Genotypic Variation in the Concentration of β - American Chemical

Jun 1, 2015 - genotypes differing in seed β-ODAP concentration were grown in pots .... were ground to powder using a mini ball mill (FZ102, Zhongxing...
0 downloads 0 Views 487KB Size
Download PDF
Article pubs.acs.org/JAFC

Genotypic Variation in the Concentration of β‑N‑Oxalyl‑L‑α,βdiaminopropionic Acid (β-ODAP) in Grass Pea (Lathyrus sativus L.) Seeds Is Associated with an Accumulation of Leaf and Pod β‑ODAP during Vegetative and Reproductive Stages at Three Levels of Water Stress Jun-Lan Xiong,†,‡ You-Cai Xiong,*,† Xue Bai,† Hai-Yan Kong,† Rui-Yue Tan,† Hao Zhu,† Kadambot H. M. Siddique,‡ Jian-Yong Wang,† and Neil C. Turner*,‡,§ †

State Key Laboratory of Grassland Agroecosystems, Institute of Arid Agroecology, School of Life Sciences, Lanzhou University, Lanzhou 730000, Gansu Province, China ‡ The UWA Institute of Agriculture, The University of Western Australia, M082, Perth, Western Australia 6009, Australia § Centre for Plant Genetics and Breeding, The University of Western Australia, M080, Perth, Western Australia 6009, Australia ABSTRACT: Grass pea (Lathyrus sativus L.) cultivation is limited because of the presence in seeds and tissues of the nonprotein amino acid β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), a neurotoxin that can cause lathyrism in humans. Seven grass pea genotypes differing in seed β-ODAP concentration were grown in pots at three levels of water availability to follow changes in the concentration and amount of β-ODAP in leaves and pods and seeds. The concentration and amount of β-ODAP decreased in leaves in early reproductive development and in pods as they matured, while water stress increased β-ODAP concentration in leaves and pods at these stages. The net amount of β-ODAP in leaves and pods at early podding was positively associated with seed β-ODAP concentration at maturity. We conclude that variation among genotypes in seed β-ODAP concentration results from variation in net accumulation of β-ODAP in leaves and pods during vegetative and early reproductive development. KEYWORDS: β-ODAP amount, reproductive phase, seed yield, vegetative phase, water stress

■

studies have reported that seed β-ODAP concentration increased with water stress,25,28,29 while other studies reported that water deficit had no effect on β-ODAP concentration in seeds, while reducing biomass and grain yield.30,31 Several studies have focused on the accumulation and catabolism of β-ODAP in grass pea during growth. In general, β-ODAP concentration in vegetative tissues decreased, but it increased in reproductive tissues during their growth and development.27,32,33 Addis and Narayan32 reported high β-ODAP amounts in roots and shoots at the seedling stage [8 days after sowing (DAS)], but the βODAP was found primarily in the pod shell and immature seeds at podding and only in the seeds at maturity. Jiao et al.27 indicated that the highest concentration of β-ODAP occurred in 6-day-old shoots and then decreased sharply, so that only mature seeds contained β-ODAP when the plants were senescing. Previous work has also shown that variation in tissue β-ODAP at early growth stages can affect the amount and concentration of βODAP in seeds at maturity. Seeds with low- and high-β-ODAP concentrations planted in a nutrient-rich field showed that grass pea with high β-ODAP concentrations in young shoots had relatively high β-ODAP concentrations in mature seeds.27 Kuo et al.33 studied the incorporation of the radioactive precursor of β-

INTRODUCTION Grass pea (Lathyrus sativus L.), an annual legume grown for forage and seed, has a long history of cultivation in arid and semiarid areas because it is easy to establish and can withstand extreme environments from drought to flooding.1−6 Grass pea seeds contain up to 25% protein, include several essential and nonprotein amino acids, and are often the only option for poor illiterate families to avoid starvation when other crops fail to grow.7−9 However, grass pea contains a neuro-excitatory nonprotein amino acid, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which has been identified as one of the main factors causing neurolathyrism in humans and animals when consumed for long periods (over 2−3 months) and in large quantities.10−14 Nevertheless, in small quantities, β-ODAP also has some beneficial effects on health. For example, β-ODAP is the same chemical as decichine, which is extracted from longevitypromoting Ginseng roots and is reported to be hemostatic and to increase platelet numbers in vivo.15 β-ODAP biosynthesis in grass pea is regulated by multiple genes, and its concentration and amount is affected by both the genotype and environment.16−19 Grass pea germplasm exhibits considerable variability in β-ODAP concentration with traditional varieties containing 0.5−2.5% β-ODAP in the seed and some accessions containing less than 0.1% β-ODAP.20−24 Drought, salinity, and heavy metals can influence the concentration and amount of β-ODAP in seeds or other tissues,25−27 but the effect of water stress is not clear. Numerous © XXXX American Chemical Society

Received: April 8, 2015 Revised: June 1, 2015 Accepted: June 1, 2015

A

DOI: 10.1021/acs.jafc.5b01729 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 1. Accession Numbers, Country of Origin, Days from Sowing to 50% Flowering and Maturity, Plant Height, Seed Coat Color, and Seed β-ODAP Concentration [% of seed dry weight (DW)] of Seven Grass Pea (Lathyrus sativus L.) Genotypes Measured in Plants Grown in the Field in 2012 accession no.

origin

days to 50% flowering

days to maturity

plant height (mm)

seed coat color

seed β-ODAP concentration (% DW)

