myo-Inositol is an essential component in the biosynthesis of an array of derivatives ranging from simple inositol phosphates to complex membrane-associated products with important cellular functions. It can be isomerized and (or) methylated to form a variety of species-specific epimers and methyl ethers. A number of these, including Ins, have been noted to accumulate mostly in osmotically challenged plants and have since been recognized as osmoprotectant metabolites [1].

Ins is also central to the biosynthesis of a number of antinutritional components such as sucrose galactosides (e.g. RFO) and inositol polyphosphates such as PhA (also known as InsP₆). The synthesis of PhA predominates in developing seeds, and constitutes the major storage form of seed phosphorus. Both Ins and its bound phosphates are released by hydrolysis upon germination.

The antinutritional properties of PhA reside in its strong binding affinity for positively charged species such as essential minerals (e.g. iron and zinc) and proteins, significantly lowering their bioavailability to humans and animals. As a consequence, the presence of high levels of PhA in canola seed hinders the full exploitation of the pure meal and underrates its potential as a major crop worldwide.

In contrast, PhA has been accredited as an effective antioxidant with antitumor properties and risk reduction of certain types of cancer [2]. Besides decreasing uncontrolled cellular proliferation, PhA is also thought to cause differentiation of malignant cells resulting in reversion to the normal phenotype [3]. Further, PhA has been shown to play a critical role in many cellular events such as signaling [4], apoptosis [5, 6], neuroprotection [7], as well as functioning as enzyme cofactor [8]. The antioxidant properties of PhA have also been shown to inhibit free radical formation and lower lipid peroxidation, making it a very efficient natural food and feed preservative [9]. Moreover, PhA has been shown recently to protect developing seeds against oxidative stress [10]. Because of the seemingly paradoxical and unique roles of PhA, and in view of the agronomic value placed on low phytate-containing seeds and (or) meals, total elimination of PhA was not our intended goal.

Phytic acid biosynthesis constitutes a uniquely complex process, consisting of a primary substrate, Ins, and a number of interjecting secondary Ins polyphosphate substrates from other sources and pathways [11, 12]. In the primary pathway, the de novo synthesis of Ins involves the oxidative cyclization of glucose 6-phosphate (G-6-P) to L-myo-inositol-1-phosphate (L-Ins-1-P) by the action of a single enzyme, L-myo-inositol-1-phosphate synthase (MIPS). It has been shown that by affecting the production of this enzyme directly or indirectly through mutagenesis in maize [13, 14] and soybean [15] or by genetic engineering methods in rice [16, 17], soybean [18] and canola [19] PhA accumulation can be reduced by 20–94.5% with a concomitant increase in inorganic phosphate (P_i). Of the various transgenic approaches reported, MIPS-RNAi [18] and MIPS-cosuppression [19] transgenics yielded the lowest levels of PhA in the corresponding mature seeds (94.5 and 44%, respectively). However, the RNAi approach reportedly has resulted in hindered seed development [18]. Thus, in view of the fact that Ins is a key substrate in the biosynthesis of many essential cell components, we decided to assess the consequences of its metabolic diversion versus its complete elimination through MIPS down-regulation, and compare the effects associated with each approach. We reasoned that while metabolic diversion of Ins may not interfere with its biosynthesis, it could preferably limit its participation in PhA production.

The first MIPS product, L-Ins-1-P, appears to be in quasi equilibrium with free Ins due to the activity of a two-enzyme system, in which L-myo-inositol-1-phosphate monophosphatase converts L-Ins-1-P to free Ins, while myo-inositol-1-kinase regenerates it (Scheme 1). Depending on the rigidity of the requirements for free Ins vs. L-Ins-1-P in the developing seed, the relative abundance of each substrate is ultimately determined by the differences in the kinetics of the two opposing reactions. We hypothesized that continual exclusion of free Ins from this quasi equilibrium by metabolic diversion through methylation, could drive the quasi equilibrium in the direction of free Ins. This would subsequently limit the participation of both substrates (Ins and L-Ins-1-P) in PhA biosynthesis. Towards this end, we studied the effect of over-expressing the gene encoding IMT (EC 2.1.1.129) from M. crystallinum[20] on seed PhA accumulation in transgenic B. napus under two different seed-specific promoters, napin and phaseolin [21]. We also examined the effect of enhancing the translational context of IMT on its gene product accumulation and PhA reduction during seed development.

Although the M. crystallinum IMT (McIMT) has been used to study the osmoprotective properties of methylated cyclitols in transgenic plants [22–25], the current investigation is the first example of seed-specific McIMT-mediated metabolic diversion to reduce phytic acid biosynthesis in seed crops through in vivo methylation of Ins.

