can also improve the efficiency ofpropagation (Xu et al., 1997; Cairney et al., 2000). Unlikeangiosperm embryos with attached cotyledons as seed storageorgans, the diploid conifer embryo is surrounded by the unat-tached haploid female gametophyte (FG). This FG tissue isabsent in LP embryogenic tissue culture.

In previous work, we have shown that extracts from early-stageFGs stimulate growth and multiplication of early-stage somaticembryos, whereas water extracts from late-stage FGs containsubstance(s) inhibitory to early-stage somatic embryo growth(De Silva et al., 2008). We now report the identification of theinhibitory substance as InsP6 on the basis of liquid chromato-graphy-mass spectrometry (LC-MS), liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS), exactmass, and one- and two-dimensional 1H, 31P, and 13C NMRanalyses. Our findings constitute the first evidence that InsP6

inhibits cell proliferation in plants under growth conditionswhere phosphorus is not limited. Many aspects of the function ofInsP6 in plants have remained undefined (Turner et al., 2002;Raboy, 2003). We therefore anticipate that our results willhelp to stimulate additional investigations into the roles playedby inositol phosphates in cellular growth and development inplants.

Materials and Methods

Chemicals

Tris base, deuterium oxide (min 99.96 atom% D), Dowex resin50WX8-200 and phytic acid dodecasodium salt hydrate fromrice were obtained from Sigma (St. Louis, MO, USA). Phyticacid water solution (50% w ⁄ w) was obtained from Chromadex(Chromadex, Irvine, CA, USA). High-performance liquid chro-matography-grade acetonitrile, sodium chloride, sodium phos-phate monobasic and dibasic, ammonium acetate, and formicacid were purchased from Fisher Scientific (Pittsburgh, PA,USA). Muco-inositol hexakisphosphate (muco-InsP6) was gener-ously gifted by Dr Alan Richardson from the collection of the lateDr Dennis Cosgrove. There is some uncertainty as to the bariumstoichiometry in this muco-InsP6 material, and because of the verylimited quantity available to us we were unable to carry out anyexperiments to resolve this uncertainty. The range of muco-InsP6

concentrations tested was such that, even if the barium stoichiom-etry in the sample was as low as zero or as high as six, the testedconcentrations spanned the range over which InsP6 itself exhibitsinhibitory activity.

Female gametophyte collection and water extraction

Loblolly pine cones were collected weekly throughout thesequence of embryo development from multiple open-pollinated

mother trees over 10 yr. Seeds from Union Camp tree UC5-1036, located in a seed orchard near Bellville, GA, were collectedduring the years 1996–1998. Seeds from grafts of tree S4PT6were collected in Louisiana in 1997 and 1998 and from Texas in2002, 2003 and 2005. Cones were shipped on ice and receivedwithin 24–48 h of collection and processed within 2 wk ofreceipt. Cones were pried open, seeds were isolated, and FGswere collected and stored under liquid nitrogen. FG collectionand water extraction were done using procedures described previ-ously (Pullman & Buchanan, 2006; De Silva et al., 2008).Extracts were obtained from late-stage FGs (Stages 9.10–9.12).The resulting water extract solution was filtered with a 0.2 lmsyringe filter (Pall, East Hills, NY, USA), and lyophilized over-night to dryness. In some experiments, FGs were obtained fromfull-term dry seeds from tree S4PT6 of year 2006; no differencesin fractionation or bioactivity profiles were observed between FGextracts from these seeds and those from Stage 9.10–9.12 seeds.

Early-stage somatic embryogenic multiplication bioassay

A previously described staging system (Pullman & Webb, 1994)was used to evaluate morphological development in zygotic andsomatic embryos. Somatic embryos at stage 2 were isolated byforceps from suspension culture and placed on 2 ml of multipli-cation medium 1250 contained in 24-well plates. Forty replicatebioassays were then carried out, for each fraction or control treat-ment tested, using the following protocol. Each FG extract frac-tion, corresponding to 20 FGs in our general bioassay protocol,was ultrafiltered, lyophilized, dissolved in 2 ml of deionizedwater, adjusted to pH 5.7, sterilized with a 0.2 lm syringe filter,and topically applied to the stage 2 somatic embryos at 50 ll perwell. Embryos were grown in the dark at 23–25�C and after 4–7 wk the diameter of the embryogenic tissue was measured witha dissecting microscope using a calibrated eyepiece reticle. Typi-cally a single embryo, c. 1 mm in size, grows into a mass of multi-ple embryos c. 5–9 mm in diameter depending on culturegenotype, medium contents, and time. All the data were evalu-ated by multifactor analysis of variance. The significantdifferences between means of each treatment were determined bythe multiple range test at 95% level of significance. Both analyseswere performed using Statgraphics Plus Version 4.0 (Manugistics,Rockville, MD, USA). This bioassay system was used to studysomatic embryogenesis in our earlier research (Pullman & Johnson,2002; Pullman et al., 2003c, 2006; De Silva et al., 2008).

Liquid chromatography isolation

All chromatographic steps described here were carried out at4�C. All collected fractions described here from a given columnwere pooled based on the absorbance monitored at 215, 254 and280 nm. A Superdex 75 10 ⁄ 300 GL gel filtration column(10 · 300 mm, GE Healthcare, Piscataway, NJ, USA) was pre-equilibrated with 150 mM NaCl for 118 min at 0.5 ml min)1.Lyophilized powder corresponding to the water extract of 30 FGswas dissolved in 250 ll of 150 mM NaCl, centrifuged at16 000 g for 10 min, and the supernatant was applied to this

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column and eluted at a flow rate of 0.5 ml min)1 using 150 mMNaCl. Fractions collected in multiple tubes (0.5 ml per tube)were pooled with the following designations: F1, from 15.0 to27.0 min (6.0 ml); F2, from 27.0 to 34.0 min (3.5 ml); F3,from 34.0 to 55.0 min (10.5 ml); and F4, from 59.0 to77.0 min (9.0 ml). All fractions were ultrafiltered (MilliporeYM1 membrane, 1000 Da cutoff) and the retentates lyophilized.Fractions were bioassayed as described earlier.