IF 225 IF 2156 Dingxi IF 1942 IF 1347 IF 1312 IF 1310

Slovakia Bangladesh China Bangladesh Syria Syria Syria

65.0 59.0 64.5 58.0 61.0 63.0 63.0

105.0 93.0 107.8 90.5 95.0 99.0 100.0

416 320 423 374 352 339 355

cream light brown cream light brown light brown dark brown dark brown

2.53 1.81 1.72 1.66 1.58 1.51 1.22

ODAP, β-isoxazolinon-alanine, and found that there was no difference in the incorporation of the precursor in seeds of highand low-β-ODAP genotypes, suggesting that the differences in βODAP among genotypes was likely the result of differences in the biosynthesis and amount of the precursor of β-ODAP near maturity. It is therefore not clear whether the accumulation or catabolism of β-ODAP at vegetative and reproductive stages is associated with the final concentration of β-ODAP in seeds at maturity. As the basis for genotypic variation in the amount and concentration of β-ODAP in seeds and the effect of water stress on this genotypic variation is not known, an experiment on the accumulation and catabolism of β-ODAP in vegetative and reproductive tissues of seven grass pea genotypes varying in seed β-ODAP concentration was conducted at three levels of water availability. We hypothesized that variation in amount and concentration of β-ODAP in seeds is determined by the accumulation and/or catabolism of β-ODAP in vegetative and reproductive tissues at the early reproductive phase and that the variation in the amount and concentration of β-ODAP in seeds with varying water availability is determined as a result of the processes of accumulation and catabolism in these tissues. The purpose of the present study was to (1) track the variation in βODAP concentration and amount in leaves and pods of seven grass pea genotypes at flowering, podding, and maturity; (2) examine whether water stress affected the distribution of βODAP at these stages; and (3) determine the relationship between amount and concentration of β-ODAP in seeds in the vegetative and early reproductive stages at three levels of water availability.

■

seedlings per pot. Nitrogen (0.68 g of NH4NO3) and phosphorus (1.58 g of KH2PO4) were supplied as a solution to each pot before sowing. Three water treatments were imposed: (1) Well-watered (WW): pots were watered daily in the late afternoon to 80% FC throughout the growing period. (2) Moderate water-stressed (WS): pots were kept at 80% FC until 35 DAS when soil water content (SWC) was allowed to fall to 50% FC and maintained at 50% FC to maturity by daily watering in the late afternoon until maturity. (3) Severe WS: pots were kept at 80% FC until 35 DAS when the SWC was allowed to fall to 35% FC and maintained at 35% FC by daily watering in the late afternoon to maturity. Days to 50% flowering and maturity were recorded and dry matter of different organs measured at flowering (54 DAS), early podding (75 DAS), and maturity (103 DAS). At flowering and podding, plants were cut off at soil level and separated into stems and leaves at flowering and leaves, stems, and pods at early podding. They were then dried in a forced-draft oven at 80 °C for 24 h, and dry weights (DW) were recorded. Fresh leaf (0.5 g) and pod (0.5 g) material at flowering and podding stages were sampled for measurement of β-ODAP concentration. At maturity, plants were cut off at soil level and separated into stems, leaves, pod walls and grain and then dried in a forced-draft oven at 80 °C for 24 h; DW was then recorded. Total grain number was counted and individual grain DW (seed size) was calculated as grain DW/total grain number. A subsample (about 1.0 g) of air-dried seeds was collected at maturity to measure β-ODAP concentration. β-ODAP amount for each plant organ was calculated as product of the β-ODAP concentration and the DW of each organ. There were 21 different treatment combinations (7 genotypes, each with 3 water treatments), with 15 pots per treatment, giving a total of 315 pots. At both flowering (54 DAS) and early podding (75 DAS), 3 of the 15 pots per treatment were harvested for DW measurements and 3 for β-ODAP concentration, while at maturity the three remaining pots were harvested. The experiment was arranged as a complete randomized block design with three replicate blocks. β-ODAP Concentration. β-ODAP concentration was analyzed using the methods of Wang et al.35 and Yan et al.36 Dry seeds (0.5 g) were ground to powder using a mini ball mill (FZ102, Zhongxing Ltd. Beijing, China) or fresh leaves (0.5 g) or fresh pods (0.5 g) were homogenized with 4.5 mL of ethanol/water (7:3, v/v) and extracted for 24 h at room temperature. The mixture was centrifuged for 0.25 h at 150 000g with 0.5 mL of supernatant dried under vacuum. A 100 μL of 0.5 M NaHCO3 solution was used to dissolve the residue, and then 100 μL of the reagent (100 mg of 1-fluoro-2,4-dinitrobenzene in 10 mL of acetonitrile) was added to the mixture for derivatization which was completed in 0.75 h at 60 °C. After the mixture was cooled to room temperature, 0.8 mL of 0.01 M KH2PO4 solution was added to the above reaction mixture; then the mixture was vortexed for 10 s and filtered through a 0.45 μm membrane filter. A 20 μL sample of the product was taken for high-performance liquic chromatography (HPLC) analysis using a HPLC system (Agilent 1200, Waldbronn, Germany) to detect βODAP concentration. The chromatographic conditions were set as follows: Phenomenex C 18 column of 3.9 × 150 mm; mobile phase, acetonitritle/0.1 M KH2PO4, 17/83 (v/v); column temperature, 40 °C; flow rate, 1/60 mL/s; detection wave, 360 nm. The mobile solutions were filtered through a 0.45 μm membrane filter and degassed before use in the HPLC system. ODAP (containing 80% β-ODAP, and 20% α-

