plant lnositol monophosphatase 1s a lithium … plant cell, vol. 7, 2175-2185, december 1995 o 1995...

The Plant Cell, Vol. 7, 2175-2185, December 1995 O 1995 American Society of Plant Physiologists

Plant lnositol Monophosphatase 1s a Lithium-Sensitive Enzyme Encoded by a Multigene Family

Glenda E. Gillaspy, James S. Keddie, Kenj i Oda, a n d Wi lhe lm Gruissem’

Department of Plant Biology, 111 Koshland Hall, University of California-Berkeley, Berkeley, California 94720-3102

myo-lnositol monophosphatase (IMP) is a soluble, Li+-sensitive protein that catalyzes the removal of a phosphate from myo-inositol phosphate substrates. IMP is required for de novo inositol synthesis from glucose Cphosphate and for breakdown of inositol trisphosphate, a second messenger generated by the phosphatidylinositol signaling pathway. We cloned the IMP gene from tomato (LelMP) and show that the plant enzyme is encoded by a small gene family. Three different LelMPcDNAs encode distinct but highly conserved IMP enzymes that are catalytically active in vitro. Similar to the single IMP from animals, the activities of all three LelMPs are inhibited by low concentrations of LiCI. LelMP mRNA levels are developmentally regulated in seedlings and fruit and in response to light. lmmunoblot analysis detected three proteins of distinct molecular masses (30, 29, and 28 kD) in tomato; these correspond to the predicted molecular masses of the LelMPs encoded by the genes. lmmunoreactive proteins in the same size range are also present in several other plants. lmmunolocalization studies indicated that many cell types within seedlings accumulate LelMP proteins. In particular, cells associated with the vasculature express high levels of LelMP protein; this may indicate a coordinate regulation between phloem transport and synthesis of inositol. The presence of three distinct enzymes in tomato most likely reflects the complexity of inositol utilization in higher plants.

INTRODUCTION

myo-lnositol (inositol) is a six-member carbon ring structure required for normal cell growth and other critical cell functions (for reviews, see Loewus and Loewus, 1983; Berridge, 1987; Loewus, 1990; Majerus, 1992). Reduction of cellular inositol levels has been linked to inhibition of cell division in plant tissue culture cells (Biffen and Hanke, 1990) as well as developmental alterations in mice and Xenopus (Busa and Gimlich, 1989; Cockcroft et al., 1992; Maslanski et al., 1992). In animal cells, inositol is primarily incorporated into inositol phospholipids that also function as precursors for the phosphatidylinositol (Pl) signaling pathway (for review, see Michell, 1986). In addition to inositol phospholipid synthesis, inositol is used for other functions in plant cells, including auxin conjugation and storage, phosphorus storage in the form of inositol hexakisphosphate (IP6), and biosynthesis of the cell wall compounds glucuronic acid, arabinose, and xylose (Loewus and Loewus, 1983; Loewus, 1990). Another important role for inositol in plant physiology is adaptation to stress; several studies have linked accumulation of both inositol and its derivatives with increased survival under salt conditions (Loewus and Loewus, 1983; Vernon and Bohnert, 1992; Bohnert et al., 1995).

lnositol synthesis from glucose 6-phosphate is a two-step process involving conversion of glucose 6-phosphate to inositol 1-phosphate, which is catalyzed by the enzyme inositol-l-phos-

To whom correspondence should be addressed

phate synthase, and dephosphorylation of inositol 1-phosphate to yield free inositol (Loewus and Loewus, 1983; see Figure 1). This last step is catalyzed by the enzyme inositol monophosphatase (IMP), a soluble, Li+-sensitive protein (for review, see Parthasarathy et al., 1994). IMP can also dephosphorylate other inositol phosphate compounds, such as the breakdown products of the signaling molecule inositol trisphosphate (IP,) (Chen and Charalampous, 1966; Eisenberg, 1967; Hallcher and Sherman, 1980); thus, IMP is required for both de novo synthesis and recycling of inositol. Therefore, it can be considered a potential regulatory point for all pathways that utilize free inositol

An important role for IMP in the regulation of signaling and development has emerged from studies using Li+ as an IMP inhibitor. Li+ can act as an effective inhibitor of the animal PI pathway by blocking synthesis of inositol for incorporation into membrane inositol phospholipids (Berridge et al., 1982, 1989; Sherman et al., 1986; see Figure 1). The disruption of the PI signaling pathway by Li+ causes severe alterations in embryonic development. For example, treatment of Xenopus embryos with Li+ results in dorsalization (Kao et al., 1986) and a tran- sient decrease in IPs levels (Maslanski et al., 1992). These effects can be prevented or rescued by cointroduction of inositol with Li+ (Busa and Gimlich, 1989), implying that a decrease in inositol levels is sufficient to cause altered signaling (referred to as the “inositol depletion hypothesis”; Berridge et al., 1989).

2176 The Plant Cell

I \ release \

\Glucose-6-P lnsP

\ \

\ \ \ '.

UDP-Glucukonate Ins-IAA lnsPs

cell wall components and transpori storage Biosynthesls of Auxln storage Phosphorous

Figure 1. lnositol Synthesis and Utilization in Plant Cells.

De novo synthesis of inositol is catalyzed by IMP action on inositol phosphate (InsP). Li+ inhibition of IMP can lead to a reduction in free inositol levels, which may affect many plant functions, including cell wall biosynthesis (through UDP-glucuronate), auxin storage and transport (through auxin-inositol conjugation [lns-IAA]), and phosphorous storage (through hexakisphosphate [lnsPs]). A scavenger pathway of inositol synthesis exists that makes use of lnsP generated by the PI signaling pathway: a putative receptor (R) responding to externa1 signals may signal phospholipase, C (PLC) and its associated G protein (G) to convert phosphatidyli~ositol-4,5 bisphosphate (PIPp) into the second messengers inositol trisphosphate (InsP3) and diacylglycerol (DAG).

Because plants use inositol for diverse cell functions, it is difficult to separate the role of inositol in signaling pathways (for reviews, see Trewavas and Gilroy, 1991; Cote and Crain, 1993; Drgbak, 1993; Gross and Boss, 1993) from its more general role in plant metabolism. Data in the literature suggest that Li+ is not a useful tool in regulating either inositol synthesis or PI signaling, dueto the apparent lack of Li+ sensitivity of plant IMP (Gross and Boss, 1993). To understand how the pool of free inositol is regulated in plant cells and to explore the possibility of a role for IMP in the regulation of the plant PI pathway, we have cloned the plant IMP genes and characterized the enzymes. In this study, we report that, in contrast to information currently available for animal and human IMP, plant IMP is encoded by a family of three genes in tomato that are differentially expressed. The predicted protein products are conserved and share significant sequence homology with the single IMP from animals. Like animal IMP, our studies confirm that all three plant enzymes are sensitive to Li+ in vitro. Together, our work establishes the presence of Li+- sensitive IMPs and increased complexity of inositol synthesis in plants. Along with the recently cloned Arabidopsis gene encoding phospholipase C (Hirayama et al., 1995; Yamamoto et al., 1995), the IMPgenes can now be used as molecular tools to augment physiological studies on the plant PI signaling pathway.