A Mono S strong cation exchange column (5 · 50 mm, GEHealthcare) was used for the second chromatography step. Bio-active fraction ‘F2’ (i.e. the fraction eluting from the previousSuperdex 75 column from 27.0 to 34.0 min) was dissolved in300 ll of 20 mM sodium phosphate buffer, pH 2.5 (eluent A),applied to the Mono S column, and eluted at 1.5 ml min)1. Thebinary gradient elution was monitored spectrophotometrically.Eluent B was eluent A containing 2 M NaCl. The gradientstarted at 0% B for 9 min, 0– 50% B over 27 min, 50–100% Bfor 2 min, and 100% B for 18 min. Fractions collected in multi-ple tubes (1.0 ml per tube) were pooled with the designations:F2-S1, from 0.7 to 2.0 min (2.0 ml); F2-S2, from 2.0 to 3.3 min(2.0 ml); F2-S3, from 13.3 to 21.3 min (12.0 ml); F2-S4, from21.3 to 27.3 min (9.0 ml); and F2-S5, from 37.5 to 41.5 min(6.0 ml). All fractions were ultrafiltered (YM1) and the retentateslyophilized. Fractions were bioassayed as described earlier.

The third chromatography was achieved on a Mini Q stronganion exchange column (4.6 · 50 mm, GE Healthcare). Thebioactive fraction ‘F2-S1’ (i.e. the fraction eluting from 0.7 to2.0 min on the previous Mono S column) was dissolved in300 ll of 20 mM Tris-Cl buffer at pH 8.0 (eluent A) and loadedon the pre-equilibrated Mini Q column. Eluent B was eluent Acontaining 2 M NaCl. The flow rate was 0.5 ml min)1, and theelution profile was monitored spectrophotometrically. A binarygradient was applied: 0% B for 23 min, 0–30% B over 58 min,30–100% B for 4 min, and 100% B for 4 min. Fractions col-lected in multiple tubes (0.5 ml per tube) were pooled with thefollowing designations: F2-S1-Q1, from 2.1 to 6.1 min (2.0 ml);F2-S1-Q2, from 6.1 to 11.1 min (2.5 ml); F2-S1-Q3, from 25.4to 30.4 min (2.5 ml); F2-S1-Q4, from 30.4 to 36.4 min(3.0 ml); F2-S1-Q5, from 36.3 to 43.3 min (3.5 ml); and F2-S1-Q6, from 49.4 to 58.3 min (4.5 ml). Each fraction wasultrafiltered (Millipore YM3 membrane, 3000 Da cutoff) andthe retentates were bioassayed as described earlier.

Final purification was accomplished on a Superdex Peptide10 ⁄ 300 GL gel filtration column (10 · 300 mm, GE Health-care) pre-equilibrated with 100 mM ammonium acetate buffer,pH 5.5, at 0.5 ml min)1 for 138 min before sample loading.The lyophilized (without ultrafiltration) bioactive fraction ‘F2-S1-Q6’ (i.e. the fraction that had eluted from 49.4 to 58.3 minon the previous Mini Q column) was dissolved in 150 ll of thesame buffer, applied to the column, and eluted at a flow rate of0.2 ml min)1 for 158 min. The elution was monitored spectro-photometrically. Fractions collected in multiple tubes (0.2 mlper tube) were pooled with the following designations: F2-S1-Q6-D1, from 62.1 to 80.1 min (3.6 ml); F2-S1-Q6-D2, from80.1 to 90.1 min (2.0 ml); F2-S1-Q6-D3, from 90.1 to95.1 min (1.0 ml); F2-S1-Q6-D4, from 95.1 to 98.1 min

(0.6 ml); and F2-S1-Q6-D5, from 98.1 to 100.1 min (0.4 ml).All fractions were lyophilized and bioassayed as described earlier.The bioactive fraction ‘F2-S1-Q6-D2’ (i.e. the fraction elutingfrom 80.1 to 90.1 min) was further characterized by LC-MS andNMR. It should be noted that in our hands InsP6 behaves indialysis at low ionic strength as if it had a higher molecular massthan predicted from its formula. Similar observations havebeen reported by others (Hanakahi et al., 2000; Irigoin et al.,2002), and this phenomenon has been studied in detail byVan der Kaay & Van Haastert (1995).

LC-MS and LC-MS-MS characterization under positivepolarity

The mass spectrometer was an Applied Biosystems ⁄ MDS SCIEX4000 QTRAP (Linear Ion Trap Quadrupole) LC-MS-MS sys-tem (Applied Biosystems, Foster City, CA, USA). The electro-spray ionization (ESI) interface was operated in positive modeat 5500 V. A Vydac C8 capillary column (5 l, 300 A,0.3 · 250 mm, Deerfield, IL, USA) was coupled to a ShimadzuLC-10ADVP. Full-scan mass spectra were recorded from m ⁄ z500 to 2000 amu at 1000 amu s)1 by the linear ion trap as anenhanced mass spectrometric (EMS) scan, with declusteringpotential at 90 V. The total flow rate was 0.6 ml min)1, whichwas split to 5 ll min)1 to the column. Mobile phase A consistedof 2% acetonitrile and 98% water, and mobile phase B was 80%acetonitrile and 20% water, both containing 0.1% formic acid asthe ion pair reagent. The elution protocol was a 1 min columnpre-equilibration with 100% A, followed by a 10.0 ll sampleinjection, a 5 min sample load and washing with 100% A. Thegradient was 105 min in total: 0% B for 12 min, 0–40% B over63 min, 40–100% B for 5 min, 100% B for 8 min, back to 0%B for 5 min, and re-equilibration for 12 min.