MATERIALS AND METHODS

Plant Materials and Growth Conditions. Seven genotypes of grass pea (six genotypes from the International Centre for Agricultural Research in Dry Areas and one local cultivar, Dingxi, from China) were planted on April 4, 2013 at the Yuzhong Experiment Station of Lanzhou University, Yuzhong, Gansu province, China (35°51′ N, 104°07′S; altitude, 1620 m). The genotypes had similar phenologies, differing by 6 days in time to flowering; plant height varied from 320 to 420 mm; seed coat color varied from cream to dark brown; and seed β-ODAP concentration varied more than 200% when the genotypes were planted in the field at the Yuzhong Experiment Station of Lanzhou University in 2012 (Table 1). The experimental site has a typical semiarid climate with an accumulated rainfall of 202.5 mm, a mean temperature of 14.5 °C, and a mean relative humidity of 58% during the growing season (March−July). Ten uniform seeds of the seven genotypes were each sown in 45 plastic pots (0.26 m diameter, 0.30 m high) containing 8.0 kg of a dry silty-loam loess soil (haplic greyxems34) and vermiculite mixture (soil:vermiculite = 2:1, v/v) with a field capacity (FC, the percentage of water in the soil when the soil is fully saturated, then allowed to drain for 48 h) of 30.8%; the pots were placed in a rainout shelter that was open when rain was not forecast. Ten DAS, the plants were thinned to five B

DOI: 10.1021/acs.jafc.5b01729 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 2. Days to 50% Flowering [Days after Sowing (DAS)], Days to Maturity (DAS), Leaf Dry Weight (DW, g/plant), Pod DW (g/plant), Aboveground DW (g/plant), Grain DW (g/plant), Grain Number (/plant), and Individual Grain Dry Weight (DW, g/ seed) in Seven Grass Pea (Lathyrus sativus L.) Genotypes Subjected to Three Water Regimes [80% field capacity (FC), 50% FC, and 35% FC]a leaf DW water 80% FC

50% FC

35% FC

days to 50% flowering

days to maturity

pod DW

above ground DW

grain DW

grain number

individual grain DW

flowering

podding

podding

maturity

maturity

maturity

maturity

IF 225 IF 2156 Dingxi IF 1942 IF 1347 IF 1312 IF 1310 mean IF 225 IF 2156 Dingxi IF 1942 IF 1347 IF 1312 IF 1310 mean IF 225 IF 2156 Dingxi IF 1942 IF 1347 IF 1312 IF 1310 mean

50 43 55 44 47 44 48 47 48 42 52 43 46 43 47 46 46 42 52 43 46 43 47 46

99 91 103 91 94 91 94 95 91 87 93 88 91 87 90 90 89 84 91 84 89 84 84 86

1.6 1.0 1.4 1.0 1.1 1.3 1.1 1.2 1.1 0.8 1.0 0.8 0.8 1.0 0.7 0.9 0.9 0.5 0.7 0.6 0.5 0.8 0.7 0.7

2.2 0.8 2.3 1.0 1.3 1.5 1.3 1.5 1.0 0.6 1.3 0.5 0.9 0.5 0.7 0.8 0.5 0.3 0.8 0.4 0.4 0.5 0.5 0.5

1.6 2.2 1.9 2.2 2.1 2.3 2.1 2.1 1.7 1.2 2.1 2.0 1.7 1.7 2.2 1.8 1.1 1.2 0.6 1.2 1.1 1.6 1.5 1.2

7.9 4.1 9.9 5.0 5.9 6.9 5.4 6.4 4.5 2.8 5.6 3.3 4.1 3.5 3.3 3.9 2.7 1.8 2.3 1.9 2.0 2.4 2.7 2.3

3.1 2.2 4.5 2.7 2.9 3.2 2.7 3.0 2.0 1.5 2.7 1.9 2.3 1.8 1.9 2.0 1.2 1.0 0.9 1.0 1.1 1.3 1.5 1.1

18.1 17.7 22.6 20.9 27.5 26.9 24.3 22.6 13.3 14.8 12.7 16.8 22.5 13.1 19.5 16.1 7.7 10.1 4.8 10.6 13.7 12.2 14.8 10.6

0.182 0.126 0.179 0.104 0.102 0.100 0.105 0.128 0.106 0.073 0.141 0.082 0.054 0.087 0.055 0.085 0.119 0.077 0.235 0.076 0.051 0.058 0.047 0.095

LSD0.05(W) LSD0.05(G) LSD0.05(W×G)

**1.0 ***1.6 n.s.

***0.9 ***1.5 ***2.5

***0.04 ***0.07 *0.12

***0.1 ***0.2 ***0.3

***0.2 **0.3 **0.5

***0.09 ***0.14 ***0.80

***0.2 ***0.3 ***0.5

***1.6 ***2.4 *4.2

***0.013 ***0.019 ***0.034

genotype

a

Least significant differences (LSD) for each column are given: LSD0.05(W) for water treatments, LSD0.05(G) for genotypes, LSD0.05(W×G) for water treatment by genotype interactions (n.s., no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

ODAP, provided by Prof. Zhixiao Li, School of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu Province, China) was used as a standard for quantitative analysis. Statistical Analyses. Data were analyzed by two-way ANOVA (water treatments and genotypes) using Genstat 12.1 (VSN International Ltd., Hemel Hempstead, UK). Where significant effects were found, least significant difference (LSD) values at P = 0.05 are presented. Linear regressions between the amount or concentration of β-ODAP in the seeds and the amount of β-ODAP in the leaves and pods during growth were analyzed by Origin 8.0 (Microcal Software Inc. Northampton, Massachusetts, United States). Ordinary least-squares (OLS) regression analysis was used to test linear Y − linear X relationships, and OLS slope heterogeneity among water treatments was tested using the Standardized Major Axis Tests and Routines (SMATR) (version 2.0, NSW, Australia).37