RESULTS

Tomato IMP 1s Encoded by a Small Gene Farnily

A cDNA for tomato IMP (LelMP for Lqlopefsicon esculentum jnositol monophosphatase) was isolated from a young fruit cDNA expression library as described in Methods and was used to rescreen tomato cDNA and genomic libraries. This approach resulted in the isolation of severa1 cDNAs and genomic DNAs encoding three distinct LelMP proteins (Fig- ure 2). Based on the size of the cDNAs and the lengths of contiguous open reading frames, the proteins encoded by the LelMP genes appear to be full length. All three cDNAs encode proteins with significant amino acid sequence similarity with the known IMP enzymes from humans and Xenopus (Diehl et al., 1990; McAllister et al., 1992; Wreggett, 1992). Alignment of the LelMP amino acid sequences with the sequence for the Xenopus IMP reveals 434% amino acid sequence identity and 70% sequence similarity. The predicted amino acid sequences

1 46 L e I M P l WRNGSLEEF LG VAVLMAK RAGEIIRKGF HETKWHKG QVDLVTE LeIMPZ M.. . VD .. I E . .. K ...... H.. YKS .. I E ... V . . . . . . LeIMP3 .. Q...V.Q. .D ... E. .. K ...... E . . YK .... E . . . M . . . . . .

HunonIMP UAWWQ.C MDY ..TL.R Q...VVCEAI KNEMN.ML.S S P . . . . . A b4EDRWQ.C MDFL..SI.R K..SVVCAAL K.OVS1MV.T SLAPA .... A XenopusIMP

47 mtif A - .... L e I M P l W C E D L I F NHLKQHFPSH KFIGETSAA TGDFDLTDEP LeIHPZ ...... V . . . ..... C. . . . ....... T.. AS.N.E ..... LeIMP3 ....... F . . ..... R.... ....... T. . C.N.E .....

H m n I H P ..QKV.KHLI SSI.EKY ... S ..... SV.. GEKSI ... N. XenopusIHP ..QKV.EH.I SSI.EKY ... S ..... SV.. GAGST ... N.

97 1 A7 Le IMP1 FPSV CVSIGLTIGK IPTVGVVYDP I IDELFTGIN GKGAYLNGKP LeIHPZ . . F . ........ E. K.V ..... N. ....... AIY . R . . F , . . . S LeIMP3 ..F. ........ E. K ....... N. ......... D .... F . ....

. . F . A. ... FAVN. KIEF .... SC VEGKMY.ARK .... FC..QK

. . F . A. ... FAVH. QVEF .... SC VEDKMY..RK ... SFC..QK HumonIMP

XenopusIHP

148 196 L e I H P l IKVSSQSELV KSLLGTEVGT TRDNLTVEll T RRINNLL FKVRSLRMCG LeIMPZ .R . . .E .Q . . .A.VA.. . . . N..KAI.DA. . G . . R V I ........ S . LeIMP3 .......... .A..A..A.. N..K.V.DA. . G...S.. ..........

HumanIHP LQ..Q.EDIT .... V . . L . S S.TPE..RMV LSNMEK.FC IP.HGI.SW. XenopusIMP LQ..G.KDIT . . H I I . . L , S N.NPEF1K.V SLSNMER.LC IPIHGIRAV.

197 m n t i f R 747 ....... - _.I

VLFDPSGSEF L e I H P l TCALDLCWA CGRLELFYLI GYGGP LeIHPZ .... N..G.. .... D . . . E . EF ... LVL ....... LeIHP3 .... N..G.. .... D...EL EF ... FV ........

HumonIHP TA.VNH.L.. T.GADAY.EH . IHC . .H.VT.GP. XenopusIHP TA.VNM.1.. T.GADAY.EM .LHC T I L .AT. GL.

248 L e I M P l DITSQRV AA TNPHLKEAFV EALQLSEWSZ LeIWP2 .L.AR.. . . . . A . . . D . . I N..NE.Z LeIMP3 .L.AR.. . . . . A . . . D . . I K..NEZ

HumonIMP . L H . R . . I . . N .R I .A .R IA KEI.VIPLQR DDEDZ XenopusIMP . L H . C . I I S . SSREIA.RIA KEL.I IPLER DDGKSTNZ

Figure 2. Alignment of Predicted Amino Acid Sequences from Tomato, Human, and Xenopus IMP Genes.

The alignment was obtained using the Pileup program of the Genetics Computer Group (Madison, WI) software package and the human (McAllister et al., 1992) and Xenopus (Wreggett, 1992) sequences. Dots indicate amino acid identity with the LelMPI predicted protein. Motifs A and B were previously identified by Neuwald et al. (1991). Residues involved in metal or sulfate binding (Bone et al., 1992) are underlined.

lnositol Monophosphatase Genes 21 77

LeIMPl BtfMP ECSuhB NcQax BtIPP ScHAL2

LeIMPl BtIMF EcSuhB NcQax BtIPP ScHALZ

LeIMPl BtIMP EcSuhB NcQan BtIPP ScHAL2

60-92 DLVTETDKACEDLLIFNHLKQHFPSHK FIGEET 40-72 . . . . A..QKV.KM..TSIKEK Y...S . . . . . S 31-69 .F..NV...A.AVI.DTIR.S Y.0.T I.T..S 60-92 .I..Q..EDV.AFIKSAINTR Y...D . . . . . _ 46-80 .FK.LA.VLVQEVIKE.MENK . . GLGKK . . . . . S 42-76 SP..TGDY.AQTII.NAIKSN . . DD.W.....S

motif A 86-113 WIVDPVDGTTNFVHGFPSVCVSIGLT 86-113 . . I . . I . . . . . . . . . . . F.A . . . . FV 82-108 .VI..I......IKRL.HFA...AVR

103-129 .V...I...V.YT.L..MF....AFL 151-177 . . . . I.S.YQYIK.SADITPNQ.IF 139-166 .CL..I...KG.LR.EQFAVCLALIV

motif B 207-243 CWVACGRLELFYLIGYGGP WDVAGGAVIVKEAGG VLFD 201-238 .L..A.AADAY.EM.IHC . . . . A..IIVT.... . .L. 193-230 ~Y..~..vDG.FE..LRP . . F.A.ELL.R . . . . I vs. 143-186 AYT.M.SFDIWWEG.CWE . . . . A.IA.LQ . . . . LITSANPPE. 297-315 .VI L.LADIYIFSEDTTFK ..SCAAHA.LRAM.. GMV. 269-312 .LL.L.LADVYLRLPIKLSYQEKI..H.A.N...H.... IHT.

Figure 3. Alignment of IMP-Related Proteins.

Regions of tomato LelMPl, the bovine (Bos taufus) IMP (BtlMP; Diehl et al., 1990), the SuhB product of E. coli (EcSuhB; Yano et al., 1990), the qa-x product of N. crassa (NcQax; Geever et ai., 1989), the bovine inositol polyphosphate 1-phosphatase (BtlPP; York and Majerus, 1990), and the HAL2 product from yeast (ScHAL2; Glaser et al., 1993) are aligned. Dots indicate amino acid identity with the LelMPl predicted protein.

of LelMP1 and LelMP2 are more similar to that of LelMP3 (77% identity) than they are to each other (67% identity).

