Nutritionalrich and Stresstolerant Crops by Saccharopine Pathway Manipulation

REVIEW

Nutritional-rich and stress-tolerant crops by saccharopine pathway manipulation Paulo Arruda1,2 & Izabella Pena Neshich1

1Centro de Biologia Molecular e Engenharia Gene´ tica, Universidade Estadual de Campinas, Campinas, Sao Paulo, Brazil 2Departamento de Gene´ tica e Evoluc¸a˜ o, IB, Universidade Estadual de Campinas, Campinas, Sao Paulo, Brazil

Keywords Abstract Amino acid metabolism, lysine catabolism, nutritional enhancement, saccharopine Lysine is a limiting essential amino acid in cereals, a major food source for pathway, stress tolerance humans and animals. Although cereals can synthesize lysine from aspartate, the pathway is feedback regulated in a manner that restricts lysine accumulation. Correspondence Another step that restricts lysine accumulation in seeds is the saccharopine Paulo Arruda, Centro de Biologia Molecular e pathway for lysine catabolism. This pathway converts lysine to a-aminoadipic- Engenharia Gene´ tica, Universidade Estadual d-semialdehyde (AASA) by the bifunctional enzyme lysine-ketoglutarate de Campinas, 13083-970, Campinas, Sao reductase/saccharopine dehydrogenase (LKR/SDH). Then, AASA is converted to Paulo, Brazil. Tel: +55-19-35211137; a d Fax: +55-19-35211089; aminoadipic acid (AAA) by -aminoadipic- -semialdehyde dehydrogenase E-mail: [email protected] (AASADH). The downregulation of LKR/SDH in seeds results in the over- accumulation of free lysine to levels that meet human nutritional requirements. Funding Information However, the saccharopine pathway is also involved in stress response in plants, This study was funded by FAPESP – 2010/ animals, and bacteria. In these organisms, the gene encoding LKR/SDH is 50114-4. Izabella Pena Neshich received PhD upregulated under osmotic, salt, and oxidative stress conditions. The role of the fellowship from FAPESP – 2012/00235-5. saccharopine pathway in stress response is not well understood, but the over- Received: 26 June 2012; Revised: 10 July expression of AASADH results in stress-tolerant plants and animal cells. The 2012; Accepted: 12 July 2012 saccharopine pathway may thus act either by producing osmolytes, such as pipecolic acid and proline, or by signaling compounds that regulate stress-response doi: 10.1002/fes3.9 genes. In this review, we discuss the potential use of the saccharopine pathway to engineer nutritional-rich and stress-tolerant crops.

Introduction levels of free threonine, but have a minimal effect on the free lysine levels (Bright et al. 1982). Likewise, genetically Lysine is synthesized in plants and bacteria by the dihydro- modified plants overexpressing a bacterial DHDPS that is dipicolinate branch of the aspartate pathway (Azevedo less sensitive to feedback inhibition by lysine overproduce et al. 1997). In plants, however, and in cereals in particu- both threonine and lysine but accumulate high levels of lar, this pathway is regulated by complex feedback loops saccharopine, indicating high rates of lysine degradation operated by the amino acid end products in a manner through the saccharopine pathway (Falco et al. 1995). that restricts the accumulation of soluble lysine (Azevedo These results indicate that lysine catabolism plays a criti- and Arruda 2010). Lysine inhibits both aspartate kinase cal role in regulating the free lysine level in plant seeds (AK), the first enzyme in the pathway, and dihydro- and point to a direction to increase the levels of soluble dipicolinate synthase (DHDPS), the first enzyme in the lysine in seeds by engineering plants with downregulated dihydrodipicolinate branch (Galili 1995; Avin-Wittenberg lysine-ketoglutarate reductase/saccharopine dehydrogenase and Galili 2012). Regulation of these two key steps in the (LKR/SDH). However, the saccharopine pathway has also pathway fine-tunes the amount of soluble lysine, keeping been shown to be implicated in stress response in plants the amino acid at low levels (Galili 1995; Angelovici et al. and animals. Plants submitted to salt and/or osmotic 2009, 2011). Mutant plants expressing an AK that is less stress increase their expression of LKR/SDH (Deleu et al. sensitive to feedback inhibition by lysine accumulate high 1999), but the role of the enzyme in stress response has