DAS); at 50% FC, the time to maturity decreased by 5 days, and at 35% FC it decreased by a further 4 days (Table 2). Plant Dry Weight, Grain Yield, and Yield Components. In the WW treatment (80% FC), the local cultivar, Dingxi, had the most aboveground DW and grain DW at maturity with 9.9 and 4.5 g/plant, respectively, of the seven grass pea genotypes, followed by IF 1312 and IF 225 (aboveground DW 6.9−7.9 g/ plant and grain DW 3.1−3.2 g/plant); then IF 1942, IF 1310, and IF 1347 (aboveground DW 5.0−5.9 g/plant and grain DW 2.7− 2.9 g/plant); with the least aboveground DW and grain DW at maturity observed in IF 2156 (4.1 and 2.2 g/plant, respectively) (Table 2). In the WW treatment, the seven genotypes ranked differently for pod DW (including seed) at early podding (75 DAS), leaf DW at flowering (54 DAS), and early podding (75 DAS) when compared to the seed DW at maturity (Table 2). Reducing available water to 50% FC significantly decreased aboveground DW and seed DW at maturity by 40% and 33%, respectively, on average across genotypes, and reducing of available water to 35% FC reduced aboveground DW and seed DW by a further 41% and 45%, respectively (62−65% less than in the WW treatment) (Table 2). There was a highly significant interaction between genotypes and water treatments, with aboveground DW and seed DW of the local cultivar, Dingxi,

■

RESULTS Phenology. In the WW treatment, the seven genotypes reached 50% flowering in 43−55 DAS (mean, 47 DAS) with IF 2156, IF 1942, and IF 1312 flowering first and the local cultivar, Dingxi, flowering last (Table 2). Water stress reduced the time to 50% flowering by 1−2 days at both 50% FC and 35% FC (Table 2). In the WW treatment, maturity varied from 91 DAS in the early flowering genotypes to 103 DAS in Dingxi (mean, 95 C

DOI: 10.1021/acs.jafc.5b01729 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Relationship between grain dry weight (DW) and grain number in seven grass pea (Lathyrus sativus L.) genotypes. The lines are the fitted ordinary least-squares (linear) regressions (regression data shown).

Table 3. Leaf β-ODAP Concentration [% Dry Weight (DW)] at Flowering [54 Days after Sowing (DAS)] and Early Podding (75 DAS), Pod β-ODAP Concentration (% DW) at Early Podding (75 DAS), and Seed β-ODAP Concentration (% DW) at Maturity (103 DAS) in Seven Grass Pea (Lathyrus sativus L.) Genotypes Given Three Water Regimes [80% Field Capacity (FC), 50% FC, and 35% FC]a

decreasing by 40−43% at 50% FC and by 77−80% at 35% FC, compared to 30−39% and 44−50% at 50% FC and 35% FC, respectively, in genotype IF 1310 (Table 2). Leaf and pod DW at podding decreased with decreasing water availability in a similar manner and percentage to seed DW (Table 2). The reduction in leaf and pod DW in the local cultivar, Dingxi, was greater than that in IF 1310 as water availability decreased from 80% FC to 35% FC (Table 2). The large reduction in seed DW with water stress in Dingxi (Table 2) was associated with a large decrease (44% at 50% FC and 79% at 35% FC) in seed number (Table 2). This contrasts with the smaller decrease in seed numbers in IF 1310 (20% at 50% FC and 39% at 35% FC) with water stress (Table 2). The reduction in seed DW with water stress was associated with a decrease in grain number in all genotypes (Table 2, Figure 1). Seed size (individual grain DW) decreased in all genotypes when the water treatment decreased from 80% FC to 50% FC, but it increased in some genotypes when the water stress treatment decreased to 35% FC (Table 2), such that the reduction in seed DW with WS was not associated with individual grain DW (data not shown). β-ODAP Concentration in Leaves, Pods, and Seeds. βODAP concentration in leaves at flowering varied significantly among genotypes, from 0.79% in IF 1310 to 1.58% in IF 1347 in the WW treatment (Table 3). In all genotypes the concentration of β-ODAP in the leaves decreased between flowering and podding, which varied with genotype (Table 3). Water stress increased β-ODAP concentration in the leaves at both flowering and early podding, but the only significant interaction was at flowering when the severe stress treatment (35% FC) induced a significant increase in β-ODAP concentration in IF 225, IF 2156, and IF 1347 (Table 3). At maturity when plants senesce, β-ODAP can be detected only in seeds and not in the leaves, stems, roots, or pod walls.27,32 Therefore, we measured β-ODAP concentration in whole pods (including developing seeds) at early podding but only in seeds at maturity. In the WW treatment, IF 1312 had the highest pod βODAP concentration at 2.51%, while IF 1310 had the lowest at 0.65% at podding; there was a significant reduction (more than 1% decrease on average across genotypes) in β-ODAP concentration in seeds at maturity compared with pods at early podding (Table 3). The concentration of β-ODAP in seeds varied from 0.85% in IF 1312 to 0.22% in IF 1310 in the WW treatment (Table 3). In all genotypes except IF 1312, water stress (50% FC) significantly increased β-ODAP concentration in pods

β-ODAP concentration water 80% FC

50% FC

35% FC

pods

seeds

flowering

podding

podding

maturity

IF 225 IF 2156 Dingxi IF 1942 IF 1347 IF 1312 IF 1310 mean IF 225 IF 2156 Dingxi IF 1942 IF 1347 IF 1312 IF 1310 mean IF 225 IF 2156 Dingxi IF 1942 IF 1347 IF 1312 IF 1310 mean

1.33 1.50 1.17 1.28 1.58 1.32 0.79 1.28 1.96 1.61 1.13 2.20 1.84 1.30 1.24 1.61 2.10 2.48 1.21 1.84 3.16 1.67 1.20 1.95