Amino acid sequences that have been previously identified as characteristic of metal and sulfate binding sites and of putative Li+-sensitive domains are conserved in each LelMP protein (Bone et al., 1992). The LelMP proteins also share homology with the SuhB (extragenic suppressor of cold sensitivity; Yano et al., 1990) and AmtA (ammonium transporter; Fabiny et al., 1991) proteins of Escherichia coli, the qa-x protein of Neurospora crassa (quinic acid cluster; Geever et al., 1989), the qutG protein of Aspergillus nidulans (quinate utliza- tion cluster; Hawkins et al., 1988), the HAL2 protein from yeast (halotolerance; Glaser et al., 1993), and the inositol polyphosphate 1-phosphatase (IPP) protein from animals (York and Majerus, 1990) (see Figure 3). These diverse proteins are enzymes that have been implicated in the synthesis or degradation of phosphorylated messenger molecules (Neuwald et al., 1991), but specific substrates have been identified for only three of the enzymes. Matsuhisa et al. (1995) have recently demon- strated that the suhB gene encodes an active IMP and appears to have a function in cold-sensitive growth processes in bacteria. The yeast HAL2 gene imparts tolerance for both NaCl and LiCl when overexpressed (Glaser et al., 1993) and was recently shown to encode a Li+-sensitive 3'(2'),5'-bisphosphate nucleotidase (Murguia et al., 1995). The bovine IPP catalyzes the removal of 1'-phosphate from both inositol 1,3,4-P3 and inositol 1,4-P2 substrates and thus is also involved in the recycling of inositol (Majerus, 1992). These proteins from bacteria, yeast, and animals, along with the LelMP proteins, share significant amino sequence homology in four domains, of which motifs A and B are most likely responsible for Li+ sensitivity (Neuwald et al., 1991).

To determine the number of LelMP genes in the tomato ge- nome, analysis of tomato genomic DNA was performed using each LelMP cDNA as a probe. Each cDNA probe produced

a diagnostic hybridization profile at high stringency; at low stringency, the cross-hybridization profile included additional bands diagnostic for the other two cDNAs (results not shown). We have completed the analysis and sequence for the genomic loci of LelMP7 and LelMP2, and similar work is in progress for LelMP3 (J. Keddie, K. Oda, G. Gillaspy, and W. Gruissem, unpublished results). Three genes for conserved IMPs have also been identified in Arabidopsis (J. Kudla, G. Gillaspy, and W. Gruissem, unpublished results). From this analysis, we con- cluded that the IMP enzyme in tomato is encoded by a small gene family of three related genes in plants.

LelMP Gene Products Are Li+-Sensitive IMPs

Based on their amino acid sequence homology with the animal IMP, we tested the LelMP proteins for their ability to catalyze the removal of a phosphate from an inositol 1-phosphate substrate. Bacteria containing a P-galactosidase (P-Gal)-LelMP gene fusion on a phagemid were induced with isopropyl P-o-thiogalactopyranoside (IPTG) for 2 hr, and protein extracts were prepared from the induced cells along with the appropri- ate control cells (including E. colicells expressing the human IMP). Protein gel blot analysis of the protein extracts was performed using an anti-LelMP1 antibody generated against a glutathione S-transferase (GST)-LelMP1 fusion protein. Fig- ure 4A shows that the molecular masses of the LelMP proteins expressed from LelMP7, LelMP2, and LelMP3 cDNAs in bac- teria1 cells range between 34 and 30 kD. After subtracting the 4-kD P-Gal peptide, the apparent molecular masses of the LelMP1, LelMP2, and LelMP3 proteins (30, 29, and 28 kD, respectively) are in close agreement with the molecular masses of the predicted LelMP translation products. The bacterial protein extracts contain different amounts of immunoreactive LelMP proteins that approximately correlate with the amount of protein produced from each LelMP cDNA as detected by Coomassie Brilliant Blue G staining of a duplicate gel. This result suggests that the anti-LelMP1 antibody cross-reacts efficiently with each of the LelMP proteins but not with the human IMP.

The activity of the bacterial protein extracts in removing a phosphate from the 14C-labeled inositol-1-phosphate substrate was measured under various conditions (Figure 46). Protein extracts prepared from E. coliXL-1 Blue control cells expressing the P-Gal peptide alone provided the baseline for activity (E2) and were not stimulated or inhibited by anyadditions. The results in Figure 48 confirm that each LelMP cDNA encodes an active IMP. The relative activities of LelMP1, LelMP2, and LelMP3 extracts differed, but this result is most likely a reflec- tion of the amount of LelMP protein that was present in each extract (see Figure 4A) rather than a difference in specific activities of the enzymes. LelMP activities were reduced after the addition of unlabeled inositol 1-phosphate, demonstrating efficient substrate competition and the specificity of the enzyme assay (results shown for LelMPl only). Enzyme activities were dependent on the presence of Mg2+ (results not shown)

2178 The Plant Cell

*?*

49 kD-

32 kD

27 kD

.01 .1 1mMLiCI

Figure 4. LeIMP Expression and Activity in E. coli.

(A) Protein gel blot analysis. Proteins prepared from IPTG-induced ex-tracts of E. coli XL-1 Blue containing LeIMP cDNAs (IMP1, IMP2, orIMP3), the human IMP (hIMP), or control plasmid (E2) were analyzedusing anti-LelMP1 antiserum. Numbers at left indicate the migrationof size markers.(B) LeIMP activity assays Protein extracts from (A) were assayed asdescribed in Methods. IMP activity is expressed as counts per min-ute. -Ca, no addition of 1 mM final CaCI2; + IP, addition of unlabeledinositol 1-phosphate to 100 mM.(C) Sensitivity of LeIMP to LiCI. IMP activity was assayed in the pres-ence of increasing concentrations of LiCI as given in (B). Activity isexpressed as a percentage of maximal activity.

and were differentially stimulated by increasing concentrationsof Ca2* to 1 mM (Figure 4B). It is possible that the effect ofCa2+ on the LeIMP enzymes is nonspecific in the bacterialprotein extracts because the human IMP was also stimulatedby Ca2+ under these conditions (the human IMP is reportedlyinhibited by micromolar levels of Ca2*; Hallcher and Sherman,1980). Plant PI pathway enzyme activities have been notedpreviously to be sensitive to changes in Ca2+ concentrations(Joseph et al., 1989; Memon et al., 1989).

Increasing concentrations of LiCI resulted in the inhibitionof each LeIMP enzyme activity in the bacterial protein extracts(Figure 4C). No reduction in activity was measured after theaddition of up to 10 mM NaCI or choline chloride (results notshown); therefore, inhibition by Li+ is specific. The concen-tration of Li+ required to inhibit the tomato enzymes in thebacterial protein extract is similar to that required to inhibit thehuman enzyme (Figure 4C), and our results are in agreementwith the published data (0.3 mM for half-maximal inhibition;McAllister et al., 1992). Our results are in contrast to earlierexperiments with plant protein extracts. In these experiments,no Li+-sensitive IMP activity could be detected (Cumber et al.,1984; Joseph et al., 1989), or very high Li+ concentrations(>50 mM) were required to inhibit IMP enzyme activity (Loewusand Loewus, 1982). Based on the similarity in amino acidsequences, enzymatic activities, and Li+ sensitivity, we con-cluded that the proteins encoded by the LeIMPI, LelMP2, andLelMP3 cDNAs are functional plant homologs of the animalLi+-sensitive IMP enzyme.

LeIMP Genes Have Different Temporal and SpatialExpression Patterns

Expression of the LeIMP genes in E. coli confirmed their IMPactivity and established that, in contrast to animals and hu-mans, tomato has multiple genes for this enzyme. To gaininsights into the expression of the LeIMP genes, we analyzedthe temporal and spatial accumulation patterns of LeIMPmRNAs. RNA gel blot analysis using LeIMPI and LelMP2cDNAs as probes showed a low-abundance 1.2-kb band withthe LeIMPI cDNA probe, but no RNA signal could be detectedwith the LelMP2 cDNA probe (data not shown). We thereforeused RNase protection as a more sensitive assay to measureLeIMPI-, LelMP2-, and Le/MP3-specific mRNA levels (Figure 5).