The relative incorporation of ³H-Ins in the different fractions of developing B. napus seeds, presented in Figure 1, depicts an image of the dynamics of Ins participation in phospholipid and PhA biosynthesis at early to mid stages of development. The data suggests the occurrence of horizontal interconversions between ³H-Ins and its polar and non-polar phosphorylated variants. Evidence of this is seen at 20 and 30 DAP where substantial shifts seem to occur across the hexane- and acid-soluble fractions.

The observed decrease in the relative contents of the acid-soluble fraction in the period 20–25 DAP reflects the utilization of myo-inositol in the biosynthesis of non-polar compounds (e.g. phospholipids). This is supported by the observation that a parallel increase in the relative amounts of label incorporation appeared in the hexane-soluble fraction during the same period. After 30 DAP, the relative rates of incorporation in the acid-soluble fraction increased again at the expense of the non-polar fraction, reflecting the rapid accumulation of PhA during this period (Figure 2). This is likely, since hydrolysis of Ins-containing phospholipids is known to lead to increased phytate accumulation in seeds of B. napus [11].

Typically, the newly forming seed coat is made up of four layers of cells, the outermost epidermal and palisade cell layers (which develop from the outer integument of the ovule) and parenchyma and endothelial cell layers (which are derived from the inner ovule integument) [27]. At 5 DAP the majority of seed inner space is filled with differentiating integument and a small amount of endosperm. That the majority of Ins at 15–20 DAP is localized in the seed coat of dissected seeds (Figure 3B) and that the bulk of the seed at 5–15 DAP is primarily made up of this tissue may be construed as evidence that the seed coat is the primary site of Ins biosynthesis in the B. napus seed. In support of this is the abundant supply of sucrose, which is transported through the phloem, apoplastic region and the seed coat during this early stage [28, 29] and which, through hydrolysis, acts as source of G-6-P, the precursor for Ins.

As the embryo expands through its initial globular, heart, torpedo and bent cotyledon stages, the endosperm proportionally shrinks (Figure 3C), and Ins becomes localized mainly in the embryo and seed coat (Figure 3B). That the seed coat is the tissue where Ins is most abundant at the early stages of seed development is of special importance since it plays a major role in the biosynthesis of mucilage, other seed coat polysaccharides and sugar acids through its participation in the oxidative pathway [30]. PhA synthesis started at very early stages (<10DAP) and reached its maximum levels at about 35 DAP (Figure 2). Distribution analysis of PhA accumulation between cotyledons and embryo axes revealed that from 20 to 75 DAP more than 80% of PhA is accumulated in cotyledons (80% at 20–25 DAP, increasing up to 90% after 30 DAP) (data not shown). At 25–30 DAP, as the Brassica embryo expanded, higher accumulation of the non-polar derivatives (needed for membrane biogenesis) occurred (Figure 1). This period (25–30 DAP) appears to be marked by a slower decline in Ins levels, nearly reaching a plateau (Figure 3A), while, in parallel, PhA levels continued to rise at a relatively slower rate. After 25 DAP and through Ins appears to have reached a stable level in embryo, and more so in seed coat which could account for its apparent slower decline between 25 and 30 DAP (Figure 3B). At this stage the decline in Ins levels was accompanied by a concomitant rise in PhA levels (Figure 2) at the expense of the non-polar components (Figure 1) as the seed approached desiccation through the onset of RFO biosynthesis (30-35DAP) (unpublished results).

Since the initial phosphorylation steps of free Ins commence either with the reconstitution of L-Ins-1-P or through other positional phosphate esters, our strategy was to investigate the effect of competitive metabolic shunt of Ins on its phosphorylation and subsequent PhA and RFO accumulation in canola seeds. To accomplish this, we have explored the conversion of Ins into ononitol (1-D-4-O-methyl-myo-inositol), by methylation at the D-4 position, through the action of the IMT enzyme.

The observed variations in PhA reduction levels do not necessarily reflect different IMT expression levels since all selected lines displayed almost uniform levels of the IMT protein at the time of sampling as shown by Western blot analysis (Figure 4A and 4B). That the recorded ranges of PhA reduction appear to be similar with either promoter (Figure 6) suggests one of two possible scenarios: a) A certain threshold may exist at which a steady balance between supply and removal of Ins is reached, which confines PhA reduction levels within the observed limits; b) The temporal appearance of IMT activity under these promoters may not be in synchrony with the highest point of Ins accumulation, which is presumably reached in less than 10 DAP (Figure 3A), leading to the early onset of PhA synthesis. The latter scenario is likely, since the IMT protein was not detected until 25 DAP in lines Ph1-18 and Ph2-15 (Figure 4A) in contrast to PhA accumulation which is shown to be in progress at 15–20 DAP (Figure 2). A similar result was obtained when a MIPS antisense transcript was expressed in transgenic rice driven by the glutelin GluB-1 promoter [17].