0.58 0.45 0.79 1.09 1.38 0.73 0.76 0.83 0.68 1.01 0.89 1.08 1.65 1.12 0.97 1.06 0.97 0.97 1.01 1.04 2.22 0.89 1.79 1.27

1.64 1.67 1.85 1.80 1.59 2.51 0.65 1.67 2.11 4.23 2.44 2.13 2.27 1.96 0.96 2.30 4.03 3.54 7.22 3.76 4.02 2.72 1.20 3.78

0.59 0.77 0.57 0.58 0.68 0.85 0.22 0.61 0.80 0.78 0.76 0.75 0.75 0.67 0.29 0.69 0.81 0.88 0.90 0.75 0.86 1.02 0.52 0.82

LSD0.05(W) LSD0.05(G) LSD0.05(W×G)

***0.22 ***0.34 **0.59

**0.23 ***0.35 n.s.

***0.40 ***0.61 ***1.05

***0.09 ***0.15 n.s.

genotype

leaves

a

Least significant differences (LSD) for each column are given: LSD0.05(W) for water treatments, LSD0.05(G) for genotypes, LSD0.05(W×G) for water treatment by genotype interactions (n.s., no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

D

DOI: 10.1021/acs.jafc.5b01729 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 4. Leaf β-ODAP Amount (mg/plant) at Flowering [54 Days after Sowing (DAS)] and Early Podding (75 DAS), Pod β-ODAP Amount (mg/plant) at Early Podding (75 DAS), and Seed β-ODAP Amount (mg/plant) at Maturity (103 DAS) in Seven Grass Pea (Lathyrus sativus L.) Genotypes Given Three Water Regimes [80% Field Capacity (FC), 50% FC, and 35% FC]a

at early podding and increased it further at 35% FC (Table 3). Similarly, water stress increased the concentration of β-ODAP in seeds at maturity, but the low seed β-ODAP genotype, IF 1310, was consistently low in all three water treatments (Table 3). It is notable that the ranking for seed β-ODAP concentration in the seven genotypes in the moderate WS (50% FC) treatment (Table 3) was the same as that observed in the field in 2012 (Table 1). Contrary to previous observations,38−40 there was no association between seed size (individual grain weight, Table 2) or seed color (Table 1) with the β-ODAP concentration in the seed (Table 3) in the limited number of genotypes used in this study. β-ODAP Amount in Leaves, Pods, and Seeds. As the amount of β-ODAP in the tissues is a product of β-ODAP concentration and tissue DW, it is a measure of the net accumulation, synthesis minus catabolism, and redistribution of β-ODAP by the plant. In the WW treatment, IF 225 had the greatest, while IF 1310 had the least, amount of β-ODAP in the leaves at flowering (Table 4). Over all the genotypes, water shortage significantly reduced the amount of β-ODAP in the leaves at flowering from 15.6 mg/plant at 80% FC to 14.1 mg/ plant at 50% FC to 12.4 mg/plant at 35% FC, and there was a significant genotype by water interaction in the leaves of IF 1942 that had more β-ODAP at 50% FC than at 80% FC (Table 4). Nevertheless, IF 225 had the most and IF 1310 had the least βODAP in the leaves at flowering in all three levels of water supply (Table 4). Generally, the amount of β-ODAP decreased in the leaves between flowering and early podding, more so at 35% FC than 50% FC and less so at 80% FC (Table 4). In the WW treatment, IF 1312 had the most and IF 1310 had the least β-ODAP in pods at early podding and in seeds at maturity (Table 4). The amount of β-ODAP in pods at early podding generally increased from 80% FC to 50% FC to 35% FC, except in IF 2156, Dingxi, IF 1312, and IF 1310 (Table 4), but the amount of β-ODAP in seeds at maturity decreased with increasing water stress such that the amount of β-ODAP in mature seeds was about 54% of that in pods at podding at 80% FC, 36% at 50% FC, and 23% at 35% FC (Table 4). IF 1310 had the least β-ODAP in seeds at maturity in all soil water treatments (Table 4). Changes in β-ODAP Amount in Leaves and Pods between Different Stages of Growth. In the WW treatment (80% FC), there was a significant reduction in β-ODAP in the leaves between flowering and early podding in IF 225, IF 2156, IF 1942, and IF 1312 (Table 5). The average reduction across the seven genotypes was greater for all genotypes at 50% (5.9 mg/ plant) and 35% FC (6.5 mg/plant) than at 80% FC (3.6 mg/ plant) (Table 5). In contrast, the amount of β-ODAP in the pods at early podding increased relative to the amount in the leaves at flowering in all seven genotypes and all three water treatments (Table 5). The increase in β-ODAP in pods was greater with decreased water availability; the mean increase for all grass pea genotypes was 19.2 mg/plant at 80% FC, 24.9 mg/plant at 50% FC, and 28.0 mg/plant at 35% FC (Table 5). The amount of βODAP in pods decreased from early podding to maturity (at maturity, the seed β-ODAP amount is the same as pod β-ODAP amount as there is no β-ODAP in the pod wall) in all seven genotypes in all water treatments (Table 5). The reduction in βODAP in pods increased as the water deficit became more severe; the mean reduction for the seven genotypes was 16.1 mg/ plant at 80% FC, 24.9 mg/plant at 50% FC, and 31.2 mg/plant at 35% FC (Table 5). Water shortage did not significantly affect the

β-ODAP amount water 80% FC

50% FC

35% FC

genotype

leaves flowering

pods

seeds

podding

podding

maturity

IF 225 IF 2156 Dingxi IF 1942 IF 1347 IF 1312 IF 1310 mean IF 225 IF 2156 Dingxi IF 1942 IF 1347 IF 1312 IF 1310 mean IF 225 IF 2156 Dingxi IF 1942 IF 1347 IF 1312 IF 1310 mean