Liquid chromatography-mass spectrometry-mass spectrometryspectra of the precursor ion at m ⁄ z 661 (obtained from F2-S1-Q6 ultrafiltered against an YM3 3000 Da cutoff membrane)under positive polarity were acquired subsequent to the LC-MSon the QTRAP. Enhanced product ion (EPI) scans were per-formed subsequent to the EMS scan at a scanning rate of1000 amu s)1 over the mass range m ⁄ z 50–700 with collisionenergies at both 40 and 80 eV. Data acquisition and instrumentcontrol were performed by Analyst software (version 1.4.2).

Exact mass and MS-MS analyses under negative polarity

Exact mass analysis under negative polarity was obtained fromthe final active fraction from the Superdex Peptide column (F2-S1-Q6-D2).

Exact mass measurement under negative ion polarity was con-ducted on an Applied Biosystems ⁄ MDS SCIEX QSTAR� XL(hybrid quadrupole time-of-flight mass spectrometer, TOF-MS)LC-MS system, with F2-S1-Q6-D2 and phytic acid standarddirectly infused with an automated chip-based nanoelectrosprayTriVersa� NanoMate system (Advion, Ithaca, NY, USA). TheNanoMate was controlled by the ChipSoft software (version7.1.1). The ESI chip used was D-chip (4.1 lm ID). A low

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delivery pressure of 0.15 psi of nitrogen gas and a voltage of)1.6 kV were applied to generate a nanoelectrospray plumefrom a given nozzle when infusing the sample of interest.TOF-MS spectra were recorded over the mass range of m ⁄ z 300–2000. The declustering potential was held at )150 V and thefocusing potential at )200 V. Data acquisition and QSTARinstrument control were performed by Analyst QS software(version 1.1).

Tandem mass spectra of the precursor ion at m ⁄ z 659 undernegative polarity were acquired subsequent to the exact massmeasurement on the QSTAR XL interfaced with the NanoMatesystem. The spectra were recorded from m ⁄ z 50–700 with colli-sion energies at both 40 and 80 eV.

InsP6 standard preparation for NMR characterization

Phytic acid dodecasodium salt hydrate from rice (300.2 mg) wasconverted to InsP6 using the cation exchange resin Dowex50WX8-200 hydrogen form. The eluate was collected, dividedinto 10 aliquots and lyophilized to dryness. Each aliquot of thelyophilized product was redissolved in 500 ll of deuterium oxide(min. 99.96 atom% D) and the pH adjusted to 8.22 by additionof concentrated NaOD.

One-dimensional NMR characterization

Nuclear magnetic resonance spectra of the final purified activefraction (F2-S1-Q6-D2) were recorded in D2O at 500.13 MHzfor 1H with 32 scans, 202.46 MHz for 31P with 64 scans and125.77 MHz for 13C with 16 386 scans using a multinucleiprobe maintained at 280 K on a Bruker DRX 500 spectrometer.F2-S1-Q6-D2 was dissolved in 500 ll of D2O (min. 99.96atom% D) and lyophilized to dryness, repeated three times. Thefinal lyophilized product was redissolved in 200 ll D2O (min.99.96 atom% D) and transferred to a symmetrical 5 mm NMRtube (Shigemi, Allison Park, PA, USA) matched with D2O (insert4.1 · 190 mm; outer tube 4.52 mm ID, 4.965 mm OD · 180mm). A 5-mm-OD NMR tube (Wilmad, Buena, NJ, USA)

designed for 500 MHz NMR was used for the InsP6 standardunder the same conditions as those of F2-S1-Q6-D2, except forfewer scans. 1H, 31P, and 13C chemical shifts were reported inppm and assignments for both F2-S1-Q6-D2 and the InsP6 stan-dard were made with reference to the 4.67 ppm water peak. Cou-pling constants were determined using the MestRe-C softwarepackage and spectra were analyzed by MestReNova software(Mestrelab Research, Santiago de Compostela, Spain):• F2-S1-Q6-D2: 1H NMR analysis d 4.59 (d, 1, H2), 4.09 (q,2, H4 ⁄ 6), 3.82 (q, 1, H5), 3.76 (t, 2, H1 ⁄ 3). 31P NMR analysis d2.54 (1, P5), 2.29 (2, P1 ⁄ 3), 2.06 (1, P2), 1.63 (2, P4 ⁄ 6). 13CNMR analysis d 77.40 (1, C5), 75.89 (2, C4 ⁄ 6), 74.51 (1, C2),73.39 (2, C1 ⁄ 3).• InsP6 standard: 1H NMR analysis d 4.56 (d, 1, H2), 4.08 (q,2, H4 ⁄ 6), 3.79 (q, 1, H5), 3.74 (t, 2, H1 ⁄ 3). 31P NMR analysis d2.72 (1, P5), 2.54 (2, P1 ⁄ 3), 2.30 (1, P2), 1.75 (2, P4 ⁄ 6). 13CNMR analysis d 77.47 (1, C5), 75.77 (2, C4 ⁄ 6), 74.72 (1, C2),73.38 (2, C1 ⁄ 3).

Two-dimensional NMR characterization

1H-31P heteronuclear single quantum correlation (HSQC)experiments were carried out using the standard Bruker programhsqcetgpsi2 modified for 31P gradient strength. The parameterswere 1.5 s recycle delay, 1024 data points in F2, 128 incrementsin F1, globally optimized alternating phase rectangular pulse(GARP) 31P decoupling during acquisition, and shifted sine-squared apodization before Fourier transformation.

1H-13C HSQC experiments were carried out using the stan-dard Bruker program hsqcetgpsi2. The parameters were 1.5 srecycle delay, 1024 data points in F2, 128 increments in F1,GARP 13C decoupling during acquisition, and shifted sine-squared apodization before Fourier transformation.