The phaseolin promoter was chosen based on its reported early transcriptional activation in transgenic systems such as tobacco (15–16 DAP) [31, 32]. The napin promoter which is native to Brassica was chosen with a view to comparing the effect of temporal expression differences of the two promoters on PhA accumulation. However, because IMT transcription from the phaseolin promoter did not commence as early as in the case of transgenic tobacco, it was not possible to assess such differences. The complex architecture of the phaseolin promoter has been shown to play a major role in spatial regulation of this promoter in transgenic systems [33]. Further, seed-specific transcriptional regulatory regions in the same promoter have been identified which affect its activation in a temporally dependent manner [34, 35]. Therefore, it is possible that the phaseolin promoter, when expressed in different systems, could be affected by elements that may impose temporal expression variations. This is suggested by the fact that, in our hands, the transcriptional activation of phaseolin-IMT was triggered in canola at a later time point (20 DAP, Figure 4A) than in tobacco, indicating that this promoter could be influenced by developmentally regulated programs in a host-specific manner.

Attempts at enhancing the IMT translation efficiency through the modification of its translational context [36, 37] resulted in somewhat improved translation levels at 20 DAP under the phaseolin promoter when a dA nucleotide was positioned at the -3 position and a dC nucleotide at each of positions -2 and -1 of the initiation codon (transgenic line Ph3-19, Table 1 and Figure 4A). Translation levels at subsequent stages in the same line also appear to have been relatively enhanced. However, in spite of the protein level enrichment at 20 DAP in line Ph3-19, this did not substantially improve the overall PhA reduction, confirming the need for a more temporally and (or) spatially synchronized IMT expression and Ins production, as opposed to early enhancement of IMT translation. Nevertheless, while the apparent compartmentalization of Ins in the seed coat may shield it from IMT action in the embryo, seed-specific promoters are known to drive gene expression in the inner layer of seed coats and there is active transport of Ins from seed coat to embryo [38].

Absence of ononitol in wild-type HPLC chromatograms and failure of the wild-type protein extracts to produce ononitol, together with the Western analysis results suggest possible absence of native IMT-like activity in B. napus.

In mature transgenic seeds, the level of Ins was not changed significantly (Figure 8) but presumably during maturation was partitioned among PhA, galactinol and ononitol biosyntheses (Figure 5B). Accordingly, PhA formation was decreased in concert with ononitol accumulation. This pathway perturbation (Scheme 1) would have lowered the production of galactinol as well, and consequently, RFO accumulation [40] with the notable increase in sucrose levels (Figure 8). Curiously, the decrease in raffinose levels was accompanied by an almost proportionate rise in stachyose. Since sucrose levels remained high in transgenic seeds, the inverse modulation in the levels of raffinose and stachyose could indicate raffinose as a possible galactosyl donor for the subsequent chain elongation in a raffinose:raffinose galactosyltransfer manner, in which a molecule of raffinose would release its sucrose moiety after each galactosyl-residue transfer to another raffinose molecule (Figure 9B) [41, 42]. This is conceivable in view of the limited availability of galactinol (Figure 8). Alternatively, since methylated derivatives of galactinol are known to take part in RFO synthesis in some plants [43], and although not hitherto proven in B. napus, we propose that ononitol, the methylated product of IMT, may be utilized to form the corresponding galactosylononitol (methylated galactinol), which could still participate, in part, in RFO-chain elongation to the higher oligomer, stachyose (Figure 9A). Since this does not account for the observed increase in sucrose levels, we postulate that both routes may be working concertedly. The differential extent of stachyose and sucrose accumulation (Figure 8) could be indicative of the different kinetics of the two routes (Figure 9A and 9B).

Early stages of Brassica seed development appear to be dominated by reciprocal interconversions of polar Ins phosphates and non-polar species. Although Ins is shown to be produced in significant amounts in the endosperm as well as seed coat during the early stages, no PhA accumulation occurs in those tissues. Instead, PhA accumulation appears to be mainly restricted to the embryo throughout seed development and maturation.

In addition to lowering PhA levels in the developing seeds, the competitive methylation of Ins resulted in changes in the distribution and accumulation patterns of seed carbohydrates, leading to enhancement of the digestible and metabolizable energy profile of the meal as demonstrated by the higher content of the nutritionally useful sugar, sucrose. While the ratio of raffinose to stachyose was altered, the overall balance of RFO appears to be unaffected. No deleterious effects were encountered as a result of Ins methylation in developing Brassica seed.

It is evident from this study as well as previous ones that one of the answers to reducing phytate in crop seeds to a level which allows an appreciable beneficial effect on phosphorous and microelements bioavailability without adversely affecting yield and phenotype may be found in a model which combines the additive effects of more than one mechanism acting concertedly with separate independent contributions. Such an approach would allow selective inhibition of Ins phosphorylation at different steps for optimum phytate reduction. Nonetheless, the current strategy, if adopted, could potentially raise the canola market value as well as that of other crops.