20.6 15.5 15.9 13.4 17.6 17.3 8.9 15.6 21.6 12.8 11.5 17.6 14.1 12.4 8.6 14.1 18.0 12.7 8.4 11.0 15.6 13.1 8.1 12.4

12.8 3.7 18.2 10.5 18.3 10.6 10.0 12.0 7.0 6.4 11.5 5.2 14.2 6.1 6.7 8.2 5.3 3.3 8.0 4.0 8.1 4.3 8.7 6.0

27.0 36.6 35.9 39.6 33.0 57.6 13.7 34.8 36.4 49.5 51.4 42.6 37.8 34.0 21.0 39.0 45.9 41.7 44.8 44.9 44.8 42.8 17.9 40.4

18.5 17.3 26.1 15.7 19.9 27.5 6.0 18.7 16.2 12.0 20.5 14.5 17.6 11.8 5.5 14.0 9.9 8.4 8.3 7.6 9.5 13.1 7.7 9.2

LSD0.05(W) LSD0.05(G) LSD0.05(W×G)

***1.3 ***2.0 **3.5

***1.8 ***2.7 n.s.

**3.1 ***4.7 ***8.2

***1.6 ***2.5 ***4.3

a

Least significant differences (LSD) for each column are given: LSD 0.05(W) for water treatments; LSD 0.05(G) for genotypes; LSD0.05(W×G) for water treatment by genotype interactions (n.s., no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001)

sum of β-ODAP in leaves and pods at the early podding stage, but there was significant variation among genotypes from 68.2 mg/ plant in IF 1312 to 23.7 mg/plant in IF 1310 in the WW treatment (Table 5). IF 1310 consistently had the least β-ODAP in leaves and pods at 23.7 to 27.7 mg/plant, while the genotype with the greatest amount varied from IF 1312 at 80% FC to Dingxi at 50% FC to Dingxi and IF 1347 at 35% FC (Table 5). Relationship between the Amount or Concentration of β-ODAP in Seeds at Maturity and the Amount of β-ODAP in Tissues during Growth. There was no significant (P > 0.05) association between the amount of β-ODAP in leaves at either flowering or podding and the amount or concentration of βODAP in seeds at maturity (data not shown). However, there was a significant (P < 0.05) positive correlation between the sum of leaf and pod β-ODAP amount at early podding and seed βODAP concentration at maturity in all three water treatments (Figure 2a). There was no significant difference in the slope among the three water treatments, but the concentration of βODAP in seeds at 35% FC was higher than that of the other two water treatments (Figure 2a). There was also a positive E

DOI: 10.1021/acs.jafc.5b01729 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 5. Changes in Leaf β-ODAP Amount (mg/plant) and Pod β-ODAP Amount (mg/plant) between Flowering (F), Podding (P), and Maturity (M), and the Sum of Leaf and Pod β-ODAP Amounts (mg/plant) at P (75 DAS) in Seven Grass Pea (Lathyrus sativus L.) Genotypes Given Three Water Regimes [80% Field Capacity (FC), 50% FC, and 35% FC]a water 80% FC

50% FC

35% FC

change in leaf β-ODAP amount between F and P

Change in β-ODAP amount between leaves at F and pods at P

change in pod β-ODAP amount between P and M

IF 225 IF 2156 Dingxi IF 1942 IF 1347 IF 1312 IF 1310 mean IF 225 IF 2156 Dingxi IF 1942 IF 1347 IF 1312 IF 1310 mean IF 225 IF 2156 Dingxi IF 1942 IF 1347 IF 1312 IF 1310 mean

−7.8 −11.8 +2.3 −2.9 +0.7 −6.7 +1.1 −3.6 −14.6 −6.4 −0.0 −12.4 +0.1 −6.3 −1.9 −5.9 −12.7 −9.4 −0.4 −7.0 −7.5 −8.8 +0.6 −6.5

+6.4 +21.1 +20.0 +26.2 +15.4 +40.3 +4.8 +19.2 +14.8 +36.7 +39.9 +25.0 +23.7 +21.6 +12.4 +24.9 +27.9 +29.0 +36.4 +33.9 +29.2 +29.7 +9.8 +28.0

−8.5 −19.3 −9.8 −23.9 −13.1 −30.1 −7.7 −16.1 −20.2 −37.5 −30.9 −28.1 −20.2 −22.2 −15.5 −24.9 −36.0 −33.3 −36.5 −37.3 −35.3 −29.7 −10.2 −31.2

39.8 40.3 54.1 50.1 51.3 68.2 23.7 46.8 43.4 55.9 62.9 47.8 52.0 40.1 27.7 47.1 51.2 45.0 52.8 48.9 52.9 47.1 26.6 46.4

LSD0.05(W) LSD0.05(G) LSD0.05(W×G)

***1.2 ***1.9 ***3.3

***1.9 ***2.9 ***5.1

***1.7 ***2.6 ***4.4

n.s. ***3.5 ***6.1

genotype

sum of leaf and pod βODAP amounts at P

a

Least significant differences (LSD) for each column are given: LSD0.05(W) for water treatments; LSD0.05(G) for genotypes; LSD0.05(W×G) for water treatment by genotype interactions (n.s., no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

relationship between the amount of β-ODAP in seeds at maturity and the sum of leaf and pod β-ODAP amounts at 80% FC and 50% FC, but not at 35% FC, with no increase in the amount of βODAP in seeds relative to the amount of β-ODAP in leaves and pods at podding in severely stressed plants (Figure 2b). There was also a significant (P < 0.05) positive association between the change in β-ODAP amount between leaves at flowering and pods at early podding, and seed β-ODAP concentration at maturity in all three water treatments (Figure 3a), and with the amount of βODAP in seeds at maturity in the WW and moderate WS (50% FC) treatments (Figure 3b). There was no significant (P > 0.05) relationship between the change in leaf β-ODAP between flowering and podding, nor in the change in β-ODAP amount in the pods between podding and maturity and the seed β-ODAP concentration and amount at maturity (Table 6).