ª 2012 John Wiley & Sons Ltd and the Association of Applied Biologists. This is an open access article under the terms of the Creative 1 Commons Attribution Non-Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Nutritional Rich and Stress-Tolerant Crops P. Arruda & I. P. Neshich not yet been elucidated. The third enzyme in this We also discuss the role of the saccharopine pathway in pathway, a-aminoadipic-d-semialdehyde dehydrogenase stress response and how the pathway could be manipu- (AASADH), has been shown to be upregulated when lated to produce nutritionally improved and stress- tissues or cells are submitted to osmotic treatments, sug- tolerant crops. gesting a role for the enzyme that is linked to the stress response (Rodrigues et al. 2006; Brocker et al. 2010). The Saccharopine Pathway in Diverse Recently, the saccharopine pathway has been shown to Organisms operate in Silicibacter pomeroyi, a bacteria living in high- salt environments such as seawater (Serrano et al. 2012). The saccharopine pathway is used for lysine synthesis in The saccharopine pathway genes in S. pomeroyi were fungi and lysine catabolism in plants, animals, and bacteria upregulated in bacteria submitted to osmotic stress (I. P. (Markovitz and Chuang 1987; Gaziola et al. 1997; Arruda Neshich and P. Arruda, unpubl. data). Lysine catabolism et al. 2000; Serrano et al. 2012). The ﬁrst two reactions of is also associated with increased levels of compatible sol- the pathway are catalyzed by LKR, which condenses lysine utes, such as pipecolic acid, in Corynebacterium ammoni- and a-ketoglutarate to form saccharopine, and SDH, which agenes that has been submitted to osmotic stress hydrolyses saccharopine to form AASA and glutamic acid (Gouesbet et al. 1992). Thus, understanding the role of (Fig. 1) (Arruda et al. 2000). The third reaction is catalyzed the saccharopine pathway in diverse organisms may help by AASADH, which converts AASA into a-aminoadipic to devise strategies to produce plants with increased acid (AAA) (Fig. 1) (Arruda et al. 2000). The saccharopine nutritional value and adaptability to stress environments. pathway is present in several eukaryotic organisms (Ander- In this review, we discuss the cases in which genetically son et al. 2010). In eukaryotes except fungi, the lkr/sdh gene modiﬁed crop plants resulted in increased lysine content. produces a transcript that is translated into a bifunctional

Figure 1. The saccharopine pathway for L-lysine degradation in plant, mammals, and bacteria. The indicated enzymes are LKR, lysine- ketoglutarate reductase; SDH, saccharopine dehydrogenase; AASADH, a-aminoadipic semialdehyde dehydrogenase; P5CR, pyrroline-5-carboxylate reductase. Compatible solutes proline and pipecolate are highlighted in blue.