Concentration of InsP6 dodecasodium salt equivalent tofraction F2-S1-Q6-D2

The F2-S1-Q6-D2 fraction used for the NMR analysis had a pHof 8.22 and an array of sodium adduct ions were observed down-stream of the InsP6 peak in the LC-MS spectra. Eight of the 12exchangeable hydrogens in InsP6 have a pKa below 8.22 (Barreet al., 1954; Isbrandt & Oertel, 1980), and the resulting InsP6-8Na salt has a molecular weight of 836 Da. F2-S1-Q6-D2extracted from 100 FGs weighed 0.11 mg equivalent to26.3 nmol from 20 FGs. Therefore, 26.3 nmol of the commer-cial phytic acid dodecasodium salt hydrate in 2 ml of water wasused as the equivalent of fraction F2-S1-Q6-D2. In the bioassays,this was divided equally between 40 wells, and topically appliedto the stage 2 somatic embryos at 50 ll per well, with each wellalready containing 2 ml of 1250 multiplication medium. Thefinal concentration of 0.32 lM InsP6 in each well was calculatedbased on combined volume of the medium and the 50 ll topicalapplication. A stock solution (0.53 mM) was made and severalconcentrations ranging from 0.032 to 3.2 lM were generated bydilution. The bioassays were performed as described earlier in‘early-stage somatic embryogenic multiplication bioassay’.

Bioassays with muco-inositol hexakisphosphate

Somatic embryos at stage 2 were isolated by forceps from suspen-sion culture and placed on 2 ml of multiplication medium 1250contained in 24-well plates. Fifty replicate bioassays were thencarried out, for each concentration tested, using the followingprotocol. A stock solution of muco-InsP6 (1.8 mg ml)1) wasadjusted to pH 5.7 and sterilized with a 0.2 lm syringe filter. Aseries of dilutions were made such that, when topically applied tothe stage 2 somatic embryos at 50 ll per well, the final concen-tration of muco-InsP6 ranged from 6.7 to 670 lg ml)1. Embryoswere grown and measured and all data were analyzed as describedin ‘early-stage somatic embryogenic multiplication bioassay’.

Norway spruce materials and experimental design

Briefly, seeds from F.W. Schumacher Co., Sandwich, MA, USA,were rinsed under cold tap water for 30 min, soaked overnight,

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and surface-sterilized. Shortly after sterilization, seeds were dis-sected and the integuments and nucellus removed. The FG wascarefully split, the embryo removed, and the embryo and splitgametophyte placed next to each other or the embryo was placedalone on 7 ml of initiation medium 1165 contained in six-wellPetri plates. Sixteen replications of six seeds were tested per treat-ment with no FG present or with the FG placed next to theembryo and incubated at 23–24�C in 16 h of c. 30 lmolphotons m)2 s)1 of cool white fluorescent light.

Results

Liquid chromatography isolation and identification

We established a purification protocol for the inhibitor of early-stage somatic embryo growth comprising water extraction, aSuperdex 75 gel filtration column, a Mono S strong cationexchange column, two ultrafiltrations against 1000 Da cutoffmembranes after the first two columns, a Mini Q strong anionexchange column, and a Superdex Peptide gel filtration column.For the first-step fast protein liquid chromatography (FPLC) (theSuperdex 75 column), F2 was the only fraction that inhibitedembryo growth in a statistically significant manner comparedwith the blank control (Fig. 1). The second fractionation wasachieved on a Mono S column. F2-S1 eluting during the wash ofunbound compounds was the only fraction that showed statisti-cally significant inhibition to the embryo growth (Fig. 2), inexactly the same manner as that of the F2 control. Nonretentionon a strong cation exchanger suggested that the inhibitor is highlyanionic. Thus, Mini Q was selected for the third-step chromatog-raphy isolation. Bioassay results revealed that F2-S1-Q6 inhibited

embryo growth in a statistically significant manner (Fig. 3).Therefore, F2-S1-Q6 was further purified on a Superdex Peptidecolumn. Fig. 4 shows the bioassay results of the Superdex Peptidecolumn. It is evident that F2-S1-Q6-D2 is the only subfractionthat exhibits statistically significant inhibitory activity of thebioassay.

Structure characterization by LC-MS and LC-MS-MS underpositive polarity

Liquid chromatography-mass spectrometry was performed oneach successive inhibitory fraction to investigate components, aswell as to identify the inhibitor to be pursued. One distinct ion atm ⁄ z 661 was detected in all active fractions, including F2, F2-S1,F2-S1-Q6 (ultrafiltered against 3000 Da cutoff membrane),F2-S1-Q6-D2, and in F2-S1-Q5 (36.3–43.3 min) which wassignificantly more dilute and less active than F2-S1-Q6. Inactivefractions from both the Mini Q and the Superdex Peptide col-umns did not reveal the presence of this ion in their mass spectra.These results indicate that the ion at m ⁄ z 661 corresponds to theinhibitor to be pursued. LC-MS of F2-S1-Q6-D2 revealed thatonly the ion at m ⁄ z 661 was dominantly present, with an array ofsodium adduct ions with mass difference of 22 amu (the massdifference between sodium Na+ and proton H+) being observeddownstream of the ion at m ⁄ z 661. Additional LC-MS analysesof all fractions from the Mini Q column under negative polaritywere acquired (data not shown). The m ⁄ z difference of 2 amubetween the ion at m ⁄ z 661 from the positive scan and the ion atm ⁄ z 659 from the negative scan indicate that both ions are singlycharged and the neutral monoisotopic mass of this molecule is660 Da.

Fig. 1 Superdex 75 fast protein liquid chromatography elution profile for the fractionation of female gametophyte (FG) water extract eluted with 150 mMNaCl. Elution was monitored at 215, 254 and 280 nm, and the absorbance in milliabsorbance units (mAU) at 280 nm is shown on the chromatogram. Frac-tions collected in multiple tubes (0.5 ml per tube) were pooled and bioassayed with the following designations: F1, from 15.0 to 27.0 min (6.0 ml); F2,from 27.0 to 34.0 min (3.5 ml); F3, from 34.0 to 55.0 min (10.5 ml); and F4, from 59.0 to 77.0 min (9.0 ml). The inset shows the bioassay results (usinggenotype 500) for the fractions eluted from the Superdex 75 column. Forty replicate bioassays were carried out for each fraction and also for blank controlsthat contained only the bioassay components. All the data were evaluated by multifactor ANOVA. The significant differences between means of eachtreatment were determined by the multiple range test at 95% level of significance. F2 was the only fraction inhibiting the embryo growth in a statisticallysignificant manner compared with the blank control. The chromatography and bioassays were repeated at least three times with similar results. LSD, leastsignificant difference.






