High-lysine corn generated by endosperm-specific suppression of lysine catabolism using RNAi

Because of the limited lysine content in corn grain, synthetic lysine supplements are added to corn meal-based rations for animal feed. The development of biotechnology, combined with the understanding of plant lysine metabolism, provides an alternative solution for increasing corn lysine content through genetic engineering. Here, we report that by suppressing lysine catabolism, transgenic maize kernels accumulated a significant amount of lysine. This was achieved by RNA interference (RNAi) through the endosperm-specific expression of an inverted-repeat (IR) sequence targeting the maize bifunctional lysine degradation enzyme, lysine-ketoglutarate reductase/saccharopine dehydrogenase (ZLKR/SDH). Although plant-short interfering RNA (siRNA) were reported to lack tissue specificity due to systemic spreading, we confirmed that the suppression of ZLKR/SDH in developing transgenic kernels was restricted to endosperm tissue. Furthermore, results from our cloning and sequencing of siRNA suggested the absence of transitive RNAi. These results support the practical use of RNAi for plant genetic engineering to specifically target gene suppression in desired tissues without eliciting systemic spreading and the transitive nature of plant RNAi silencing.     

Genetic manipulation of lysine catabolism in maize kernels

In plants, lysine catabolism is thought to be controlled by a bifunctional enzyme, lysine ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH). Lysine is converted to saccharopine, through condensation with alpha-ketoglutarate, by LKR, and subsequently to glutamate and alpha-aminoadipate-delta-semialdehyde by SDH. To investigate lysine catabolism in maize kernels, we generated transgenic plants with suppressed LKR/SDH activity in either endosperm or embryo. We found that the suppression of LKR/SDH in endosperm induced an increase in free lysine in developing endosperm, which peaked at 32 days after pollination. At later stages of kernel development, most of the free lysine was found in the embryo along with an elevated level of saccharopine. By combining endosperm LKR/SDH suppression with embryo LKR/SDH suppression through crosses, the saccharopine level in embryo was reduced and resulted in higher lysine accumulation in mature kernels. These results reveal new insights into how free lysine level is regulated and distributed in developing maize kernels and demonstrate the possibility of engineering high lysine corn via the suppression of lysine catabolism.     

Lysine catabolism, an effective versatile regulator of lysine level in plants

Lysine is a nutritionally important essential amino acid, whose synthesis in plants is strongly regulated by the rate of its synthesis. Yet, lysine level in plants is also finely controlled by a super-regulated catabolic pathway that catabolizes lysine into glutamate and acetyl Co-A. The first two enzymes of lysine catabolism are synthesized from a single LKR/SDH gene. Expression of this gene is subject to compound developmental, hormonal and stress-associated regulation. Moreover, the LKR/SDH gene of different plant species encodes up to three distinct polypeptides: (i) a bifunctional enzyme containing the linked lysine-ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) whose LKR activity is regulated by its linked SDH enzyme; (ii) a monofunctional SDH encoded by an internal promoter, which is a part of the coding DNA region of the LKR/SDH gene; and (iii) a monofunctional, highly potent LKR that is formed by polyadenylation within an intron. LKR activity in the bifunctional LKR/SDH polypeptide is also post-translationally regulated by phosphorylation by casein kinase-2 (CK2), but the consequence of this regulation is still unknown. Why is lysine metabolism super-regulated by synthesis and catabolism? A hypothesis addressing this important question is presented, suggesting that lysine may serve as a regulator of plant growth and interaction with the environment.     

High-lysine corn produced by the combination of enhanced lysine biosynthesis and reduced zein accumulation

Corn is one of the major crops in the world, but its low lysine content is often problematic for animal consumption. While exogenous lysine supplementation is still the most common solution for today's feed corn, high-lysine corn has been developed through genetic research and biotechnology. Reducing the lysine-poor seed storage proteins, zeins, or expressing a deregulated lysine biosynthetic enzyme, CordapA, has shown increased total lysine or free lysine content in the grains of modified corn plants, respectively. Here, by combining these two approaches through genetic crosses, the total lysine content has more than doubled in F1 progeny. We also observe a synergy between the transgenic zein reduction and the enhanced lysine biosynthesis by CordapA expression. The zein reduction plants are found to accumulate higher levels of aspartate, asparagine and glutamate, and therefore, provide excess precursors for the enhanced lysine biosynthesis.     

