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a Dipartimento di Produzione Vegetale, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy
b Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy
c Dipartimento di Genetica e Microbiologia, Università degli Studi di Pavia, Via Ferrata 1, 27100 Pavia, Italy
* Corresponding author (nielsen{at}ipvgen.unipv.it)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These mutants are quite interesting, as they may be used to obtain information on the role and mechanisms of action of myo-inositol and its derivatives in both plant and seed physiology. For instance, it has been shown that the genetic lesion causing the LR33 soybean mutation is a single base change in the third base of the codon for the amino acid residue 396, which decreases the specific activity of the seed-expressed myo-inositol 1-phosphate synthase by about 90% (Hitz et al., 2002). The LR33 mutation also confers a seed phenotype characterized by increased P and decreased phytic acid and induces a decrease in the level of raffinosaccharides, which are synthesized from myo-inositol. In cereals, although the molecular lesions have not been discovered, the lpa-2 mutations affect some of the six phosphorylation steps involved in the synthesis of phytic acid from myo-inositol. Hatzack et al. (2001) reported that D/L-Ins(1,3,4,5)P4 accumulates in lpa-2 barley mutant seeds without affecting their germination vigor. All lpa-1 mutations affect the first committed step in inositol biosynthesis, i.e., the production of myo-inositol-3-phosphate (Ins3P) from glucose-6-phosphate (G6P), catalyzed by the enzyme myo-inositol-3-phosphate synthase (Ins3P synthase, MIPS) (Loewus et al., 1990; Yoshida et al., 1999; Loewus and Murthy 2000). The product of this reaction, Ins3P, is then dephosphorylated by inositol monophosphatase to yield myo-inositol, which in addition to being phosphorylated to hexakis phosphate (phytic acid) during seed maturation, plays a central role in several metabolic processes and in signal transduction in the plant cell (Johnson and Wang, 1996; Raychaudhuri and Majumder 1996; Majumder et al., 1997; Raychaudhuri et al., 1997).
Thus, the free myo-inositol level may influence both plant growth and development and responses to variations in environmental conditions in different ways (Munnik et al., 1998; Stevenson et al., 2000). For instance, perturbation in growth patterns may occur because of alterations in cell wall extensibility caused by variations in myo-inositol-derived wall components. Likewise, tolerance to temperature stresses may be altered as a result of changes in membrane composition brought about by variations in the supply of myo-inositol-derived membrane components (for reviews see Drøbak, 1992; Loewus and Loewus, 1983). Smart and Flores (1997) investigated this by generating transgenic Arabidopsis plants overexpressing MIPS and, thus, containing elevated levels of free myo-inositol (over four fold compared with wild-type plants). Their hope was to disclose an increased salt tolerance in transgenic plants. However, the higher amount of myo-inositol did not result in salt tolerance or alteration of a number of other plant characteristics linked with putative functions of myo-inositol-derived metabolites.
Nevertheless, other approaches based on the generation of transgenic plants, in which an antisense RNA strategy allows the suppression of Ins3P synthase activity, or based on the study of MIPS defective mutants, might produce more information. These approaches might disclose the consequences of myo-inositol deficiency on both phytic acid and raffinosaccharide accumulation as well as on any process influenced by inositol-derived compounds.
The antisense technique has been applied (Keller et al., 1998) to obtain transgenic potato (Solanum tuberosum L.) plants in which MIPS activity in leaves was suppressed to below 20% of the wild-type level, leading to extremely low levels of myo-inositol, galactinol, and raffinose (approximately 7, 5, and 12% of wild-type values, respectively). These plants exhibited reduced apical dominance, altered leaf morphology, precocious leaf senescence, as well as a decrease in overall tuber yield, thus indicating a crucial role for myo-inositol in plant physiology and development.
Regarding MIPS defective mutants, lpa type 1 mutations appear to affect the activity or the amount of MIPS, so they are suitable for evaluating the effects of myo-inositol shortage on cell and plant growth. We disclosed a single, recessive mutation (named lpa241) in maize, which confers a typical lpa-1 seed phenotype: increased inorganic phosphate and decreased phytic acid, with neither accumulation of hypophosphorylated intermediates, nor major variations of total P, suggesting the occurrence of an alteration in the activity or the expression of MIPS (Pilu et al., 2003). The genetic characterization of the mutation showed that it maps in maize chromosome 1S in the same location as the lpa-1 mutant. RT-PCR analysis showed that expression of the MIPS1S gene in the lpa241 mutant is weaker than in the wild type, pointing to a mutation affecting the activity of this gene. Moreover, the same analysis showed that MIPS1S gene expression is not limited to the kernel but is also evident in the seedling (Pilu et al., 2003). In a more recent work by Shukla et al. (2004), a MIPS expression decrease was shown to occur in another low phytic acid lpa-1 type maize mutant.