Fig. 2 Mono S fast protein liquid chromatography elution profile for the fractionation of the bioactive fraction ‘F2’ (from Fig. 1) eluted with 20 mM sodiumphosphate buffer at pH 2.5. Elution was monitored at 215, 254 and 280 nm, and the absorbance in milliabsorbance units (mAU) at 215 nm is shown onthe chromatogram. Fractions collected in multiple tubes (1.0 ml per tube) were pooled and bioassayed with the following designations: F2-S1, from 0.7 to2.0 min (2.0 ml); F2-S2, from 2.0 to 3.3 min (2.0 ml); F2-S3, from 13.3 to 21.3 min (12.0 ml); F2-S4, from 21.3 to 27.3 min (9.0 ml); and F2-S5, from37.5 to 41.5 min (6.0 ml). The inset shows the bioassay results (using genotype 500) for the fractions eluted from the Mono S column and the F2 controlfrom the previous Superdex 75 column. Forty replicate bioassays were carried out for each fraction and also the blank control that contained only the bioas-say components. All the data were evaluated by multifactor ANOVA. The significant differences between means of each treatment were determined bythe multiple range test at 95% level of significance. F2-S1 was the only fraction that showed statistically significant inhibition to the embryo growth, inexactly the same manner as that of the F2 control, compared with the blank control. The chromatography and bioassays were repeated at least three timeswith similar results. LSD, least significant difference.

Fig. 3 Mini Q fast protein liquid chromatography elution profile for the fractionation of the bioactive fraction ‘F2-S1’ (from Fig. 2) eluted with 20 mMTris-Cl buffer at pH 8.0. Elution was monitored at 215, 254 and 280 nm, and the absorbance in milliabsorbance units (mAU) at 215 nm is shown on thechromatogram. Fractions collected in multiple tubes (0.5 ml per tube) were pooled and bioassayed with the designations: F2-S1-Q1 from 2.1 to 6.1 min(2.0 ml), F2-S1-Q2 from 6.1 to 11.1 min (2.5 ml), F2-S1-Q3 from 25.4 to 30.4 min (2.5 ml), F2-S1-Q4 from 30.4 to 36.4 min (3.0 ml), F2-S1-Q5 from36.3 to 43.3 min (3.5 ml), and F2-S1-Q6 from 49.4 to 58.3 min (4.5 ml). The inset shows the bioassay results (using genotype 500) for the fractions elutedfrom the Mini Q column and the F2-S1 control from the previous Mono S column. Forty replicate bioassays were carried out for each fraction and also theblank control that contained only the bioassay components. Both F2-S1-Q5 and F2-S1-Q6 had statistically significant inhibition to the embryo growth, withF2-S1-Q6 being the most significant and selected for further purification. The chromatography and bioassays were repeated at least three times with similarresults. LSD, least significant difference.

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Liquid chromatography-mass spectrometry-mass spectrometryspectra of the precursor ion at m ⁄ z 661 under positive polaritywere then acquired on the QTRAP. The precursor ion (M+H)+

at m ⁄ z 661 was subjected to collision energies of 40 and 80 eV.At 40 eV, the protonated ion at m ⁄ z 661 yielded a series ofprominent ions at m ⁄ z 643, 625, 607, 581, 563, 545, 527, 483,465, 447, 429, 385, 367, 349, 287, 269, 259, 189, 179, 161,and 99. At 80 eV, further dissociation intensified the low m ⁄ zfragment ions, whereas the abundances of the high m ⁄ z fragmentions decreased tremendously. Ions higher than m ⁄ z 447 were notobserved.

One-dimensional NMR characterization

The 13C NMR spectra of F2-S1-Q6-D2 revealed four distinctpeaks at 77.40, 75.89, 74.51 and 73.39 ppm with an intensityratio of 1 : 2 : 1 : 2; this indicates the presence of six carbons withtwo independent pairs of equivalent carbons and two nonequiva-lent individual carbons. A search within the spectral database fororganic compounds (National Institute of Advanced IndustrialScience and Technology, 2007) based on 13C NMR spectra found

only one match as myo-inositol. Similar patterns were observed inboth 1H NMR (four distinct peaks at 4.59, 4.09, 3.82 and3.76 ppm with an intensity ratio of 1 : 2 : 1 : 2, Fig. 5a) and 31PNMR (four distinct peaks at 2.54, 2.29, 2.06 and 1.63 ppm withan intensity ratio of 1 : 2 : 1 : 2) spectra of F2-S1-Q6-D2.

The pH of F2-S1-Q6-D2 was measured at 8.22 and all itsspectra were recorded at this value. The InsP6 standard was pre-pared from its dodecasodium salt through cation exchange resinand the pH adjusted to the same value using NaOD, in order tocircumvent the generation of extra NaCl. The chemical structureof InsP6 is shown in Fig. 5(c). Both the chemical shifts and split-ting patterns of F2-S1-Q6-D2 are identical to those of the InsP6

standard (Fig. 5b). Taken together, these results are consistentwith the conclusion that the inhibitory molecule in F2-S1-Q6-D2 is InsP6. The 1H chemical shift assignments shown in Fig. 5were made according to Bauman et al. (1999).