A novel composite locus of Arabidopsis encoding two polypeptides with metabolically related but distinct functions in lysine catabolism

Both plants and animals catabolize lysine via saccharopine by two consecutive enzymes, lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single polypeptide. We recently demonstrated that Arabidopsis plants possess not only a bifunctional LKR/SDH but in addition a monofunctional SDH enzyme. We also speculated that these two enzymes may be controlled by a single gene (G. Tang et al. Plant Cell, 1997, 9, 1305-1316). By expressing several epitope-tagged and GUS reporter constructs, we demonstrate in the present study that the Arabidopsis monofunctional SDH is encoded by a distinct gene, which is, however, nested entirely within the coding and 3' non-coding regions of the larger bifunctional LKR/SDH gene. The entire open reading frame of the monofunctional SDH gene, as well as some components of its promoter, are also parts of the translated coding sequence of the bifunctional LKR/SDH gene. These special structural characteristics, combined with the fact that the two genes encode simultaneously two metabolically related but distinct enzymes, render the LKR/SDH locus a novel type of a composite locus. Not all plant species possess an active monofunctional SDH gene and the production of this enzyme is correlated with an increased flux of lysine catabolism. Taken together, our results suggest that the composite LKR/SDH locus serves to control an efficient, highly regulated flux of lysine catabolism     

The activity of the Arabidopsis bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase enzyme of lysine catabolism is regulated by functional interaction between its two enzyme domains

Lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) is a bifunctional enzyme catalyzing the first two steps of lysine catabolism in animals and plants. To elucidate the biochemical signification of the linkage between the two enzymes of LKR/SDH, namely lysine ketoglutarate and saccharopine dehydrogenase, we employed various truncated and mutated Arabidopsis LKR/SDH polypeptides expressed in yeast. Activity analyses of the different recombinant polypeptides under conditions of varying NaCl levels implied that LKR, but not SDH activity, is regulated by functional interaction between the LKR and SDH domains, which is mediated by the structural conformation of the linker region connecting them. Because LKR activity of plant LKR/SDH enzymes is also regulated by casein kinase 2 phosphorylation, we searched for such potential regulatory phosphorylation sites using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and site-directed mutagenesis. This analysis identified Ser-458 as a candidate for this function. We also tested a hypothesis suggesting that an EF-hand-like sequence at the C-terminal part of the LKR domain functions in a calcium-dependent assembly of LKR/SDH into a homodimer. We found that this region is essential for LKR activity but that it does not control a calcium-dependent assembly of LKR/SDH. The relevance of our results to the in vivo function of LKR/SDH in lysine catabolism in plants is discussed. In addition, because the linker region between LKR and SDH exists only in plants but not in animal LKR/SDH enzymes, our results suggest that the regulatory properties of LKR/SDH and, hence, the regulation of lysine catabolism are different between plants and animals.     

Characterization of the two saccharopine dehydrogenase isozymes of lysine catabolism encoded by the single composite AtLKR/SDH locus of Arabidopsis

Arabidopsis plants possess a composite AtLKR/SDH locus encoding two different polypeptides involved in lysine catabolism: a bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) enzyme and a monofunctional SDH enzyme. To unravel the physiological significance of these two enzymes, we analyzed their subcellular localization and detailed biochemical properties. Sucrose gradient analysis showed that the two enzymes are localized in the cytosol and therefore may operate at relatively neutral pH values in vivo. Yet while the physiological pH may provide an optimum environment for LKR activity, the pH optima for the activities of both the linked and non-linked SDH enzymes were above pH 9, suggesting that these two enzymes may operate under suboptimal conditions in vivo. The basic biochemical properties of the monofunctional SDH, including its pH optimum as well as the apparent Michaelis constant (K(m)) values for its substrates saccharopine and nicotinamide adenine dinucleotide at neutral and basic pH values, were similar to those of its SDH counterpart that is linked to LKR. Taken together, our results suggest that production of the monofunctional SDH provides Arabidopsis plants with enhanced levels of SDH activity (maximum initial velocity), rather than with an SDH isozyme with significantly altered kinetic parameters. Excess levels of this enzyme might enable efficient flux of lysine catabolism via the SDH reaction in the unfavorable physiological pH of the cytosol.     