A labeled antisense RNA probe encompassing the 3' se-quence of the coding region was transcribed from each cDNAand used to confirm that the detected signals were diagnosticand specific for each mRNA. The RNA probes were then usedto determine LeIMP mRNA accumulation at different develop-mental stages and in different tomato organs. Using this assay,LeIMPI and LelMP3 mRNAs were readily detectable in mostorgans, but the LelMP2 mRNA was rare and required five-fold more RNA and extended exposures for detection (seeMethods). LeIMPI mRNA accumulation was highest in light-grown seedlings, flowers, young and mature green fruit, andcallus tissue (Figure 5A). It was measurable in root and stem

Inositol Monophosphatase Genes 2179

tissue, and accumulation decreased as fruits matured (maturegreen to breaker fruit stage). LeIMPS mRNA also accumulatedto high levels in the shoot apex and callus and was presentin root, stem, leaf, flower, and young and mature green fruit.LeIMPS was induced by light, although not to the same degreeas LeIMPI (Figure 5C). LelMP2 mRNA levels were significantlylower at all developmental stages and in all tomato organs ascompared with LeIMPI and LeIMPS mRNAs (Figure 5B). Ac-cumulation of LelMP2 mRNA was highest in seedlings, roots,and young fruit. Together, these results indicate that accumula-tion of LeIMP mRNAs is regulated by a complex developmentalprogram in seedlings and fruit and is differentially controlledin response to light during seedling development. At a first ap-proximation, the expression pattern of the LeIMP genescorrelates with established inositol requirements in proliferat-ing tissues but also suggests that the individual members ofthe LeIMP gene family may have more restricted and special-ized functions.

LeIMP Protein Accumulation Is Developmentally andLight Regulated

We used the anti-LelMP1 antibody in a protein blot analysisof different tomato organs and developmental stages to ana-lyze LeIMP protein accumulation (see Figure 6). Although theantibody used in this analysis was generated against thep-Gal-LelMP1 fusion protein, the results in Figure 4A confirmthat it cross-reacts with the LelMP2 and LeIMPS proteins aswell. Three immunoreactive proteins were detected in solubleprotein extracts prepared from different tomato organs afterseparation of the proteins by SDS-PAGE. Their apparent mo-

CA DGS LGS RT ST LF SA YF 1cm MG BR RF

1 2 DGS 12 24 RT ST LF SA FL YF MG BR CA

IrFigure 5. LeIMP mRNA Levels in Tomato.RNase protection assays were performed as described in Methods.Lane 1 contains the probe alone without RNase digestion; lane 2, theprobe alone plus RNase digestion; DGS, 6-day-old dark-grown seed-lings; 12, DGS exposed to light for 12 hr; 24, DGS exposed to lightfor 24 hr; RT, root; ST, stem; LF, leaf; SA, shoot apex; FL, flower; YF,fruit 0.5 cm in diameter; MG, mature green fruit; BR, breaker fruit; CA,callus.(A) LeIMPI mRNA levels, 16-hr exposure.(B) LelMP2 mRNA levels, 10-day exposure.(C) LeIMPS mRNA levels, 16-hr exposure.

—27 kD

Figure 6. Protein Blot Analysis of LeIMP.Soluble protein extracts were separated by SDS-PAGE, transferred tonitrocellulose, and immunoreacted with anti-LelMP1 antibody as de-scribed in Methods. Arrows indicate the three immunoreactive bandsnoted. Numbers at right indicate the migration of size markers. CA,callus; DGS, 6-day-old dark-grown seedlings; LGS, 6-day-old light-grownseedlings; RT, root; ST, stem; LF, leaf; SA, shoot apex; YF, fruit 0.5cm in diameter; 1cm, fruit 1 cm in diameter; MG, mature green fruit;BR, breaker fruit; RF, ripe fruit.

lecular masses ranged from 30 to 28 kD, which is in closeagreement with the molecular mass range for the LeIMPproteins predicted from their amino acid sequences. Three pro-teins of similar molecular masses were also detected in proteinextracts from other plants (Arabidopsis, tobacco, wheat, andspinach; data not shown). The apparent molecular mass ofthe LeIMP proteins produced by in vitro translation and by over-expression of LeIMPI in transgenic tobacco (data not shown)suggests that the 30-kD protein is LeIMPI and that the pro-teins with approximate molecular masses of 29 and 28 kD areLeIMPS and LelMP2, respectively.

The protein pattern confirms that LeIMP proteins are pres-ent at most developmental stages and in most organs analyzed,with callus tissue, light-grown seedlings, leaves, and youngfruit containing the highest protein levels. LeIMP proteins ac-cumulated to higher levels in light-grown seedlings ascompared with dark-grown seedlings and decreased duringfruit development. At these developmental stages and in callustissue, roots, leaves, and flowers, LeIMP protein accumulationcorrelated well with LeIMP RNA levels, suggesting that expres-sion is controlled primarily at the level of transcription or mRNAstability. Other organs and developmental stages, such asstems, shoot apices, and mature green fruit, did not show thiscorrelation, suggesting that other controls may be importantwith respect to LeIMP protein accumulation.

LeIMP Proteins Accumulate Differently in Specific CellTypes

To investigate the tissue-specific expression of the LeIMP pro-teins in more detail, we used the anti-LeIMP antiserato localizeLeIMP in sections of imbibed seed, light-grown seedlings,young fruit, and the shoot apices of mature plants (see Figure7). To establish the specificity in each tissue, three controlswere always included: immunoreaction with the secondary an-tibody alone (Figure 7D), anti-LelMP-depleted serum (Figure7B), and preimmune serum (data not shown). We detected

2180 The Plant Cell

Figure 7. Immunolocalization of LeIMP

Control and affinity-purified anti-LeIMP antisera were used to localize IMP proteins in longitudinal sections ([A] to [F]) or cross-sections (G) oflight-grown seedlings, as described in Methods. Sections were photographed under a light microscope using differential interference contrastoptics ([A) to [C] and [G]) or phase contrast optics ([D] to [F])-(A) Anti-LeIMP antiserum. Bar = 100 urn.(B) and (C) Anti-LelMP-depleted antiserum (B) and anti-LeIMP antiserum (C). Bars = 50 urn.(D) and (E) Secondary antiserum alone (D) and anti-LeIMP antiserum (E) Bars = 50 nm(F) and (G) Anti-LeIMP antiserum. Bar = 10 \im.p, phloem; x, xylem.

specific immunoreactivity in most of the above-mentioned tis-sues, with imbibed seed containing little or no LeIMP andlight-grown seedlings containing the highest levels. Within theseedling, LeIMP was expressed in the cotyledons, hypocotyls,and roots (Figure 7A and data not shown). Interestingly, cellsin the shoot apical meristem region of the seedling appearedto express less LeIMP than did other cell types, including pali-sade cells (Figure 7C). Similarly, the shoot apex from matureplants showed reduced levels of immunoreactivity and was

thus similar to the meristematic region staining seen in light-grown seedlings (data not shown). A subset of cells within thetips of the cotyledons had increased levels of LeIMP relativeto other nearby cells (Figure 7E). Cells associated with the vas-cular tissue reacted most strongly with the anti-LeIMP antibody(Figures 7F and 7G). These cells are organized as files withinthe vascular tissue and contain nuclei as determined by4',6-diamidino-2-phenylindole staining of adjacent serial sec-tions. Because of their number, morphology, and position, these

lnositol Monophosphatase Genes 2181

cells are most likely phloem parenchyma or differentiating sieve tube members. Embryos from imbibed seed did not show such strongly immunoreactive cells (data not shown), but vascular tissue from young fruit contained immunoreactive cells of similar morphology and spatial organization. Young fruit expressed LelMP most strongly in the pericarp, in developing seed, and in cells associated with the vascular tissue (data not shown).