Exact mass analysis and MS-MS under negative polarity

Exact mass measurements and subsequent tandem MS undernegative ion polarity of both F2-S1-Q6-D2 and the InsP6

Fig. 4 Superdex Peptide fast protein liquid chromatography elution profile for the fractionation of the bioactive fraction ‘F2-S1-Q6’ (from Fig. 3) elutedwith 100 mM ammonium acetate buffer at pH 5.5. Elution was monitored at 215, 254 and 280 nm, and the absorbance in milliabsorbance units (mAU) at215 nm is shown on the chromatogram. Fractions collected in multiple tubes (0.2 ml per tube) were pooled and bioassayed with the following designa-tions: F2-S1-Q6-D1, from 62.1 to 80.1 min (3.6 ml); F2-S1-Q6-D2, from 80.1 to 90.1 min (2.0 ml); F2-S1-Q6-D3, from 90.1 to 95.1 min (1.0 ml); F2-S1-Q6-D4, from 95.1 to 98.1 min (0.6 ml); and F2-S1-Q6-D5, from 98.1 to 100.1 min (0.4 ml). The inset shows the bioassay results (using genotype500) for the fractions eluted from the Superdex Peptide column, the blank control, the F2-S1 control from the Mono S column, and a buffer control labeledNH4Ac (6 ml of 100 mM ammonium acetate lyophilized to dryness, corresponding to the highest amount of ammonium acetate contained in any fraction).Forty replicate bioassays were carried out for each fraction and also the blank control that contained only the bioassay components. F2-S1-Q6-D2 is theonly subfraction that exhibits statistically significant inhibitory activity in the bioassay. The chromatography and bioassays were repeated at least three timeswith similar results. LSD, least significant difference.






standard were carried out on the QSTAR XL system interfacedwith an automated chip-based nanoelectrospray TriVersa�NanoMate system. As shown in the Fig. 6 (a) (inset), F2-S1-Q6-D2 exhibited exact mass (M-H)) of 658.8506 with an error ofnegative 5.3 ppm with respect to the calculated molecular massof 658.8541, and the InsP6 standard showed exact mass (M-H))

of 658. 8563 with an accuracy of 3.3 ppm, as shown in theFig. 6 (b) (inset).

Two collision energies of 40 and 80 eV were applied to theprecursor ion (M-H)) at m ⁄ z 659 from F2-S1-Q6-D2 and theInsP6 standard, respectively. MS-MS spectra of F2-S1-Q6-D2(Fig. 6a) and the InsP6 standard (Fig. 6b) under both collisionenergies were identical (MS-MS at 80 eV not shown). Further-more, MS-MS spectra acquired under negative polarity were con-sistent with those from positive mode, with each fragment ionlower by 2 amu.

Two-dimensional NMR characterization

Two-dimensional 1H-13C HSQC, 1H-31P HSQC and 1H-1HCOSY experiments were obtained for both F2-S1-Q6-D2and the InsP6 standard to assign the chemical shifts as well

as to compare the correlation patterns. The chemical shiftassignments and coupling constants are listed in Table 1 andthe corresponding 1H-13C and 1H-31P HSQC spectra areshown in Figs 7 and 8. All two-dimensional NMR of F2-S1-Q6-D2 showed identical correlation patterns to those obtainedfrom the InsP6 standard. Consistency in correlation patterns,chemical shifts and intensity ratios in all one-dimensional andtwo-dimensional NMR spectra unequivocally established thatthe inhibitory molecule isolated from FG tissue is myo-inositolhexakisphosphate.

Concentration dependence of bioactivity on InsP6

Bioassays were carried out to confirm that the InsP6 standardinhibits the early-stage somatic embryo growth. The histories ofthe genotypes used in these experiments are as follows: genotype500 was from a seed from mother tree 7–56 initiated in 2003;genotype 51 was from a seed from mother tree MWv-2 initiatedin 2005; genotype 222 was from a seed from a high-value crossinitiated in 2007; genotype 279 was from a seed from mothertree 7–56 initiated in 2006; and genotype 433 was from a seedfrom a high-value cross initiated in 2007.

Fig. 5 1H NMR spectra of F2-S1-Q6-D2 and myo-inositol hexakisphosphate (InsP6) standard. (a) 1H NMR spectrum of F2-S1-Q6-D2; (b) 1H NMRspectrum of the InsP6 standard, both recorded at pH 8.22. (c) The structure of InsP6 with the carbons numbered.

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Results for five genotypes (51, 222, 433, 279, 132) tested at noneand five concentrations of InsP6 are averaged and shown in Fig. 9.The InsP6 standard at five concentrations was found to inhibitsomatic embryo growth in a statistically significant manner. Fur-thermore, inhibition corresponding to the concentration of InsP6

actually isolated from female gametophytes (0.32 lM in the bioas-say well) was the most significant. An additional two genotypeswere tested at none and 0.32 lM. All seven genotypes testedshowed reduced growth in the bioassay with application of InsP6 at0.32 lM; differences were statistically significant at P = 0.05.

Muco-inositol hexakisphosphate

Bioassays were carried out to test whether muco-InsP6, a stereo-isomer of InsP6, also inhibits early-stage embryo growth. Results

for one genotype (652) tested at none and six concentrations ofmuco-InsP6 are shown in Table 2. It is evident from the data thatmuco-InsP6 at six concentrations did not inhibit somatic embryogrowth, whereas control experiments confirmed that, as expected,InsP6 itself did inhibit somatic embryo growth using this geno-type.

Norway spruce embryogenic tissue initiation

Several embryogenic tissue initiation protocols in conifers callfor full-term seed FG to be placed next to the embryo on theinitiation medium. With the finding that FG tissue containsInsP6 that is able to inhibit early-stage LP somatic embryogrowth, the question arose if FG tissue might inhibit initiationwhen full-term seed embryos are used for initiation. To test this

Fig. 6 Exact mass analysis and MS-MS under negative polarity of both F2-S1-Q6-D2 and InsP6 standard. (a) Tandem mass spectrum of F2-S1-Q6-D2 at40 eV and the exact mass (M-H)). (b) Tandem mass spectrum and exact mass for the InsP6 standard.