In this study, we present results of further genetic and molecular characterization experiments as well as of histological and physiological analysis performed on the lpa241 mutant, which provide new information concerning the nature and the pleiotropic effects of lpa-1 mutations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Assay for High Phosphate Levels in Maize Kernels
Seeds were individually ground in a mortar with a steel pestle. A total of 100 mg of the resulting flour was then extracted with 1 mL 0.4 M HCl at 4°C overnight. Samples were mixed briefly and 100 µL were removed and supplemented with 900 µL Chen's reagent (12 M H2SO4: 2.5% ammonium molybdate: 10% ascorbic acid: H2O [1:1:1:2,v/v/v/v]) in microtiter plates (Chen et al., 1956). In the case of high phosphate content, a dark-blue colored phosphomolybdate complex formed in 1 to 2 h.
Quantitative Determination of Seed Phosphorus and Inositol Phosphorus Fractions
Total seed P was determined following wet-ashing of flour aliquots (50–150 mg) and colorimetric assay of digested P (Chen et al., 1956). A modification of the ferric precipitation method was used for quantitative determination of the inositol P fraction (Raboy et al., 1990). Samples of dried mature seeds were ground and stored in a desiccator until analysis. Samples of flour (50–200 mg) were extracted in 3 mL 0.4 M HCl containing 0.7 M Na2SO4 with magnetic stirring (room temperature, overnight). Following centrifugation (8000 g for 10 min), 1 mL of supernatant was placed in a Corex tube and 0.5 mL of a 15 mM FeCl3: 0.2 M HCl solution was added. The mixture was placed in a boiling water bath for 30 min. The ferric phytate precipitate obtained after centrifugation (8000 g for 10 min) was washed with 0.2 M HCl, digested to completion on a hot plate with H2SO4 and H2O2 as needed, and diluted with distilled H2O. Phytic acid phosphorous in the digests was determined colorimetrically (Chen et al., 1956).
Trichloroacetic Acid (TCA) Extraction of Inositol Phosphates and TLC Analysis
Extraction was performed by suspending 40 mg portions of flour prepared as described above in a 10-fold excess of ice-cold TCA extraction buffer [10% (w/v) TCA, 5 mM NaF, 5 mM EDTA]. Samples were shaken for 1 h at 4°C and centrifuged at 8000 g for 5 min at 4°C. Pellets were resuspended in 10 fold excess of ice-cold TCA extraction buffer and shaken by magnetic stirring at room temperature for another 1.5 h. Supernatants from both extraction rounds were pooled and TCA was largely removed by four consecutive ether extractions. Inositol phosphates analysis by TLC was performed as described by Rasmussen and Hatzack (1998).
DNA Isolation and Southern Analysis
Genomic DNA isolation and Southern analysis were performed as previously described by Sambrook et al. (1989). Genomic DNA was extracted from leaves of lpa241 homozygous and wild-type plants and digested with ApaI, EcoRI, KpnI, MluI, SmaI, SalI, MpsI, and PvuII restriction enzymes. The filters were probed with a 3' MIPS1S probe, a 925-bp amplified fragment.