DlSCUSSlON

We have established that plants contain conserved Li+- sensitive IMPs that catalyze the conversion of inositol phosphates into inositol. In animals, this reaction is critical for synthesis of inositol phospholipids and the PI signaling pathway. Unlike the single gene for IMP reported from animals, plant IMP is encoded by a small gene family in tomato and Arabidopsis. Considering the increased complexity of inositol utilization in plants, a gene family for IMP was not unexpected. The presence of multiple genes could indicate redundant functions for the enzymes in the catalytic conversion of inositol phosphates into inositol in plants. Alternatively, based on the apparent differences in the expression pattern of their genes, we favor a model in which specific IMP isoforms function in different cell types or spatially distinct cellular compartments.

IMP Activity and Substrate Specificity

Animal IMP (Hallcher and Sherman, 1980) and the previously reported lily pollen IMP (Loewus and Loewus, 1982) have been shown biochemically to form dimers of -60 kD in their catalytically active form. Our preliminary data suggest that the LelMPs also exist as active dimers (G. Gillaspy and W. Gruissem, unpublished results). A potentially interesting consequence of multiple gene products is the possibility of heterodimer formation in plant tissues in which two or more IMPs are expressed. It is not clear whether heterodimeriza- tion could alter enzyme activity andlor specificity.

All three LelMPs expressed in E. coli have similar activity profiles in vitro, although there were differences in the ability of Caz+ to stimulate activity. These differences may reflect al- ternate regulation of the enzymes, potentially through the release of Ca2+ generated by the PI pathway. A more detailed characterization of the purified enzymes is required, however, to conclude that the differences in Ca2+ sensitivity are of potential physiological or regulatory relevance.

Because several inositol phosphate-metabolizing enzymes with different substrate specificities have been reported, it is also of interest to determine the in vivo substrate specificity of IMF? In general, IMP hydrolyzes those monophosphates that are equatorially located within the inositol ring but has limited activity on other inositol phosphate substrates. We have ex- amined LelMP activity using only inositol 1-phosphate, which represents one of a few possible substrate pools in the cell. lnositol4-phosphate is another substrate that results from the

breakdown of IP3 (Michell, 1986) and that can be utilized efficiently by animal IMP (Eisenberg, 1967; Ragan et al., 1988). The proposed product of IPG breakdown in plants, inositol 2-phosphate (I-2-P; Cosgrove, 1980), potentially constitutes another significant inositol phosphate pool in plant cells. Animal IMP does not utilize I-2-P efficiently, and we have not determined the activity of LelMP proteins expressed in E. coli with this substrate. The IMP-like enzyme activity previously purified and characterized from lily pollen catalyzes the removal of a phosphate from several different inositol phosphate substrates, including I-2-P (Loewus and Loewus, 1982), but we do not know at present how this biochemical activity relates to activities of the cloned IMPs.

IMP-Related Proteins

The discovery of IMP-related proteins is interesting in the context of their amino acid homologies, mutant phenotypes, proposed functions, and substrate specificities. It is noteworthy that the bacterial SuhB protein can utilize inositol 1-phosphate most efficiently as a substrate and may have a function in cold- sensitive growth (Matsuhisa et al., 1995). The HAL2 gene in yeast (Glaser et al., 1993) encodes a 3’(2’),5’-bisphosphate nucleotidase enzyme (Murguia et al., 1995). HALP confers Li+ and Na+ tolerance when overexpressed, and an enzymatic activity similar to HALP has been identified in tomato leaves (Murguia et al., 1995). IPP from animals catalyzes the removal of 1 ’-phosphate from I-1,3,4-P3 and I-1,4-P2 substrates (York and Majerus, 1990; Majerus, 1992). This enzyme functions im- mediately upstream of IMP in the IPs breakdown pathway. The substrates for three other IMP-related proteins, the AmtA protein of E. coli(Fabiny et al., 1991), the qa-x protein of Neurospora (Geever et al., 1989), and the qutG protein of Aspergillus (Hawkins et al., 1988), have not been identified, but it has been suggested that they are phosphate messenger molecules (Neuwald et al., 1991). Thus, we cannot excludeat the present time that one or all of the LelMPgene products have specificities for substrates other than inositol monophosphates.

Effects of Li+

Our results confirm that the activity of each LelMP is inhibited by LiCl in vitro. A similar sensitivity to Li+ has been determined for the animal and human IMPs. Of all known enzymes, there is only a small select group of enzymes that have been reported to share this characteristic, including the mammalian inositol polyphosphate 1-phosphatase (Inhorn and Majerus, 1987; York and Majerus, 1990), yeast HAL2 (Murguia et al., 1995), and fructose 1,6-bisphosphatase (Black et al., 1972). Except for fructose 1,6-bisphosphatase, which shares structural similarity with IMP, all other enzymes share amino acid homologies in conserved domains identified as motifs A and B in Figure 3. It has been proposed that these conserved domains are responsible for the Li+ sensitivity of the enzymes (Neuwald et al., 1991). The biological role of Li+ has been

2182 The Plant Cell

studied for many years, and more recent studies have shown that Li+ produces striking developmental alterations in many organisms, including Xenopus (Nieuwkoop, 1973), sea urchin (Livingston and Wilt, 1990), and Dictyostelium (Peters et al., 1989). In humans, Li+ therapy is used in the clinical treatment of certain bipolar disorders (e.g., manic depression). The ef- fectiveness of this treatment rests on a model in which Li+ decreases brain inositol levels via reduction of IMP activity (Berridge et al., 1982, 1989; Sherman et al., 1986).

In most plants, Li+ is present in only trace amounts, although Li+ can accumulate to high levels in many plant species when it is present at elevated concentrations in the soil or in tissue culture medium (for review, see Anderson, 1990; G. Gillaspy and W. Gruissem, unpublished results). Long-term exposure to Li + has pleiotropic effects on plants, including phytotoxicity and changes in biomass (Anderson, 1990). We have observed both hypertrophy along seedling stems and severe developmental abnormalities in tomato seedlings ger- minated in the presence of Li+ (G. Gillaspy, K. Oda, and W. Gruissem, unpublished results). Li+ toxicity has been a significant agricultura1 problem in citrus orchards of the southwestern United States where Li+-contaminated water is used for irri- gation, causing Li+ buildup in the soil (Hilgenan et al., 1970; Aldrich et al., 1974). It is not currently known whether any of these biological effects of Li+ on plants involves the IMP enzyme, but our work establishes this as a formal possibility.

IMP and Synthesis of lnositol

Inositol-containing compounds are very abundant in plant cells (for review, see Loewus and Loewus, 1983; Drebak, 1993). It is likely that conjugated and phosphorylated forms of inositol are sequestered into individually regulated pools. For example, bean embryos store high concentrations of inositol as phytic acid, but de novo synthesis of inositol is induced during germination before the phytic acid pool is utilized, suggesting a need for free inositol (Sasaki and Taylor, 1986). The exis- tente of multiple plant IMPs may allow the cell to regulate such hypothetical inositol pools independently. Alternatively, each plant IMP may regulate synthesis of inositol as an intermediate for specific end products. The fact that LelMf genes encode three distinct IMPs that are developmentally regulated is con- sistent with either view.