Table 1 Myo-inositol hexakisphosphate (InsP6) 1H-, 31P- and 13C-NMR chemical shift assignments and coupling constants

1Hchemical shift Proton position Integration Multiplicity J (Hz)

F2-S1-Q6-D2 4.59 2 1 d J2-P2 (8.80)4.09 4 ⁄ 6 2 q J4-3,5,P4 (9.05)

J6-1,5,P6 (9.05)3.82 5 1 q J5-4,6,P5 (9.05)3.76 1 ⁄ 3 2 t J1-6,P1 (8.30)

J3-4,P3 (8.30)

InsP6 Standard 4.56 2 1 d J2-P2 (9.05)4.08 4 ⁄ 6 2 q J4-3,5,P4 (9.54)

J6-1,5,P6 (9.54)3.79 5 1 q J5-4,6,P5 (9.54)3.74 1 ⁄ 3 2 t J1-6,P1 (8.80)

J3-4,P3 (8.80)

31PChemical Shift Phosphate Position Integration

F2-S1-Q6-D2 2.54 5 12.29 1 ⁄ 3 22.06 2 11.63 4 ⁄ 6 2

InsP6 Standard 2.72 5 12.54 1 ⁄ 3 22.30 2 11.75 4 ⁄ 6 2

13CChemical Shift Carbon Position Integration

F2-S1-Q6-D2 77.40 5 175.89 4 ⁄ 6 274.51 2 173.39 1 ⁄ 3 2

InsP6 Standard 77.47 5 175.77 4 ⁄ 6 274.72 2 173.38 1 ⁄ 3 2

Fig. 7 1H-13C heteronuclear single quantum correlation(HSQC). (a) 1H-13C HSQC of F2-S1-Q6-D2. (b) 1H-13CHSQC of the myo-inositol hexakisphosphate (InsP6) stan-dard. Carbon atoms are numbered as shown in Fig. 5(c).

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hypothesis, the procedure indicated in Pullman et al. (2003c)was used to initiate embryogenic tissue from mature Norwayspruce embryos with FG tissue present or absent. After 9–10 weeks, explants were evaluated for the presence of embryo-genic tissue (Table 3). Embryogenic tissue formation in Norwayspruce was increased from 23.8 to 56.1% (differences were sta-tistically significant) when the FG tissue was not present.

Discussion

We report here the first evidence of inhibition of plant somaticembryonic growth by InsP6. The highly anionic inhibitor was

purified from late-stage LP FG tissue by water extraction, two gelfiltrations and two ion exchange FPLC chromatographies, andthe final bioactive molecule was then fully characterized as tostructure and purity.

Liquid chromatography-mass spectrometry of the final activefraction indicated that the active molecule is homogeneousand has a neutral monoisotopic mass of 660 Da. The LC-MS-MS spectrum under positive mode of F2-S1-Q6 at a collisionenergy of 40 eV was identical to those obtained at 30 and35 eV by Hsu et al. (2003). A series of prominent ions atm ⁄ z 643, 625, 607, 581, 563, 545, 527, 483, 465, 447, 429,385, 367, 349, 287, 269, 259, 189, 179, 161, and 99 weregenerated by successive losses of H2O (18 amu), HPO3

(80 amu), or H3PO4 (98 amu) from the precursor ion at m ⁄ z661, with some fragment ions arising from multiple pathways.Identical exact mass and fragmentation patterns obtained fromhigh-resolution exact mass measurement and MS-MS analysisunder negative mode clearly identified the purified compound

Table 2 Effect of muco-inositol hexakisphosphate (muco-InsP6) on early-stage somatic embryo growth

muco-InsP6

concentration(lg ml)1)

Diameter(mm)

Calculatedmuco-InsP6

concentrationrange (lM)1

0 6.8 ± 0.3 06.7 5.7 ± 0.4 0.023–0.05241 6.4 ± 0.4 0.14–0.3250 6.4 ± 0.4 0.17–0.3967 6.5 ± 0.4 0.23–0.5292 6.3 ± 0.4 0.32–0.71670 6.0 ± 0.4 2.3–5.2

Diameter values are followed by their standard error. No treatment wasstatistically different from any other group, ANOVA P > 0.05.1Calculated muco-InsP6 molar concentration ranges for barium stoichio-metry from zero to six (see ‘Materials and Methods’ section).

Table 3 Effect of female gametophyte presence or absence on initiationof Norway spruce embryogenic tissue from full-term seeds

Treatment % initiation1

1165 + FG 28.3 a1165 No FG 56.1 b

FG, female gametophyte.1Sixteen replications of six explants were tested per treatment. Analysesfor initiation are based on arcsine transformation �(%). Values followed bythe same letter are not statistically different by the multiple range test atP = 0.05.

Fig. 8 1H-31P heteronuclear single quantum correlation(HSQC). (a) 1H-31P HSQC of F2-S1-Q6-D2; (b) 1H-31PHSQC of the myo-inositol hexakisphosphate (InsP6)standard. Carbon atoms are numbered as shown inFig. 5(c).

Fig. 9 Bioassay results averaged for five genotypes (51, 222,433, 279, 132) tested at different concentrations ofmyo-inositol hexakisphosphate (InsP6) standard. Means offive genotypes are shown along with 95% least significantdifference (LSD) intervals for each concentration of InsP6

tested.






from FG tissue as one of the isomers of inositol hexakisphos-phate.

The active molecule was then identified as the myo-isomer ofinositol hexakisphosphate on the basis of 1H-, 31P- and 13C-NMR, 1H-1H COSY, 1H-31P HSQC and 1H-13C HSQC. Inthe one-dimensional NMR spectra, the chemical shifts, splittingpatterns and intensity ratios were identical to those of the InsP6

standard, and a search within spectral database for organiccompounds based on 13C NMR spectra found only one matchas myo-inositol. The 13C-, 1H-, and 31P- NMR spectra allexhibited four distinct peaks with an intensity ratio of1 : 2 : 1 : 2, which is the pattern expected for the myo-isomer(Fig. 5c). Indeed, Angyal & Odier (1982) compared the 13CNMR spectra of a series of diastereomeric inositols, and onlythe myo- stereoisomer exhibited this 1 : 2 : 1 : 2 intensity ratio.All the two-dimensional NMR of the isolated moleculeshowed identical correlation patterns to those obtained from themyo-inositol hexakisphosphate standard. Taken together, theseresults unequivocally establish that the inhibitory moleculeisolated from FG tissue is myo-inositol hexakisphosphate.Furthermore, we find here that topical application of the muco-InsP6, a stereoisomer of InsP6, to early-stage somatic embryosdoes not inhibit embryonic growth in a statisticallysignificant manner, demonstrating the stereo specificity ofInsP6’s inhibition.