■

increase in the amount of β-ODAP in pods at early podding compared with leaves at flowering, but not with the marked reduction in the amount of β-ODAP in seeds at maturity compared with pods at early podding, suggesting that the concentration of β-ODAP in seeds is likely the result of synthesis of β-ODAP or its precursor, β-isoxazolinon-alanine, in the early reproductive phase and the possible transfer of β-ODAP from leaves to pods and seeds. Genotypes ranked on the basis of their seed β-ODAP concentration in the field in 2012 had the same ranking as the ranking at the moderate stress (50% FC) treatment in the rainout shelter. However, the concentration of β-ODAP in seeds showed a genotype by environment interaction in that the rankings differed in the WW (80% FC) and severe WS (35% FC) treatments. Effect of Water Stress on Biomass Accumulation, Grain Yield, and Yield Components. Reducing water supply to 50% FC and 35% FC had a marked effect on the growth of grass pea plants, reducing leaf DW at flowering and early podding as well as aboveground DW and seed DW at maturity by 40% and 33%, respectively, at 50% FC, and by 65% and 62%, respectively, at 35% FC. The reduction in grain DW by water stress in the seven genotypes was largely due to a reduction in grain number, with grain size largely unaffected; grain size even increased in the large-seeded genotype Dingxi which had a large reduction in grain number under severe water shortage. This result is consistent with other results with grass pea; water stress reduced

DISCUSSION

Grass pea differs genotypically in the concentration of β-ODAP in seeds at maturity. In this study we confirmed this and showed that the concentration of β-ODAP in seeds increased with water shortage but that a low β-ODAP genotype maintained the lowest β-ODAP concentration at all levels of water availability. We have shown that the variation in seed β-ODAP concentration at maturity is associated with the net (synthesis minus catabolism) amount of β-ODAP in pods and leaves at early podding in all three water treatments. This was associated with a marked F

DOI: 10.1021/acs.jafc.5b01729 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

seeded (0.18 g/seed) Lathyrus sativus L. genotype Dingxi.41 On the other hand, Lathyrus sativus L. cv. Ceora which has small seeds at 0.12 g/seed, produced smaller seeds (0.09 g/seed) and fewer seeds (from 269.5 to 124.0 per plant) when subjected to terminal water stress.31 These results are consistent with those of Cocks et al.28 who showed that a large-seeded (0.19 g/seed) grass pea accession, SEL 526, maintained its seed size, while a small-seeded (0.12 g/seed) accession had smaller seeds (0.09 g/ seed) with increasing severe water stress. Effect of Water Stress on β-ODAP Concentration and Amount. In this study, water stress increased β-ODAP concentration in leaves, pods, and seeds in all seven genotypes, more so with lower water supply. This result is consistent with previous results showing that water shortage increased β-ODAP concentration in different tissues of grass pea28,29,42 but does not agree with observations by Gusmao et al.30 and Kong et al.31 that terminal drought induced no change in β-ODAP concentration of seeds. However, while the concentration of β-ODAP increased in this study, the amount of β-ODAP in the leaves at flowering and podding, and in the seeds at maturity, significantly decreased when the water supply decreased from 80% FC to 35% FC because of the reduction in DW in leaves, pods, and seeds with water shortage. Accumulation and Catabolism of β-ODAP during the Vegetative and Reproductive Period. Jiao et al.27 showed that the concentration of β-ODAP in leaves gradually declined with age. In the present study, leaf β-ODAP concentration also decreased from flowering to early podding. As there was little change in leaf DW over this period, this represents a loss in βODAP by either catabolism or translocation to the developing pods. The concentration of β-ODAP in seeds at maturity was lower than that in pods (containing seeds) at podding in all seven grass pea genotypes. This loss in β-ODAP concentration was associated with a large loss in the amount of β-ODAP in seeds at maturity relative to that in pods at podding, particularly in the severe WS treatment (35% FC). This suggests that there was considerable catabolism of β-ODAP in the senescing and maturing grass pea plants. The reduction in the amount of β-ODAP in leaves from flowering to early podding was not associated (P > 0.05) with the concentration of β-ODAP in seeds at maturity, but there was a significant positive relationship between the increased amount of β-ODAP in leaves at flowering and pods at early podding, suggesting that variation in seed β-ODAP concentration may be related to differences in synthesis of new β-ODAP in pods at early podding rather than any transfer from leaves to pods, particularly as the amount of β-ODAP in pods at the podding stage was far higher than the amount of β-ODAP in leaves at flowering or podding. The amount and concentration of β-ODAP in seeds at maturity was positively associated with the sum of leaf and pod βODAP amounts at podding, further suggesting that the amount of leaf and pod β-ODAP at this early reproductive stage jointly affected the final β-ODAP concentration in seeds. This was true in the genotype, IF 1310, which had the lowest amount and concentration of β-ODAP in seeds amount at maturity and the lowest amount of β-ODAP in leaves and pods at podding of seven grass pea genotypes. This is consistent with the observations by Kuo et al.33 that genotypes differing in seed βODAP concentration differ in the biosynthesis of its precursor, not in the conversion of the precursor to β-ODAP; we suggest that the synthesis and accumulation of β-ODAP and its precursor in the vegetative and early reproductive stages determines the

Figure 2. Relationship between (a) seed β-ODAP concentration and (b) seed β-ODAP amount at maturity and sum of leaf and pod β-ODAP amounts at podding in seven grass pea genotypes maintained at three water regimes: well-watered (80% field capacity, FC), moderate water stress (50% FC), and severe water stress (35% FC). The line is the fitted ordinary least-squares (linear) regression (regression data shown).

