Genomics and biochemical analyses reveal a metabolon key to β-L-ODAP biosynthesis in Lathyrus sativus

Edwards, Anne; Njaci, Isaac; Sarkar, Abhimanyu; Jiang, Zhouqian; Kaithakottil, Gemy; Moore, C. J.; Cheema, Jitender; Stevenson, Clare E. M.; Rejzek, Martin; Novák, Petr; Vigouroux, Marielle; Vickers, Martin; Wouters, Roland H. M.; Paajanen, Pirita; Steuernagel, Burkhard; Moore, Jonathan D.; Higgins, Janet; Swarbreck, David; Martens, Stefan; Kim, Colin Y.; Weng, Jing-Ke; Kilian, Benjamin; Kumar, Shiv; Loose, Matthew; Yant, Levi; Macas, Jiří; Wang, Trevor L.; Martin, Cathie; Emmrich, Peter M. F.

doi:10.1038/s41467-023-36503-2

Cited by 20 publications

(17 citation statements)

References 79 publications

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“…However, overexpression of the pointmutated LsSAT2 in L. sativus or achievement of single base transition/transversion at target sites via plant base editing or prime editing is still expectant to facilitate the genetic site validation of LsSAT2 activity and further crop improvement. Very recently, studies verifying the links of an oxalyl-CoA synthetase (acyl-activating enzyme 3, AAE3) 41 and β-ODAP synthase (BOS) 42 to β-ODAP biosynthesis in L. sativus and the presence of β-ODAP in Pisum sativum L. 43 provide an interesting clue to reveal insights into β-ODAP biosynthesis in these two sister species via comparative genomics. 41,44−46 ■ ASSOCIATED CONTENT * sı Supporting Information…”

Section: ■ Discussionmentioning

confidence: 99%

“…sativus or achievement of single base transition/transversion at target sites via plant base editing or prime editing is still expectant to facilitate the genetic site validation of LsSAT2 activity and further crop improvement. Very recently, studies verifying the links of an oxalyl-CoA synthetase (acyl-activating enzyme 3, AAE3) and β-ODAP synthase (BOS) to β-ODAP biosynthesis in L. sativus and the presence of β-ODAP in Pisum sativum L .…”

Section: Discussionmentioning

confidence: 99%

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Critical Sites of Serine Acetyltransferase in Lathyrus sativus L. Affecting Its Enzymatic Activities

Song

Zhang

et al. 2023

J. Agric. Food Chem.

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LsSAT2 (serine acetyltransferase in Lathyrus sativus) is the rate-limiting enzyme in biosynthesis of β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP), a neuroactive metabolite distributed widely in several plant species including Panax notoginseng, Panax ginseng, and L. sativus. The enzymatic activity of LsSAT2 is post-translationally regulated by its involvement in the cysteine regulatory complex in mitochondria via interaction with β-CAS (β-cyanoalanine synthase). In this study, the binding sites of LsSAT2 with the substrate Ser were first determined as Glu290, Arg316, and His317 and the catalytic sites were determined as Asp267, Asp281, and His282 via site-directed/truncated mutagenesis, in vitro enzymatic activity assay, and functional complementation of the SAT-deficient Escherichia coli strain JM39. Furthermore, the C-terminal 10-residue peptide of LsSAT2 is confirmed to be critical to interact with LsCAS, and Ile336 in C10 peptide is the critical amino acid. These results will enhance our understanding of the regulation of LsSAT2 activities and the biosynthesis of β-ODAP in L. sativus.

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Section: ■ Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Critical Sites of Serine Acetyltransferase in Lathyrus sativus L. Affecting Its Enzymatic Activities

Song

Zhang

et al. 2023

J. Agric. Food Chem.

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“…Phylogenetic analysis of these BAHD-AT genes revealed that a particular clade has expanded in the legumes significantly. This clade encompasses nine members displaying different tissue-specific expression in grass peas (Edwards et al, 2023). Among them, BAHD-AT3 displays higher expression over the other isoforms of the clade, with its highest abundance in the late pods (Edwards et al, 2023).…”