Cloning and Sequence Analysis
The MIPS1S double stranded genomic sequence was determined by sequencing amplified products. The polymerase chain reaction (PCR) was performed in a 50-µL volume containing about 50 ng of genomic DNA; 1x polymerase buffer; 2.5 mM MgCl2; 200 µM each of dATP, dCTP, dGTP, and dTTP; 0.1 µM of each primer; and 1 unit of Pfu DNA polymerase (Stratagene, La Jolla, CA). After the first denaturation step (5 min at 94°C), the reaction mix underwent 33 cycles of denaturation at 94°C for 45 s, annealing at 66°C for 1 min, and extension at 72°C for 2 min. A final extension at 72°C for 10 min was performed to complete the reaction. The 3618-bp genomic PCR fragment obtained from homozygous lpa241 was subcloned into a Blunt II-TOPO vector (Invitrogen, Carlsbad, CA). The alignment was performed using the ClustalV package (Higgins et al., 1992). Restriction site analyses were performed by Webcutter 2.0. The sets of primers used were set 1: Zm-85 (upstream primer 5'-AGCCTCCTTCCTCCTCTCAC-3', position-85) and Zm1580 (downstream primer 5'-GTTCCCTTCCAGCAGCTAAC-3', position +1580); set 2: Zm1302 (upstream primer 5'- GCTCTTGGCTGAGCTCAGCA-3', position +1302) and Zm1580 (downstream primer 5'-GTTCCCTTCCAGCAGCTAAC-3', position +1580). Set 1 was used to clone 3618 bp lpa241 genomic sequences and Set 2 was used for PCR amplifications of sequences and cleaved amplified polymorphic sequence (CAPS) cosegregation analysis
CAPS Cosegregation Analysis
The F2 segregating populations were used to perform segregation analysis. The F2 seeds were obtained from selfing F1 (ACR Lpa241/lpa241 x B73) plants. The seeds were cut into two halves and one half was crushed and assayed for the HIP (high inorganic phosphate) phenotype, while the other half was used for DNA extraction (Dellaporta et al., 1983). The PCR reaction was performed in a 25-µL volume containing about 50 ng of genomic DNA; 1x polymerase buffer; 2.5 mM MgCl2; 200 µM each of dATP, dCTP, dGTP, and dTTP; 0.1 µM of each primer (Set 2); and 0.25 unit of Taq DNA polymerase. After the first denaturation step (5 min at 94°C) the reaction mix underwent 33 cycles of denaturation at 94°C for 45 s, annealing at 64°C for 1 min, and extension at 72°C for 1 min. Ten microliters of PCR products were digested by incubation with MscI as recommended by the enzyme supplier. The DNA fragments were fractionated by electrophoresis in 2.5% (w/v) agarose gels in Tris Borate EDTA (TBE) buffer.
Histological Analysis
For light microscopy studies, mature, dry, wild-type, and mutant seeds were soaked in water for 18 h and subsequently fixed for 24 h in freshly prepared 3:1 100% ethanol:glacial acetic acid at 4°C. The fixed material was placed in 70% (v/v) ethanol and stored at 4°C until processed. After dehydration in an ethanol series and embedding in Paraplast Plus (Ted Pella, Inc. and Pelco International, Redding, CA), sections were cut at 15 µm, serially arranged, and stained with saphranine-fast green as described by Ruzin (1999). Phytic acid-containing particles (globoids) were stained with toluidine blue O (0.05% TBO in PO4 buffer, pH 5.5) before removal of the paraffin from the sections as described by Ruzin (1999).
Embryo Rescue
Mature dry seeds were sterilized with 5% (v/v) sodium hypochlorite for 15 min and then rinsed in sterile distilled water overnight. Embryos were removed aseptically and transferred to Murashige and Skoog (MS, 1962) salt mixture (pH 5.6) containing 2% (w/v) sucrose, solidified with 0.8% (w/v) agar (Plant agar, Duchefa, Haarlem, the Netherlands) in which myo-inositol (100 mg L–1) was added or omitted. Cultures were incubated in a growth chamber at 25°C with a 14/10 light/dark photoperiod and monitored for germination percentage and seedling elongation after 14 d. The light source consisted of four cool white (F36T12/CW/HO) fluorescent lamps from GTE SYLVANIA (Lighting Products Group, Danvers, MA). The distance between light sources and seeds was 50 cm. The light intensity was 0.785 µmol m–2 s–1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To test this hypothesis (i.e., MIPS1S candidate gene for lpa-1 mutation), the MIPS1S double stranded genomic sequence was determined by sequencing subcloned amplified products. The comparison of the MIPS1S wild-type genomic sequence (GenBank accession AF323175) with our lpa241 MIPS1S clones showed the presence of 10 single nucleotide mismatches: five fell within introns (A 482 to T, T 659 to C, G 1870 to A, T 2028 to C, A 2111 to G), two represented silent mutations (C 3487 to A, C 3503 to T), two fell in the 3' UTR region (A 3575 to G, C 3585 to G), and only one caused the substitution of alanine 150 residue to valine (C 1653 to T) (Fig. 1). To verify whether the polymorphisms were caused by the EMS treatment, we also sequenced the polymorphic region of the MIPS1S gene from the original ACR Lpa241 line. Alignment obtained by sequencing amplified products from genomic DNA showed that the EMS treatment did not cause a mutation in the analyzed gene region, clearly indicating that the ten mismatches represented line polymorphisms (data not shown).