We demonstrate here that topical application of InsP6 to early-stage somatic embryos from several different genotypes varying ingenetic background indeed inhibits embryonic growth in a statis-tically significant manner. Further, we demonstrate a practicalapplication of our findings, in that presence of full-term seed FGtissue inhibits embryogenic tissue initiation and should beremoved to facilitate culture initiation. Medium 1250 contains9.33 mg l)1 Na2EDTA to buffer divalent metal ions. Therefore,we do not believe that the inhibitory activity of InsP6 is the resultof metal chelation. Further, when Pullman et al. (2003b) modi-fied a similar salt mixture for improvement of embryo develop-ment and maturation by comparing control and raised mediumphosphorous through addition of extra KH2PO4, somaticembryo yield differences were not statistically significant betweentreatments.

Myo-inositol hexakisphosphate is ubiquitous and the mostabundant inositol phosphate derivative in eukaryotic cells. It isknown for its anticancer activity in reducing the proliferation ofmalignant cells (Shamsuddin et al., 1992; Shamsuddin & Yang,1995; Ferry et al., 2002). Additionally, InsP6 increases differenti-ation of malignant cells leading to reversion to the normal pheno-type with decreased production of tumor markers (Shamsuddin& Vucenik, 2005). Some evidence has begun to emerge thatInsP6 may also function as a signaling molecule in plant cells.Lemtiri-Chlieh et al. (2000) reported that the plant stress hor-mone, abscisic acid, increases InsP6 in intact guard cells of Sola-num tuberosum and that InsP6 inhibits the inward rectifying K+

current of S. tuberosum and Vicia faba guard cell protoplasts in aCa2+-dependent manner. Subsequently (Lemtiri-Chlieh et al.,2003), they showed by laser uncaging of InsP6, in V. faba guardcell protoplasts loaded with calcium-sensitive dye, that InsP6

causes release of Ca2+ from internal stores. It should also be notedthat Tan et al. (2007) have recently reported that InsP6 is a cofac-tor in the transport inhibitor response 1 protein (TIR1) thatsenses and becomes activated by the phytohormone auxin. How-ever, reduction of proliferation in plant cells by InsP6 has notbeen reported to date, and many aspects of the function of InsP6

in plants have remained undefined (Turner et al., 2002; Raboy,2003). Our findings constitute the first report that InsP6 inhibitscell proliferation in plants.

Is it possible that inhibition of somatic embryo growth inplants by InsP6 and InsP6’s anticancer activity occur via similarmechanisms? In JB6 epidermal cells, it has been shown that InsP6

inhibits epidermal growth factor-induced phosphatidylinositol-3kinase (PtdIns 3-kinase), thereby impairing epidermal growthfactor- or phorbol ester-induced cell transformation and activatorprotein 1 activation (Huang et al., 1997). PtdIns 3-kinases arewidely distributed in eukaryotic cells, and they are involved in anumber of cellular processes, including activation of intracellularsignaling molecules such as rac, ras, rab, mitogen-activated pro-tein kinase, protein kinase B ⁄ Akt (Vanhaesebroeck & Waterfield,1999), protein kinase C and JNK ⁄ p38 kinase (Leevers et al.,1999; Meijer & Munnik, 2003; Amin et al., 2007). Turning toplants, PtdIns 3-kinase homologs have been cloned in soybean(Hong & Verma, 1994), Arabidopsis thaliana (Welters et al.,1994) and Brassica napus (Das et al., 2005), and expression ofantisense PtdIns 3-kinase AtVPS34 mRNA results in severeinhibition in growth and development of second-generationtransformed plants. Recently, both PtdIns 3-kinase and PtdIns 4-kinase activities have been observed during the induction ofsomatic embryogenesis in Coffea arabica (Ek-Ramos et al.,2003), and the products of both kinase activities were detected inthe somatic-embryo extracts. Moreover, growth of these somaticembryos was inhibited when a kinase inhibitor was included inthe induction medium during the first differentiated stage(Ek-Ramos et al., 2003). Taken together, these facts are notinconsistent with the notion that inhibition of PtdIns kinase maybe a common feature of InsP6’s activity as an inhibitor of somaticembryo growth in plants and as an anticancer agent, but at thispoint the evidence must be regarded as circumstantial. In thisregard, we have carried out a BLAST database search on anexpressed sequence tag library of LP somatic embryos (Cairneyet al., 2006), and we have identified one singleton (Gene Banknumber DR688191) that shows 83% identity in amino acidsequence to that of PtdIns 3-kinase AtVPS34. Clearly, additionalstudies will be needed to fully elucidate the mechanisms by whichInsP6, (and perhaps other inositol phosphates as well) regulatecellular growth and development in plants. Such studies couldwell lead to significant improvements in the technology ofsomatic embryogenesis in plants.

Acknowledgements

This work was supported by the National Research Initiative ofthe USDA Cooperative State Research, Education and ExtensionService, grant number 2003-35103-12924, and also through TheConsortium for Plant Biotechnology Research, Inc. by DOE

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Prime Agreement No. DEFG36-02GO12026, and by the Mons-anto Company as a member of the CPBR. We thank the Instituteof Paper Science and Technology at Georgia Tech for a fellow-ship award to D.W. We also thank Drs Kristi Burns, YanfengChen, Zhen Zhou, and Michael Foster for many technicaldiscussions and recommendations. Muco-InsP6 was provided as agenerous gift by Dr Alan Richardson from the collection of thelate Dr Dennis Cosgrove.

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Myo-inositol hexakisphosphate, isolated from female gametophyte tissue of loblolly pine, inhibits growth of early-stage somatic embryos