Section: Figurementioning

confidence: 99%

“…This clade encompasses nine members displaying different tissue-specific expression in grass peas (Edwards et al, 2023). Among them, BAHD-AT3 displays higher expression over the other isoforms of the clade, with its highest abundance in the late pods (Edwards et al, 2023). Therefore, we hypothesized that loss-of-function mutation of this gene might significantly reduce the β-ODAP content in grass pea seeds.…”

Section: Figurementioning

confidence: 99%

Designer grass pea for transgene-free minimal neurotoxin-containing seeds with CRISPR-Cas9

Saha

Shee

Sahid

et al. 2023

Preprint

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Grass pea seeds are consumed as food in several South Asian and Sub-Saharan African nations. However, the presence of the neurotoxic compound N-oxalyl-L-diamino propionic acid (β-ODAP) has restricted its cultivation. Although various cultivars with low β-ODAP levels have been developed, their cultivation is still limited due to the risk of neurolathyrism from long-term grass pea seed ingestion. In this study, we employed the CRISPR-Cas9-mediated gene editing technique to generate grass pea seeds with zero or minimal β-ODAP levels. We targeted the BAHD-AT3 gene that encodes a key enzyme in the β-ODAP biosynthesis pathway. We developed bahd-at3 knock-out lines using three gRNAs targeting different regions of this gene and characterized them. Cas9-free independent lines from each event carrying the desired on-target mutation were selected and backcrossed twice with the wild-type to eliminate any off-target mutation present therein. Various agronomical parameters were analyzed from the backcrossed mutant lines and they displayed no phenotypic abnormalities. Interestingly, the seed β-ODAP content ranged between 0.001 % - 0.002 % of dry weight which is 99 % lower than the wild-type. Together, our study reports the development of transgene-free, genome-edited grass peas with insignificant levels of β-ODAP in seeds for safer food in the future.

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“…The genome of Lathyrus sativus has largely been unexplored as the genome sequences were not available until recently (Edwards et al,2023;Rajarammohan et al, 2023). Only few -omics studies have been carried out to understand the underlying mechanisms of ODAP synthesis (Almeida et al, 2014(Almeida et al, , 2015Hao et al, 2017;Verma et al, 2022;Xu et al, 2018;Yang et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

An efficient hairy root system for genome editing of a β-ODAP pathway gene inLathyrus sativus

Verma

Kaur

et al. 2023

Preprint

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Grass pea (Lathyrus sativus) is an ideal legume crop for resource-poor farmers, having resistance to various biotic and abiotic stresses. The seeds of this plant are rich in protein and are the only known dietary source of L-homoarginine. Moreover, it thrives with minimal inputs making it a promising crop in grain legume breeding programs with immense potential for food security. Despite these advantages, the global area under its cultivation has decreased because of the presence of an antinutrient compound, β-N-oxalyl-L-α,β-diamino propionic acid (β-ODAP), which results in neurolathyrism both in humans and animals. Multiple efforts in the past have resulted in the development of improved varieties with low ODAP. Still, due to variations in response to the environment, stable low-ODAP lines have not been developed for large-scale cultivation. In this paper, we report in planta characterization of Oxalyl-CoA Synthetase (OCS) involved in the oxalylating step leading to β-ODAP production. We established a hairy root transformation system for Lathyrus and demonstrated the genome editing of LsOCS. Further, we show that oxalate accumulates in these hairy roots due to loss-of-function of the OCS gene. This is the first report of functional analysis of a Lathyrus gene in Lathyrus. The hairy root genome editing system we developed can be used as a quick system for functional studies of Lathyrus genes.

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Genomics and biochemical analyses reveal a metabolon key to β-L-ODAP biosynthesis in Lathyrus sativus

Cited by 20 publications

References 79 publications

Critical Sites of Serine Acetyltransferase in Lathyrus sativus L. Affecting Its Enzymatic Activities

Critical Sites of Serine Acetyltransferase in Lathyrus sativus L. Affecting Its Enzymatic Activities

Designer grass pea for transgene-free minimal neurotoxin-containing seeds with CRISPR-Cas9

An efficient hairy root system for genome editing of a β-ODAP pathway gene inLathyrus sativus

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