An intriguing finding is the light-regulated expression of the genes in seedlings that likely results in a temporal up-regulation of inositol synthesis in support of rapid growth-associated processes. The immunolocalization experiments support this hypothesis because LelMP expression in young fruit is localized to those regions that we have previously shown to contain dividing and expanding cells (Gillaspy et al., 1993). However, our current results clearly show that the shoot apical meristem is no ta primary site of LelMP expression. Other cells, including specific cells associated with the vascular tissue, express higher levels of LelMP than do apical meristem cells. Our de-

tection of those cells with a high LelMP level-most likely phloem parenchyma cells or differentiating sieve tube members-is important with respect to our current understanding of inositol synthesis and transport. LelMP is required for the last step of inositol synthesis, and the phloem contains both transported inositol and its derivatives as well as transported auxins that are most likely conjugated to inositol (Salisbury and Ross, 1978; Reinecke and Bandurski, 1987; for review, see Hoad, 1995). It is possible, therefore, that coordinate regulation of synthesis, differentiation, and transport of inositol occurs within these cells.

The IMP genes reported here, together with the recently cloned gene for inositol-1-phosphate synthase (Johnson, 1994; Johnson and Sussex, 1995), should now provide useful tools to dissect the regulation and critical functions of inositol synthesis in the plant cell.

METHODS

Plant Materials

Tomato DNA and RNAs were isolated from greenhouse-grown Lycopef- sicon esculentum cv VFNT Cherry LA1221. Light induction experiments were performed by germinating seed on filter paper (No. 1; Whatman International, Maidstone, England) and water in total darkness for 6 days followed by a 6- to 24-hr exposure to light. The young fruit cDNA library was prepared from mRNA of 3- to 8-mm fruit using a Stratagene Uni-ZAP XR kit.

cDNA Library Screening

Active phosphatase clones were obtained by screening phage for their ability to use 5-bromo-4-chloro-3-indoyl phosphate as a substrate at neutra1 pH. Phage from a tomato young fruit cDNA library were plated onto €scherichia coli XL-1 Blue cells and were induced to express fusion proteins by overlaying nitrocellulose filters containing 10 mM isopropyl P-D-thiogalactopyranoside (IF'TG). Filters containing transferred proteins from plaques were rinsed in 10 mM Tris, pH 8.0, 150 mM NaCI, and 5 mM MgCI2 severa1 times; 5-bromo-4-chloro-3-indoyl phosphate and nitro blue tetrazolium were added, and color development was allowed to proceed at room temperature for 30 min. Approximately 106 plaques were screened by this assay, and one plaque was shown to be active. The positive phage was plaque purified, and phage DNA was excised in vivo to recover pBluescript SK+ plasmid according to Stratagene's protocol. In a separate screen, a similar clone was obtained, establishing the reproducibility of the screening procedure. 60th cDNA clones contained an identical 1.1-kb insert: This cDNA was used as a randomly primed probe (Stratagene Prime-lt kit) to rescreen the library as described in Barkan et al. (1986) to obtain a full-length LelMPl (for tomato inositol monophosphatase) cDNA. Phage were purified and excised as given above. The cDNA for LelMfl was used to screen a tomato genomic library, resulting in the isolation of LelMf2. The coding region of LelMP2 was used to screen the young fruit cDNA library, resulting in cDNAs encoding LelMP2 and LelMP3. These sequences have been deposited in GenBank with ac-


cession numbers U39444 for LelMPl, U39443 for LelMP2, and U39059 for LelMP3.

lsolation of the Human IMP cDNA and Construction of pGalactosidase Fusion Genes

Oligo(dT) was used in reverse transcription of 1.6 pg of HeLa cell RNA with the Superscript enzyme (Gibco BRL). Primers derived from the 5' and 3'ends of the published sequence of the human cDNA (McAllister et al., 1992), 5'-TTCCCGGGGATGGCTGATCCTTGGTCAG-3' and 5'- AAGTCGACTTAATCTTCGTCGTCTCG-3; were then used to amplify reverse-transcribed RNA. This mixture was heated to 94°C for 2 min, and the above-mentioned primers, deoxynucleotide triphosphates, 1.5 mM MgCI2, and 5 units of Taq polymerase were added to a 100-pL reaction. This was amplified for 39 cycles consisting of 1 min at 94% 2 min at 55"C, and 2 min at 72°C. The resulting 1-kb cDNA fragment was subcloned into the TAvector (Invitrogen, San Diego, CA). The cDNA insert was digested with Smal and Sal1 enzymes and subcloned into pBluescript SK+ vector, resulting in a P-galactosidase (P-GaI)-hlMP gene fusion. The 8-GaCLelMP2 gene fusion was constructed by am- plifying the 5' and 3' ends of a genomic clone and subcloning this fragment into pBluescript SK+ vector. The P-Gal-LelMP3 construct used in all experiments has a deletion of six amino acids at the N ter- minus; we subsequently isolated a full-length cDNA encoding a protein with a similar activity profile.

IMP Activity

Enzyme activity was determined as described by Ragan et al. (1988). E. coli strain XL-I Blue containing LelMP constructs, the human IMP cDNA, or control plasmid (E2, pBluescript SK+) was induced with 10 mM IPTG for 2 hr. Extracts were made according to the Stratagene k-ZAP protocol. Equal amounts of protein were assayed in a final vol- ume of 0.3 mL containing 50 pCi of ~-'~C-inositol I-phosphate (American Radiolabeled Chemicals, Inc., St. Louis, MO) at room temperature. Reactions were terminated after 1 hr by the addition of 0.05 mL of 10% (w/v) trichloroacetic acid. The mixture was diluted with 1.0 mL of water and applied to a Dowex AGI X8 column (formate form; Bio-Rad). The column was washed with 5.0 mL of water, and the ra- dioactivity of the eluate (containing free inositol) was determined by scintillation counting. Three to five determinations were made for each sample, and the mean and standard deviation were calculated.

probes were constructed by subcloning the 3'sequence of each LelMP cDNA coding region into pBluescript SK+ vector. T7 polymerase transcription of LeIMP templates resulted in the synthesis of a 569- nucleotide LelMPl probe, an 870-nucleotide LelMP2 probe, and a 759- nucleotide LelMP3 probe. lncorporation of label into these probes was .V4Oo/o as determined by trichloroacetic acid precipitation. LelMPl and LelMP3 antisense probes (100,000 cpm) were hybridized to 3 pg of total RNA prepared from various tissues, and the LelMP2 antisense probe (100,000 cpm) was hybridized to 15 pg of total RNA.

Protein Gel Blot Analysis

Preparation of protein extracts and protein blot analysis were performed as described by Schuster and Gruissem (1991). The anti-LelMP1 antibody was produced in a rabbit injected with a glutathione S-transferase (GST)-LelMP1 fusion protein in Ribi adjuvant, following the Ribi pro- toco1 (Ribi Immunochemicals, Hamilton, MT). The serum was purified by subtractive chromatography over E. coli antigen and GST columns, followed by chromatography on a GST-LelMP1 affinity column. All columns were prepared with Reactigel (Pierce, Rockford, IL) following the supplier's instructions. The resulting antiserum was specific for LelMP and was devoid of immunoreactivity toward GST or E. coli antigens.

lmmunolocalization

These experiments were performed as described previously by Gillaspy et al. (1993) with the following modifications: plant tissues that were fixed in 4% paraformaldehyde, primary rabbit anti-LelMP1 antiserum, and secondary goat anti-rabbit IgG conjugated to alkaline phosphatase (Boehringer Mannheim) were each diluted 1:300 in PBS plus 0.1% BSA and reacted for 45 min, followed by severa1 washes with PBS. The alkaline phosphatase detection reaction was allowed to proceed for 2.5 hr, and slides were mounted in Aquapolymount (Polysciences, Inc., Warrington, PA). To ascertain the specificity of these reactions, three controls were always included: secondary antibody alone, anti- LelMP-depleted serum, and preimmune serum. Anti-LelMP-depleted serum was obtained by binding purified recombinant histidine-tagged LelMPl protein to nitrocellulose and reacting the diluted anti-LelMP1 antisera with this filter to remove specific antibodies. The depleted serum was then used on sections.