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Penetrance and Expressivity of lpa241 Mutation and Correlation with Negative Pleiotropic Effects
Progeny from crosses between lpa241 mutants and four inbred lines segregated 3:1 for the HIP phenotype: high free P phenotype, demonstrating 100% penetrance of the mutation (Table 1). Quantitative determination of seed free P and phytic acid P fractions showed interfamily differences that were not significant at the 95% confidence interval, except for the phytic acid P level of the K6 family (Table 2). Then, free P was assayed in individual mature lpa241 homozygous seeds from each of the 20 Lpa241/lpa241 self-pollinated families (BC2F2) and six Lpa241/Lpa241 selfed B73 families as controls. Results demonstrate that the mutation is characterized by a remarkable intraline variability of expression (Fig. 2).
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Longitudinal sections of rehydrated mature lpa241 kernels, stained with saphranine-fast green (Fig. 6B), revealed an embryo that was smaller than the wild type (Fig. 6A) and had a minor displacement of the scutellum. It would appear that the lpa241 mutation upsets the bipolar embryonic axis by displacing the root primordium from the axis and thus introducing an asymmetry into the body plan (Fig. 6B). Furthermore, a longitudinal section of a rehydrated mature lpa241 kernel, stained with toluidine blue, showed that the wild-type scutella contained many large globoids (Fig. 7A, C, E), while the lpa241 scutella contained a few tiny globoids (Fig. 7B,D,F), thus providing visible proof of the reduction in phytic acid level.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The MIPS1S gene also has been located in the same map position between the molecular RFLP markers umc 157 and umc 76 (Larson and Raboy, 1999) as one among the seven MIPS sequences (1S, 4L, 5S, 6S, 8L, 9S, and 9L) discovered in the maize genome. Data obtained from the preliminary biochemical characterization of lpa-241, especially those regarding the lack of hypophosphorylated myo-inositol intermediates, had also indicated that lpa241 is phenotypically similar to Raboy's lpa1 and not to lpa-2 mutants. Both these pieces of evidence were thus consistent with a mutation causing a myo-inositol shortage by affecting the activity or the expression of the MIPS1S maize gene. Because no molecular proof of the direct MIPS1S involvement in the lpa-1 phenotype is yet available, no final conclusion could be drawn; however, in our previous work (Pilu et al., 2003), we showed that the MIPS1S transcript level was lower in lpa-241 than in wild-type tissues of both maturing seeds and seedlings, thus providing the first molecular demonstration, even if limited to gene expression, of the MIPS1S involvement in lpa-1 mutants.
The results from sequence comparison between lpa241 and the wild type (Fig. 1) failed to reveal any changes capable of affecting protein function. This finding is in agreement with the very recent results (Shukla et al., 2004) of a similar analysis performed on the maize lpa-1 mutant, allelic to lpa241.
We observed 100% penetrance and similar expression levels of the mutation in different F2 populations obtained after introgression in several inbred lines (Table 1 and 2). However, a high intra-line lpa241 expression variability was identified (Fig. 2). When the range of lpa241 phenotypic expression was determined by genotyping seeds by means of CAPS markers (Fig. 3) and measuring the free P level in the same kernels, we noticed that in the F2 progeny, the genotypes Lpa241/lpa241 and lpa241/lpa241 displayed variability of expression, confirming that lpa241 is not fully recessive as well as that it is closely linked to MIPS1S (Table 3). Moreover, when F2 families were classified in three arbitrary classes on the basis of the amount of free P accumulated in homozygous seeds and propagated, the F3 families showed similar phenotypic expression. The yield reductions and other observed pleiotropic effects appeared proportional to the increase in free P measured in lpa241 seed. These data support a predominant role of the MIPS1S expression level in the determinism of the lpa241 mutation. Further genetic and molecular analyses will be required to correlate the mutation strength to the MIPS1S expression level, which should be inversely proportional to the kernel phytic acid level.
Three hypothesis are possible as regards the origins of the lpa241 mutant and probably of other lpa1 alleles isolated by Raboy's group: (i) the mutation is located in the promoter of the MIPS1S gene, not yet analyzed. This hypothesis is unlikely because Southern analysis did not identify any major rearrangement or extensive methylation change in the MIPS1S region of the mutant genome. Although alteration of the MIPS1S promoter could cause the lower transcript level, it does not explain the expression variability detected in the mutant progeny unless the variation is caused by ectopic expression of one or multiple MIPS sequence(s); (ii) the mutation is not located in the MIPS1S gene but in a closely linked gene controlling the MIPS1S expression level. This might partially account for the incomplete dominance of the lpa241 mutation's character because the regulator, acting in trans, could perturb the MIPS1S expression; (iii) lpa241 is an epigenetic mutation, not determined by a change in the nucleotide sequence but rather by an alteration of the methylation pattern and/or chromatin conformation of the genome region in which MIPS1S is located. This might be at the basis of both the low transcript level and the phenotypic variability observed, and might also explain the unusually high isolation frequency (10–2 to 10–3) we registered for lpa241 and two additional lpa alleles (unpublished results).