Purification and characterization of bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase from developing soybean seeds

Both in mammals and plants, excess lysine (Lys) is catabolized via saccharopine into alpha-amino adipic semialdehyde and glutamate by two consecutive enzymes, Lys-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single bifunctional polypeptide. To study the control of metabolite flux via this bifunctional enzyme, we have purified it from developing soybean (Glycine max) seeds. LKR activity of the bifunctional LKR/SDH possessed relatively high K(m) for its substrates, Lys and alpha-ketoglutarate, suggesting that this activity may serve as a rate-limiting step in Lys catabolism. Despite their linkage, the LKR and SDH enzymes possessed significantly different pH optima, suggesting that SDH activity of the bifunctional enzyme may also be rate-limiting in vivo. We have previously shown that Arabidopsis plants contain both a bifunctional LKR/SDH and a monofunctional SDH enzymes (G. Tang, D. Miron, J.X. Zhu-Shimoni, G. Galili [1997] Plant Cell 9: 1-13). In the present study, we found no evidence for the presence of such a monofunctional SDH enzyme in soybean seeds. These results may provide a plausible regulatory explanation as to why various plant species accumulate different catabolic products of Lys.     

L-Lysine catabolism is osmo-regulated at the level of lysine-ketoglutarate reductase and saccharopine dehydrogenase in rapeseed leaf discs

We have previously shown that a gene encoding for lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) is upregulated in osmotically stressed leaf discs from Brassica napus. In plants, excess lysine is catabolised by these enzymes which are linked on a single polypeptide. These findings suggested that LKR and SDH activities could be enhanced with decreasing osmotic potential. This proposal has been assessed in this study where LKR and SDH activities were determined in desalted crude extracts from rapeseed leaf discs subjected in vitro to upshock osmotic stress using polyethylene glycol (PEG) as a non-permeant osmoticum. Results reported here demonstrated that LKR and SDH activities increased in stressed material similarly to that observed for the related mRNA levels. In addition, it was shown that both activities depend on the intensity of the external osmotic stress and the duration of the applied treatment. On the other hand, during recovery of leaf discs upshocked and then downshocked, LKR and SDH activities decreased which clearly demonstrated that lysine catabolism is osmo-regulated through these activities.     

The lysine-ketoglutarate reductase-saccharopine dehydrogenase is involved in the osmo-induced synthesis of pipecolic acid in rapeseed leaf tissues.

Higher plant responses to abiotic stresses are associated with physiological and biochemical changes triggering a number of metabolic adjustments. We focused on L-lysine catabolism, and have previously demonstrated that degradation of this amino acid is osmo-regulated at the level of lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) in Brassica napus. LKR and SDH activities are enhanced by decreasing osmotic potential and decrease when the upshock osmotic treatment is followed by a downshock osmotic one. Moreover we have shown that the B. napus LKR/SDH gene is up-regulated in osmotically-stressed tissues. The LKR/SDH activity produces alpha-aminoadipate semialdehyde which could be further converted into alpha-aminoadipate and acetyl CoA. Alternatively alpha-aminoadipate could behave as a precursor for pipecolic acid. Pipecolic acid is described as an osmoprotectant in bacteria and is co-accumulated with proline in halophytic plants. We suggest that osmo-induction of the LKR/SDH activity could be partly responsible for pipecolic acid accumulation. This proposal has been assessed in this study through pipecolic acid amounts determination in rape leaf discs subjected to various upshift and downshift osmotic treatments. Changes in pipecolic acid level actually behave as those observed for LKR and SDH activities, since it increases or decreases in rape leaf discs treated under hyper- or hypo-osmotic conditions, respectively. In addition we show that pipecolic acid level is positively correlated with the external osmotic potential as well as with the duration of the applied treatment. On the other hand pipecolic acid level is related to the availability of L-lysine and not to that of D-lysine. Collectively the results obtained demonstrate that lysine catabolism through LKR/SDH activity is involved in osmo-induced synthesis of pipecolic acid.     

