Characterization of the genetic factors affecting quinolizidine alkaloid biosynthesis and its response to abiotic stress in narrow‐leafed lupin (Lupinus angustifoliusL.)

Abstract

Quinolizidine alkaloids (QAs) are toxic secondary metabolites that complicate the end use of narrow-leafed lupin (NLL; Lupinus angustifolius L.) grain, as levels sometimes exceed the industry limit for its use as a food and feed source. The genotypic and environmental influences on QA production in NLL are poorly understood. Here, the expression of QA biosynthetic genes was analysed in vegetative and reproductive tissues of bitter (high QA) and sweet (low QA) accessions. It was demonstrated that sweet accessions are characterized by lower QA biosynthetic gene expression exclusively in leaf and stem tissues than bitter NLL, consistent with the hypothesis that QAs are predominantly produced in aerial tissues and transported to seeds, rather than synthesized within the seed itself. This analysis informed our identification of additional candidate genes involved in QA biosynthesis. Drought and temperature stress are two major abiotic stresses that often occur during NLL pod set. Hence, we assessed the effect of drought, increased temperature, and their combination, on QA production in three sweet NLL cultivars. A cultivar-specific response to drought and temperature in grain QA levels was observed, including the identification of a cultivar where alkaloid levels did not change with these stress treatments.

The role of jasmonate signalling in quinolizidine alkaloid biosynthesis, wounding and aphid predation response in narrow-leafed lupin

“…RNA extractions were conducted using 100 mg of tissue (from the uppermost leaves of aphid-infested plants or homogenised leaf material from MeJA-treated plants). RNA extraction and cDNA synthesis were carried out as described by Frick et al (2018).…”

“…Primers used in this study for quantitative real-time -PCR have been described previously (Frick et al 2018). These primers are used to amplify the quantitative real-time PCR reference gene L. angustifolius UBC9-like (LaUBC9-like; Lup018255.1), QA biosynthetic genes LaL/ODC (Lup009726.1) (Bunsupa et al 2012), LaCAO (Lup000530.1) (Yang et al 2017a) and LaHMT/ HLT (Lup022251.1) (Okada et al 2005) and candidate biosynthetic gene LaAT (Lup021586.1) (Bunsupa et al 2011).…”

“…These genes are Lup026769.1 (LaLOX4-like), Lup003340.1 (LaLOX5-like) and Lup009615.1 (LaPI-like). Primers were designed as outlined in Frick et al (2018). Primer sequences are listed in Table S1, available as Supplementary Material to this paper.…”

“…The quantitative real-time PCR was conducted according to Frick et al (2018), performed in 10 mL reactions with 5 mL SsoFast Evagreen Supermix, 4 mL 1 : 50 cDNA reaction dilution and 1 mL primer mix containing 5 mM each of forward and reverse primers, including a reference gene and non-template control on each plate. Relative gene expression was calculated using the 2 -DDCT method (Livak and Schmittgen 2001) to normalise the average C t values of technical replicates to the reference gene LaUBC9-like.…”

“…Levels of QAs in the grain must remain below 0.02% to be acceptable for food and feed purposes (Allen 1998); however, they often exceed this threshold and the environmental conditions that cause elevated QA levels are not well understood (Cowling and Tarr 2004). Although studies have investigated the role of abiotic factors on grain QA content in NLL, such as drought, temperature, soil pH and nutrient availability (Christiansen et al 1997;Gremigni et al 2003;Jansen et al 2009Jansen et al , 2012Frick et al 2018), there are fewer reports on the impact of biotic stresses (Pilar Vilariño et al 2005;Chludil et al 2009Chludil et al , 2013. This is an important consideration, as QAs protect the plant from insect pests (Wink 1992) and, therefore, their biosynthesis may be induced in response to insect herbivory, possibly explaining why grain QA content is highly unpredictable when only abiotic factors are assessed (Cowling and Tarr 2004).…”