It must be emphasized that several epigenetic silencing phenomena, consisting of heritable changes in the phenotypic expression of pigment accumulation in several tissues, have been described in maize. Their features are analogous to those we have pointed out in lpa241, such as the paramutations of the loci R, B, and, most of all, PL (Brink, 1973; Coe, 1996; Hollick et al., 2000). So, lpa241 (and probably the other lpa-1 type mutations so far isolated) might be the first case of paramutation having no connections with that pathway.
In the previous lpa241 isolation and preliminary characterization work, apart from about a 30% decrease in germination rate, no other pleiotropic effects had been identified. This is most probably due to the small size of the population at our disposal and the low growth rate and yield of the ACR line in which the mutation was originally isolated. In the course of the more extensive experiments reported above, several additional pleiotropic effects were observed including the appearance of des (defective seedling) phenotypes and a decrease of seedling growth rate and yield (Fig. 5). Some of these phenomena appear in developmental stages other than seed maturation and seem to have no relation to the decrease or lack of phytic acid accumulation. This is in agreement with the data obtained by Raboy and Dickinson (1987), who grew soybean plants under P starvation to produce seeds with a low phytic acid level. They found no effects on germination rate or on seedling growth. As MIPS1S expression is also not restricted to the maturing seed (Pilu et al., 2003), it may be conjectured that the gene corresponding to the lpa241 mutation (quite probably, MIPS1S) performs important functions throughout the life cycle of the maize plant.
The same observations were made by Keller et al. (1998), who used potato StIPS-1 cDNA (corresponding to MIPS gene) to make an antisense construct allowing the consequences of reduced MIPS level in transgenic potato plants to be verified. As expected, the myo-inositol levels in leaves and tubers of transformant plants were much lower than controls and this correlated with the occurrence of several pleiotropic effects similar to those we observed in lpa241 mutants.
These data indicate that the MIPS1S gene, although the only MIPS gene so far recognized as being involved in the synthesis of phytic acid, seems to have additional functions that, if lost, may not be fully compensated by the other MIPS genes known to be present in the maize genome (Larson and Raboy, 1999).
Further information on the consequences of lpa241 on seed maturation and germination has been provided by the histological analysis we performed to look for any morphological difference among wild-type and mutant embryos that could explain the relationship between the decrease in phytic acid level and the decrease of germination shown by the mutant seeds. Mutant embryos showed a reduction in size and a displacement of the embryo axis that caused an imperfect alignment of the shoot and root primordia (Fig. 6). Moreover, in accordance with the results obtained by TLC and HPLC analysis (Pilu et al., 2003), a dramatic reduction in globoid number and size (and thus of phytic acid amount) was evident in the mutant compared with the wild-type scutellum (Fig. 7). This finding is somewhat similar to those described by Ockenden et al. (2004) in the case of barley, and by Liu et al. (2004) in that of rice, and proves that the phytic acid reduction in lpa241 kernel is not simply caused by the reduced size of the storage tissue, the embryo, but by a specific decrease of globoid number and/or size.
Embryo-rescue experiments (Table 4) revealed that germination could be restored if excised lpa241 embryos are cultured in MS medium, although they grew slower than the wild type and some defective seedlings were observed. A possible explanation of these data is that lpa241 may induce a deficiency of a compound, which is contained in MS medium. As lpa241 mutation may cause a myo-inositol shortage during seed maturation and germination and this compound plays a central role in several metabolic processes and, as the Ins3P precursor, in signal transduction, it was conceivable that myo-inositol was the deficient nutrient. This hypothesis was refuted by the results obtained from embryo rescue experiments using MS medium lacking myo-inositol because the germination rates were not significantly lower than those previously obtained with the myo-inositol containing MS medium (data not shown). A possible alternative hypothesis might be that the removed endosperm contains a metabolite able to reduce germination.
Overall, the data presented in this paper point out new developments and perspectives in the field of lpa mutants research. Genetic analysis of the lpa241 mutant shows high expression variability and the probable epigenetic nature of the mutation. Physiological analyses showed mainly the pleiotropic effects of the MIPS shortage. Histological analyses showed a reduction of globoid number and size, and embryo abnormalities. Embryo rescue techniques provide a practical way to propagate the lpa mutants.
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ACKNOWLEDGMENTS |
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Received for publication November 8, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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