Metabolic engineering and profiling of rice with increased lysine

Lysine (Lys) is the first limiting essential amino acid in rice, a stable food for half of the world population. Efforts, including genetic engineering, have not achieved a desirable level of Lys in rice. Here, we genetically engineered rice to increase Lys levels by expressing bacterial lysine feedback‐insensitive aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS) to enhance Lys biosynthesis; through RNA interference of rice lysine ketoglutaric acid reductase/saccharopine dehydropine dehydrogenase (LKR/SDH) to down‐regulate its catabolism; and by combined expression of AK and DHPS and interference of LKR/SDH to achieve both metabolic effects. In these transgenic plants, free Lys levels increased up to ~12‐fold in leaves and ~60‐fold in seeds, substantially greater than the 2.5‐fold increase in transgenic rice seeds reported by the only previous related study. To better understand the metabolic regulation of Lys accumulation in rice, metabolomic methods were employed to analyse the changes in metabolites of the Lys biosynthesis and catabolism pathways in leaves and seeds at different stages. Free Lys accumulation was mainly regulated by its biosynthesis in leaves and to a greater extent by catabolism in seeds. The transgenic plants did not show observable changes in plant growth and seed germination nor large changes in levels of asparagine (Asn) and glutamine (Gln) in leaves, which are the major amino acids transported into seeds. Although Lys was highly accumulated in leaves of certain transgenic lines, a corresponding higher Lys accumulation was not observed in seeds, suggesting that free Lys transport from leaves into seeds did not occur.     

Lysine catabolism in Haemonchus contortus and Teladorsagia circumcincta

Catabolism of lysine through the pipecolate, saccharopine and cadaverine pathways has been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Both enzymes of the saccharopine pathway (lysine ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH)) were active in L3 and adult worms of both species. All three enzymes which catabolise lysine to α-amino adipic semialdehyde via pipecolate (lysine oxidase (LO), Δ(1)-piperideine-2-carboxylate reductase (Pip2CR) and pipecolate oxidase (PipO)) were present in adult worms, whereas the pathway was incomplete in L3 of both species; Pip2CR activity was not detected in the L3 of either parasite species. In adult worms, the saccharopine pathway would probably be favoured over the pipecolate pathway as the K(m) for lysine was lower for LKR than for LO. Neither lysine dehydrogenase nor lysine decarboxylase activity was detected in the two parasite species. Enzyme activities and substrate affinities were higher for all five enzymes in adult worms than in L3. An unexpected finding was that both LKR and SDH were dual co-factor enzymes and not specific for either NAD(+) or NADP(+), as is the case in other organisms. This novel property of LKR/SDH suggests it could be a good candidate for anthelmintic targeting.     

Synthesis of the Arabidopsis bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase enzyme of lysine catabolism is concertedly regulated by metabolic and stress-associated signals

In plants, excess cellular lysine (Lys) is catabolized into glutamic acid and acetyl-coenzyme A; yet, it is still not clear whether this pathway has other functions in addition to balancing Lys levels. To address this issue, we examined the effects of stress-related hormones, abscisic acid (ABA), and jasmonate, as well as various metabolic signals on the production of the mRNA and polypeptide of the bifunctional Lys-ketoglutarate reductase (LKR)/saccharopine dehydrogenase (SDH) enzyme, which contains the first two linked enzymes of Lys catabolism. The level of LKR/SDH was strongly enhanced by ABA, jasmonate, and sugar starvation, whereas excess sugars and nitrogen starvation reduced its level; thus this pathway appears to fulfill multiple functions in stress-related and carbon/nitrogen metabolism. Treatments with combination of hormones and/or metabolites, as well as use of ABA mutants in conjunction with the tester sugars mannose and 3-O-methyl-glucose further supported the idea that the hormonal and metabolic signals apparently operate through different signal transduction cascades. The stimulation of LKR/SDH protein expression by ABA is regulated by a signal transduction cascade that contains the ABI1-1 and ABI2-1 protein phosphatases. By contrast, the stimulation of LKR/SDH protein expression by sugar starvation is regulated by the hexokinase-signaling cascade in a similar manner to the repression of many photosynthetic genes by sugars. These findings suggest a metabolic and mechanistic link between Lys catabolism and photosynthesis-related metabolism in the regulation of carbon/nitrogen partitioning.