RNA and DNA Gel Blot Analyses ACKNOWLEDGMENTS

Total RNA was isolated from tomato tissues as described by Barkan et al. (1986), and samples of 10 or 25 pg of RNA were electrophoresed, blotted, and hybridized with randomly primed probes as described by Narita and Gruissem (1989). Analysis of tomato genomic DNA using randomly primed LelMP probes was performed as described by Narita and Gruissem (1989).

RNase Protection Assays

Assays were performed using an Ambion RNAP kit (Ambion, Inc., Aus- tin, TX) following the provided protocol. Templates for antisense RNA

We thank Ann Loraine and Jon Narita for help with the library screening and determination of phosphatase activity of clones, Kris Callan for contributing a protein blot, and Joerg Kudla for cloning and se- quencing the Arabidopsis IMPs. This research was supported by grants from the Department of Energy (No. 85ER13375) and the U.S. Depart- ment of Agriculture (No. 1-44037&25214) to W.G. G.E.G. was supported by a National Science Foundation postdoctoral fellowship; J.S.K. was supported by a Human Frontiers long-term fellowship; and K.O. was supported by a postdoctoral fellowship from the Japanese Society for the Promotion of Science.

2184 The Plant Cell

Received September 14, 1995; accepted October 30, 1995

REFERENCES

Aldrich, D.G., Vaneselow, A.P., and Bradford, G.R. (1974). Lithium toxicity in citrus. Soil Sci. 71, 291-295.

Anderson, C. (1990). Lithium in plants. In Lithium and Cell Physiol- ogy, R.O. Bach and V.S. Gallicchio, eds (New York: Springer-Verlag),

Barkan, A., Miles, D., and Taylor, W.C. (1986). Chloroplast gene expression in nuclear, photosynthetic mutants of maize. EMBO J. 5,

Berridge, M.J. (1987). lnositol trisphosphate and diacylglycerol: Two interacting second messengers. Annu. Rev. Biochem. 56, 159-193.

Berridge, M., Downes, C.P., and Hanley, M.R. (1982). Lithium am- plifies agonist-dependent phosphatidylinositol responses in brain and salivary glands. Biochem. J. 206, 587-595.

Berridge, M.J., Downes, C.P., and Hanley, M.R. (1989). Neural and developmental actions of lithium: A unifying hypothesis. Cell 59,

Biffen, M., and Hanke, D.E. (1990). Reduction in the leve1 of intracellular myo-inositol in cultured soybean (Glycine max) cells inhibits cell division. Biochem. J. 265, 809-814.

Black, W.J., Van Tol, A., Fernando, J., and Horecker, B.L. (1972). lsolation of a highly active fructose diphosphatase from rabbit mus- cle: Its subunit structure and activation by monovalent catlons. Arch. Biochem. Biophys. 151, 576-590.

Bohnert, H.J., Nelson, D.E., and Jensen, R.G. (1995). Adaptations to environmental stresses. Plant Cell 7, 1099-1 11 1.

Bone, R., Springer, J.P., and Atack, J.R. (1992). Structure of inositol monophosphate, the putative target of lithium therapy. Proc. Natl. Acad. Sci. USA 89, 10031-10035.

Busa, W.B., and Gimlich, R.L. (1989). Lithium-induced teratogene- sis in frog embryos prevented by a polyphosphoinositide cycle intermediate or a diacylglycerol analog. Dev. Biol. 132, 315-324.

Chen, I.W., and Charalampous, C.F. (1966). Biochemical studies on D-inositol I-phosphate as intermediate in the biosynthesis of inosi- to1 from glucose 6-phosphate, and characteristics of two reactions in this biosynthesis. J. Biol. Chem. 241, 2194-2199.

Cockcroft, D.L., Brook, F.A., and Copp, A.J. (1992). lnositol de- ficiency increases the susceptibility to neural tube defects of genetically predisposed (curly tail) mouse embryos in vitro. J. Ter- atol. 45, 223-232.

Cosgrove, D., ed (1980). lnositol Phosphates: Their Chemistry, Bio- chemistry and Physiology. (New York: Elsevier).

Cote, G.G., and Crain, R.C. (1993). Biochemistry of phosphoinositides. Annu. Rev. Plant Physiol. 44, 333-356.

Diehl, R.W., Potter, J., Gee, N., Ragan, C.I., Linemeyer, D., Schoepfer, R., Bennett, C., and Dixon, R.A. (1990). Cloning and expression of bovine brain inositol monophosphatase. J. Biol. Chem.

Dr#bak, B.K. (1993). Plant phosphoinositides and intracellular signaling. Plant Physiol. 102, 705-709.

Eisenberg, F.J. (1967). o-myo-lnositol i-phosphate as product of cy- clization of glucose 6-phosphate and substrate for a specific phosphatase in rat testis. J. Biol. Chem. 242, 1375-1382.

pp. 24-46.

1421-1427.

41 1-420.

265, 5946-5949.

Fabiny, J.M., Chinault, A.C., and Barnes, E.M., Jr. (1991). Ammo- nium transport of E. coli: Localization and nucleotide sequence of the amtA gene. J. Gen. Microbiol. 137, 983-989.

Geever, R.F., Huiet, L., Baum, J.A., Tyler, B.M., Patel, V.B., Rutledge, B.J., Case, N.E., and Giles, N.H. (1989). DNA sequence, organization and regulation of the qa gene cluster of Neurospora crassa. J. MOI. Biol. 207, 15-34.

Gillaspy, G., Ben-David, H. , and Gruissem, W. (1993). Fruits: A developmental perspective. Plant Cell 5, 1439-1451.

Glaser, H.U., Thomas, D., Gaxiola, R., Montrichard, Y.S., Surdin- Kerjan, Y., and Serrano, R. (1993). Salt tolerance and methionine biosynthesis in Saccharomyces cerevisiae involve a putative phos- . phatase gene. EMBO J. 12, 3105-3110.

Gross, W., and Boss, W.F. (1993). lnositol phospholipids and signal transduction. In Control of Plant Gene Expression, D.f?S. Verma, ed (Boca Raton, FL: CRC Press), pp. 17-32.

Gumber, S.C., Loewus, M.W., and Loewus, F.A. (1984). Furtherstudies on myo-inositol-I-phosphatase from the pollen of Lilium longiflorum Thumb. Plant Physiol. 76, 40-44.

Hallcher, L.M., and Sherman, W.R. (1980). The effects of lithium ion and other agents on the activity of myo-inositol-I-phosphatase from bovine brain. J. Biol. Chem. 255, 10896-10901.

Hawkins, AR., Lamb, H.K., Smith, M., Keyte, J.W., and Roberts, C.F. (1988). Molecular organization of the quinic acid utilization (QUT) gene cluster in Aspergillis nidulans. MOI. Gen. Genet. 214,224-231.

Hilgenan, R.H., Fullerr, W.H., True, L.F., Sharples, G.C., and Smith, P.F. (1970). Lithium toxicity in ‘Marsh grapefruit in Arizona. J. Am. SOC. Hort. Sci. 95, 248-251.

Hirayama, T., Ohto, C., Mizoguchi, T., and Shinozaki, K. (1995). A gene encoding phosphatidyl-specific phospholipase C is induced by dehydration and salt stress in Arabidopsis fhaliana. Proc. Natl. Acad. Sci. USA 92, 3903-3907.