2 © 2012 John Wiley & Sons Ltd and the Association of Applied Biologists P. Arruda & I. P. Neshich Nutritional Rich and Stress-Tolerant Crops polypeptide containing both enzymatic domains (Markovitz modified canola and soybean plants engineered to con- and Chuang 1987; Gonçalves-Butruille et al. 1996; Kemper stitutively express the Escherichia coli lysC gene that et al. 1999). Although almost all plants possess a single encodes AK and the Corynebacterium cordapA gene that gene encoding LKR/SDH, poplar (Populus trichocarpa) encodes DHDPS, both enzymes that are less sensitive to possesses two functional genes encoding the bifunctional feedback inhibition by lysine, increased the accumulation polypeptide and an additional gene encoding a mono- of soluble threonine and lysine (Falco et al. 1995). How- functional LKR (Anderson et al. 2010). The bifunctional ever, the accumulation of significant levels of saccharo- structure of the lkr/sdh gene can also lead to the forma- pine and AAA indicated extensive high rate of lysine tion of monofunctional enzymes (Anderson et al. 2010). through the saccharopine pathway (Falco et al. 1995). However, in bacteria such as S. pomeroyi, although the This extensive lysine degradation could be attributable genes encoding LKR and SDH are located in an operon, to the tissue targeted to overexpress the AK and DHDPS the two enzymes act as monofunctional polypeptides constructs. This issue was partially solved by the genera- (Serrano et al. 2012). One striking finding is that in tion of a genetically modified maize plant engineered to S. pomeroyi, the genes encoding LKR and SDH are colocal- overexpress the cordapA gene in an embryo-specific ized in an operon together with the genes encoding phos- manner. These maize plants showed increased accumula- phoglycerate mutase (PGM) and glutathione-S-transferase tion of soluble lysine at levels near the recommended (GST), two enzymes involved in the stress response in requirement for human nutrition, but lysine degradation diverse organisms (I. P. Neshich and P. Arruda, unpubl. still suggested the key role of the saccharopine pathway data). This genome architecture of the saccharopine in regulating lysine accumulation in the seeds (Lucas pathway suggests a close link between the saccharopine et al. 2007). To prevent lysine degradation, genetically pathway and the oxidative stress metabolism in bacteria. modified maize plants were engineered to express an endosperm-specific RNAi construct designed to target Manipulation of the Saccharopine the lkr/sdh gene transcript (Houmard et al. 2007). These Pathway to Produce Lysine-Rich plants decreased the lkr/sdh gene expression and Seeds increased the soluble lysine in the seeds 20-fold compared with the nonmodified plants. The downregulated Seeds accumulate storage proteins that are needed to sup- lkr/sdh gene expression combined with the genetically ply nitrogen compounds for the early phases of embryo modified plants overexpressing the cordapA gene dramat- development after germination. These seed storage pro- ically increased the soluble lysine in the seeds, reflecting teins compose the main elements of animal and human an increase in the total lysine to levels that meet the nitrogen nutrition (Ferreira et al. 2005). In cereals, which requirement for human nutrition (Frizzi et al. 2008; supply most of the seeds consumed in the world, how- Reyes et al. 2009) (Table 1). Thus, the combination of ever, the storage proteins are composed mostly of prola- the genetic manipulation of AK pathway and the saccha- mins that are devoid of lysine, an essential amino acid for ropine pathway resulted in plants with elevated nutri- animal and human nutrition (Shewry and Casey 1999; tional value, although how these plants will behave Shewry 2007; Tosi et al. 2011). The process of storage under stress conditions remains to be determined. In protein accumulation in maize seeds, a model for cereals, Arabidopsis there are evidences that dowregulation of is temporally coordinated with the rate of storage protein LKR/SDH affect seed germination by altering TCA cycle accumulation and amino acid metabolism (Arruda and metabolism (Angelovici et al. 2011). Silva 1983). The saccharopine pathway is a good example of how the regulatory machinery links storage protein synthesis and amino acid metabolism. In maize, the genes Table 1. Soluble lysine content in genetically modified high-lysine 1 encoding both zeins, the prolamins of maize, and LKR/ maize. SDH are regulated by Opaque-2, a transcription factor Soluble lysine (mg/g DW) that binds to the 22 kDa zein and lkr/sdh gene promoters WT GM line Reference and regulates their transcription (Schmidt et al. 1990; Kemper et al. 1999). Likewise, in rice, lkr/sdh gene expres- 0.10 4.00 Frizzi et al. 2008 sion is regulated by RISBZ1 and RPBF, two transcription 0.04 0.20 Reyes et al. 2009 factors that regulate the transcription of seed storage pro- 0.04 0.90 Reyes et al. 2009 tein genes (Kawakatsu and Takaiwa 2010). 0.04 1.60 Reyes et al. 2009 The genetic components comprising storage protein 1Data were compiled from the cited references. and lysine synthesis can be genetically manipulated to Adapted from Azevedo and Arruda 2010; WT, wild-type; GM, geneti- enhance lysine accumulation in the seeds. Genetically cally modified.