Hoad, G.V. (1995). Transport of hormones in the phloem of higher plants. Plant Growth Regul. 16, 173-182.

Inhorn, R.C., and Majerus, P.W. (1987). Effects of lithium on phosphoinositide metabolism in vivo. J. Biol. Chem. 262, 15946-15952.

Johnson, M.O. (1994). The Arabidopsis rhaliana myo-inositol l-phosphate synthase. Plant Physiol. 105, 1023-1024.

Johnson, M.D., and Sussex, I.M. (1995). li-myo-lnositol I-phosphate synthase from Arabidopsis thaliana. Plant Physiol. 107, 613-619.

Joseph, S.K., Esch, T., and Bonner, W.D. (1989). Hydrolysis of inositol phosphates by plant extracts. Biochem. J. 264, 851-856.

Kao, K.R., Masiu, R.P., and Elinson, R. (1986). Respecification of pattern in Xenopus laevis embryos-A nove1 effect of lithium. Na- ture 322, 371-373.

Livingston, B.T., and Wilt, F.H. (1990). Range and stabilityof cell fate determination in isolated sea urchin blastomeres. Development 108,

Loewus, F.A. (1990). Structure and occurrence of inositols in plants. In lnositol Metabolism in Plants, D.J. Morre, W.F. Boss, and F.A. Loewus, eds (New York: Wiley-Liss), pp. 1-11.

Loewus, F.A., and Loewus, M.W. (1983). myo-lnositol: Its biosynthesis and metabolism. Annu. Rev. Plant Physiol. 34, 137-161.

Loewus, M.W., and Loewus, F.A. (1982). myo-lnositol-I-phosphatase from the pollen of Lilium longiflorum Thumb. Plant Physiol. 70,

Majerus, P.W. (1992). lnositol phosphate biochemistry. Annu. Rev. Bio-

403-410.

765-770.

chem. 61, 225-250.


Maslanski, J.A., b s h b , L., and Busa, W.B. (1992). Lithium-sensitive production of inositol phosphates during amphibian embryonic meso- derm induction. Science 256, 243-245.

Matsuhisa, A., Suzuki, N., Noda, T., and Shiba, K. (1995). lnositol monophosphatase activity from the Escherichia colisuh6 gene product. J. Bacteriol. 177, 200-205.

McAllister, G., Whiting, P., Hammond, E.A., Knowles, M.R., Atack, J.R., Bailey, F.J., Maigetter, R., and Ragan, C.I. (1992). cDNA cloning of human and rat brain myo-inositol monophosphatase: Ex- pression and characterization of the human recombinant enzyme. Biochem J. 284, 749-754.

Memon, A.R., Rincon, M., and Boss, W.F. (1989). lnositol trisphosphate metabolism in carrot (Daucus carota L.) cells. Plant Physiol.

Michell, R.H. (1986). lnositol lipids and their role in receptor function: History and general principles. In Phosphoinositides and Receptor Mechanisms, R.W. Putney, ed (New York: Alan R. Liss, Inc.), pp. 1-24.

Murguia, J.R., Belles, J.M., and Serrano, R. (1995). A salt-sensitive 3'(2'),5'-bisphosphate nucleotidase involved in sulfate activation. Science 267, 232-234.

Narita, J.O., and Gruissem, W. (1989). Tomato hydroxymethylglutaryl- COA reductase is required early in fruit development but not during ripening. Plant Cell 1, 181-190.

Neuwald, A.F., York, J.D., and Majerus, P.W. (1991). Diverse proteins homologous to inositol monophosphatase. FEBS Lett. 294, 16-18.

Nieuwkoop, P.D. (1973). The organization center of the amphibian em- bryo: Its origin, spatial organization and morphogenetic action. Adv. Morphog. 10, 1-39.

Parthasarathy, L., Vadnal, R.E., Parthasarathy, R., and Shyamala Devi, C.S. (1994). Biochemical and molecular properties of lithium- sensitive myo-inositol monophosphatase. Life Sci. 54, 1127-1142.

Peters, D.J.M., Van Lookeren Campagne, M.M., Van Haastert, P.J., Spek, M.W., and Schaap, P. (1989). Lithium ions induce prestalk- associated gene expression and inhibit prespore gene expression in Dictyostelium discoideum. J. Cell Sci. 93, 205-210.

Ragan, I., Watllng, K., Gee, N., Aspley, S., Jackson, R., Reid, G., Baker, R., Billington, D., Barnaby, R., and Leeson, 1. (1988). The dephosphorylation of inositol 1,4-bisphosphate to inositol in liver and

91, 477-480.

brain involves two distinct Li+-sensitive enzymes and proceeds via inositol 4-phosphate. Biochem. J. 249, 143-148.

Reinecke, D.M., and Bandurski, R.S. (1987). Auxin biosynthesis and metabolism. In Plant Hormones and Their Role in Plant Growth and Development, P.J. Davies, ed (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. 24-42.

Salisbury, F.B., and Ross, C.W. (1978). Phloem transport. In Plant Physiology, J. Carey, ed (Belmont, CA: Wadsworth Publishing), pp.

Sasaki, K., and Taylor, I.E.P. (1986). myo-lnositol synthesis from [I-3H]glucose in Phaseolus vulgaris L. during early stages of germination. Plant Physiol. 81, 493-496.

Schuster, G., and Gruissem, W. (1991). Chloroplast mRNA 3' end processing requires a nuclear-encoded RNA-binding protein. EMBO J. 10, 1493-1502.

Sherman, W.R., Gish, B.G., Honchar, M.P., and Munsell, L.Y. (1986). Effects of lithium on phosphoinositide metabolism in vivo. FASEB

Trewavas, A., and Gilroy, S. (1991). Signal transduction in plant cells. Trends Genet. 7, 356-361.

Vernon, D.M., and Bohnert, H.J. (1992). A nove1 methyl transferase induced by osmotic stress in the facultative halophyte Mesem- bryanthemum crystallinum. EMBO J. 11, 2077-2085.

Wreggett, K.A. (1992). lnositol monophosphatase is a highly conserved enzyme having localized structural similarity to both glycerol-3- phosphate dehydrogenase and haemoglobin. Biochem. J. 286, 147-152.

Yamamoto, Y.T., Conkling, M.A., Sussex, I.M., and Irish, V.F. (1995). A cDNA related to animal phosphoinositide-specific phospholipase C genes. Plant Physiol. 107, 1029-1039.

Yano, R., Nagai, H., Shiba, K., and Yura, T. (1990). A mutation that enhances synthesis of sigma 32 and suppresses temperature sensitive growth of the rpoH15 mutant of E. coli. J. Bacteriol. 172,

York, J.D., and Majerus, P.W. (1990). lsolation and heterologous expression of a cDNA encoding bovine inositol polyphosphate I-phosphatase. Proc. Natl. Acad. Sci. USA 87, 9548-9552.

93-108.

J. 45, 2639-2646.

21 24-21 30.

DOI 10.1105/tpc.7.12.2175 1995;7;2175-2185Plant Cell

G E Gillaspy, J S Keddie, K Oda and W GruissemPlant inositol monophosphatase is a lithium-sensitive enzyme encoded by a multigene family.

This information is current as of March 11, 2019

Permissions 8X

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw153229

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

http://www.plantcell.org/cgi/alerts/ctmain

http://www.plantcell.org/cgi/alerts/ctmain

http://www.aspb.org/publications/subscriptions.cfm

plant lnositol monophosphatase 1s a lithium … plant cell, vol. 7, 2175-2185, december 1995 o 1995...

Documents