The Saccharopine Pathway in Stress and drought stresses (Rodrigues et al. 2006). Genetically Response modified plants that overexpressed the aldh7b1 gene under the control of the strong 35S CaMV constitutive The involvement of the saccharopine pathway during the promoter showed significant tolerance to salt, draught, stress response was first identified in plants where and oxidative stress induced by H2O2 and paraquat AASADH, also known as antiquitin (antique nature) due (Rodrigues et al. 2006). On the contrary, Arabidopsis to the highly conserved amino acid sequence among taxa aldh7B1 knockouts were shown to be susceptible to (Lee et al. 1994; Skvorak et al. 1997; Fong et al. 2006), water deficit and salt stress compared with wild-type was found as a turgor responsive in garden pea (Guerrero plants (Kotchoni et al. 2006). Similarly, rice aldh7B1 null et al. 1990). Later, rapeseed leaf discs submitted to osmo- mutants were more susceptible to abiotic stresses (Shin tic stress were shown to have inducible levels of lkr/sdh et al. 2009). The mammalian aldh7A1 gene is also impli- gene expression (Deleu et al. 1999). The activities of both cated in the stress response. The mammalian AASADH LKR and SDH were induced by osmotic shock, at levels was shown to protect cells against osmotic stress by a proportional to the intensity of the osmotic treatment mechanism that involves the generation of osmolytes (Moulin et al. 2000). The upregulation of LKR/SDH in (Brocker et al. 2010). The AASADH enzyme also pro- osmotically treated tissues may channel lysine to AAA, tects mammalian cells from oxidative stress, for example, further increasing the levels of pipecolic acid (Fig. 1) by metabolizing a number of lipid peroxidation-derived (Eduard and Jakobs 2010), which acts as an osmoprotectant (LPO) aldehydes. Overexpression of the aldh7A1 gene, (Moulin et al. 2006). Additionally, the glutamate formed in mammalian cells resulted in protection against by the hydrolysis of saccharopine (Fig. 1) could be an LPO-derived aldehydes (Chad et al. 2011). The role of additional substrate source for the synthesis of proline, AASADH in the detoxification of aldehydes was also dem- another osmoprotectant (Nanjo et al. 1999). Indeed, onstrated by the finding that mutations in the aldh7A1 gene under salt stress, proline synthesis is increased in young impaired the enzyme activity and was shown to be the cau- Arabidopsis plants due to the activation of both gluta- sal factor of human pyridoxine-dependent seizures (Mills mate and ornithine pathways whereas, in adult plants, et al. 2006). The defective enzyme is unable to efficiently only the glutamate pathway operates toward accumula- convert AASA into AAA, leading to the accumulation of tion of proline as a protective osmolyte (Roosens et al. AASA or its cyclic isomer, D1-piperideone-6-carboxilic 1998). In rapeseed leaf discs, both the pipecolic acid and acid (P6C). The accumulation of P6C inactivates pyri- proline levels increased with osmotic treatment, suggest- doxal-5-phosphate, leading to the impairment of synapsis ing that the saccharopine pathway is involved in the (Mills et al. 2006). osmo-induced synthesis of these osmolytes (Moulin et al. Additional evidence that the saccharopine pathway is 2006). The bifunctional enzyme LKR/SDH has also been broadly involved in mechanism of protection against shown to be involved in stresses induced during develop- stress was provided by the identification of the pathway mental processes in the tick (Haemaphysalis longicornis) in bacteria. In S. pomeroyi, lkr and sdh genes were shown (Battur et al. 2009). The enzyme was upregulated during to be located in an operon together with the genes encod- starvation after the tick detached from its host. The ing PGM and GST (Serrano et al. 2012; I. P. Neshich and downregulation of LKR/SDH revealed that the enzymes P. Arruda, unpubl. data), two enzymes involved in the might protect the tick from the osmotic and water deficit protection against oxidative stress (Roxas et al. 2000; imposed by the long period of starvation (Battur et al. Chaturvedi et al. 2010). Silicibacter pomeroyi lives in the 2009). salt-stressed environment of seawater. Thus, the saccharo- The third enzyme of the saccharopine pathway, pine pathway may help alleviate the effect of salt/osmotic AASADH, is encoded in plants by the gene aldehyde stress imposed by the environment on these bacteria. dehydrogenase 7B1 (aldh7B1) (Kirch et al. 2005), the Indeed, the lkr and sdh genes from S. pomeroyi were orthologue of the mammalian aldh7A1 (Skvorak et al. shown to be induced by high salt stress (I. P. Neshich 1997). AASADH converts AASA into AAA (Fig. 1), but and P. Arruda, unpubl. results). These findings revealed the enzyme purified from mammals can also use various that the saccharopine pathway is broadly associated with other aldehydes as substrates, including nonanal, hex- the stress response in organisms representing three king- anal, octanal, betaine aldehyde, and benzaldehyde (Broc- doms (Table 2). Therefore, the enzymes of this pathway ker et al. 2010). The ability of AASADH to use a range appear to be an interesting target to produce genetically of other substrates has been claimed as a major role of modified plants with an increased tolerance to diverse this enzyme in detoxifying cells from the toxic effect of stressful environments, although how these plants will aldehydes. However, similar to LKR/SDH, AASADH is behave in terms of nutritional value remains to be also induced when plants are submitted to osmotic, salt, determined.

Table 2. Gene expression studies of lkr/sdh or aldh7B1 genes associated with abiotic stress.

Plant Gene Observed effects References

Rapeseed lkr/sdh Upregulation of lkr/sdh gene in leaf discs under Deleu et al. 1999; osmotic stress Moulin et al. 2000 Rapeseed lkr/sdh Upregulation of lkr/sdh gene in leaf discs under Moulin et al. 2006 osmotic stress correlated with pipecolic acid accumulation Garden pea aldh7B1 Upregulation of aldh7B1 gene in tissues under Guerrero et al. 1990 water stress Soybean aldh7B1 Upregulation of aldh7B1 gene in tissues under salinity Rodrigues et al. 2006 and water stress Tobacco and aldh7B1 Overexpression of soybean aldh7B1 gene conferred tolerance Rodrigues et al. 2006

Arabidopsis to salinity, water deﬁcit, H2O2, and paraquat Arabidopsis aldh7B1 Upregulation of aldh7B1 gene in plantlets under dehydration, Kirch et al. 2005 salinity, and ABA treatments Arabidopsis aldh7B1 Overexpression of aldh7B1 gene conferred tolerance to osmotic and Kotchoni et al. 2006 oxidative stress. Knockout of the gene conferred susceptibility to dehydration and salt stress Rice aldh7B1 Rice aldh7B1 null mutant is susceptible to accelerated aging, toxic Shin et al. 2009 aldehyde levels, and abiotic stresses, e.g., drought, cold, and heat

Conclusions and Future Prospects pathway to protect diverse organisms against stressful conditions (Moulin et al. 2000, 2006; Rodrigues et al. One of the major challenges for humanity in the next few 2006; Battur et al. 2009; Brocker et al. 2010). The over- decades is to develop technologies that can sustain crop expression of enzymes of the saccharopine pathway to yield under stressful environments, which are gradually develop stress-tolerant plants may reduce the nutritional increasing because of soil salinization derived from agri- value of cereals with respect to the essential amino acid cultural practices. In arid or semi-arid regions, the leach- lysine. Therefore, engineering plants that overexpress ing of salt is poor because of low rainfall, which causes enzymes of the saccharopine pathway in the vegetative tis- salt accumulation at toxic levels (Pitman and Lauchli sues and downregulating LKR/SDH, specifically in the 2004). Soil salinity is considered one of the most signifi- seeds, would be desirable. Targeting seed-specific down- cant abiotic stresses, affecting 20% of the earth’s land regulation of LKR/SDH has already proven its utility for mass and nearly half of all irrigated land (Zhang et al. the development of lysine-rich maize (Frizzi et al. 2008). 2012). Although the world’s population is continuously The next step is designing constructs to overexpress the rising, stress-resistant and nutritionally valuable cereal enzymes of the saccharopine pathway in a vegetative- crops are still lacking. specific manner to produce stress-tolerant plants without Plants can protect themselves from stressful conditions affecting the nutritional value of the seeds. using a number of molecular and/or physiological mechanisms, but very few of these mechanisms have been vali- Acknowledgments dated in the field trials. In this article, we reviewed the potential utility of the saccharopine pathway for the This study was funded by FAPESP – 2010/50114-4. development of nutritionally rich and stress-tolerant Izabella Pena Neshich received PhD fellowship from plants. The saccharopine pathway works downstream of FAPESP – 2012/00235-5. Paulo Arruda is a CNPq lysine synthesis, converting this amino acid into AASA, productivity research fellow. which is further hydrolyzed by AASADH into AAA (Fig. 1). The production of genetically modified maize Conflict of Interest plants that overexpress lysine-insensitive AK and/or DHDPS in the embryo together with LKR/SDH down- None declared. regulation in the endosperm has been proven to result in maize seeds that meet the nutritional requirements for References lysine (Frizzi et al. 2008; Reyes et al. 2009) (Table 1). Anderson, O. D., D. Coleman-Derr, Y. Q. Gu, and S. Heath. However, the role of the saccharopine pathway in protect- 2010. Structural and transcriptional analysis of plant genes ing diverse organisms from salt, osmotic, and water stres- encoding the bifunctional lysine ketoglutarate reductase ses suggests an opposite direction, the upregulation of the saccharopine dehydrogenase enzyme. BMC Plant Biol. 10:113.

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