Figure 3. Relationship between (a) seed β-ODAP concentration and (b) seed β-ODAP amount at maturity and the change in β-ODAP amount between leaves at flowering and pods at early podding in seven grass pea cultivars maintained at three water regimes: well-watered (80% field capacity, FC), moderate water stress (50% FC), and severe water stress (35% FC). The line is the fitted ordinary least-squares (linear) regression (regression data shown).

the number of filled pods, grain number, grain DW, pod length, and pod wall DW, but not individual grain DW in the largeG

DOI: 10.1021/acs.jafc.5b01729 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 6. Relationships between the Change in β-ODAP Amounts in Leaves between Flowering (F) and Early Podding (P), and the Change in β-ODAP Amounts between Pods at P and Maturity (M), and Seed β-ODAP Concentration and Amount at Maturity Across Seven Grass Pea (Lathyrus sativus L.) Genotypes Maintained at Three Water Regimes [80% field capacity (FC), 50% FC, and 35% FC]a

a

X

Y

water treatment

slope

change in leaf β-ODAP amounts

seed β-ODAP concentration

80% FC 50% FC 35% FC

0.017 0.001 0.012

between F and P

seed β-ODAP amount

80% FC 50% FC 35% FC

0.154 −0.130 0.204

change in pod β-ODAP amounts

seed β-ODAP concentration

80% FC 50% FC 35% FC

0.010 0.010 0.006

between P and M

seed β-ODAP amount

80% FC 50% FC 35% FC

0.030 0.158 0.014

intercept

R2

0.543 0.681 0.743

0.21n.s. 0.00n.s. 0.08n.s.

23.19 18.42 10.20 0.501 0.490 0.642 23.46 14.56 11.12

0.01n.s. 0.02n.s. 0.11n.s. 0.19n.s. 0.09n.s. 0.08n.s. 0.00n.s. 0.03n.s. 0.00n.s.

n.s., not significant (P > 0.05).

gram (GYHY201106029-2), Natural Science Foundation of China (20110211110022 and 31070372), the Overseas Masters Program of Ministry of Education (Ms2011LZDX059), and the Fundamental Research Funds for the Central Universities of China (lzujbky-2012-k17).

concentration in seeds at maturity. The strong association between the sum of β-ODAP in leaves and pods at early podding and final concentration in seeds at maturity could also be used to predict final β-ODAP concentration in the seed by measuring the β-ODAP level in the vegetative tissues at the early reproductive stage. Water stress did not significantly affect the relationship between seed β-ODAP concentration and the sum of leaf and pod β-ODAP at podding. However, severe water stress significantly reduced the slope between the amount of βODAP in seeds and the sum of leaf and pod β-ODAP amounts compared to the WW treatment, indicating that the severe WS treatment reduced the transfer of β-ODAP from leaves and pods at the early reproductive stage to seeds at maturity. In summary, the variation among genotypes in seed β-ODAP concentration is a result of variation in the net accumulation (synthesis and catabolism) of β-ODAP during both the vegetative and reproductive phases. Water shortage affects this accumulation of β-ODAP in leaves and pods at podding differently among genotypes and hence induces differences in β-ODAP concentration in seeds at maturity. While the genotype by water supply interaction has been observed previously, the present study provides evidence that the differences among genotypes starts in the vegetative stage and is not simply determined by differences in synthesis in the late reproductive phase.

■

Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS USED β-ODAP, β-N-oxalyl-L-α, β-diaminopropionic acid; DAS, days after sowing; DW, dry weight; FC, field capacity; LSD, least significant difference; OLS, ordinary least-squares; SWC, soil water content; WS, water-stressed; WW, well-watered

■

REFERENCES

(1) Jackson, M. T.; Yunus, A. G. Variation in the grass pea (Lathyrus sativus L.) and wild species. Euphytica 1984, 33, 549−559. (2) Siddique, K. H. M.; Loss, S. P.; Herwig, S. P.; Wilson, J. M. Growth, yield and neurotoxin (ODAP) concentration of three Lathyrus species in Mediterranean-type environments of Western Australia. Aust. J. Exp. Agric. 1996, 36, 209−218. (3) Al-Doss, A. A.; Assaeed, A. M.; Soliman, A. S. Growth characters and yield of some selected lines of grass pea (Lathyrus sativus). J. King Saud Univ., Agric. Sci. 1998, 10, 67−72. (4) Campbell, C. G.; Mehra, R. B.; Agrawal, S. K.; Chen, Y. Z.; Abd ElMoneim, A. M.; Khawaja, H. I. T.; Yadov, C. R.; Tay, J. U.; Araya, W. Current status and future strategy in breeding grasspea (Lathyrus sativus). Euphytica 1993, 73, 167−175. (5) Hanbury, C. D.; White, C. L.; Mullan, B. P.; Siddique, K. H. M. A review of the potential of Lathyrus sativus L. and L. cicera L. grain for use as animal feed. Anim. Anim. Feed Sci. Technol. 2000, 87, 1−27. (6) Low, R. K. C.; Rotter, R. G.; Marquardt, R. R.; Campbell, G. C. Use of Lathyrus sativus L. (var. Seminis albi) as a foodstuff for poultry. Br. Poult. Sci. 1990, 31, 615−625. (7) Tamburino, R.; Guida, V.; Pacifico, S.; Rocco, M.; Zarelli, A.; Parente, A.; Maro, A. D. Nutritional values and radical scavenging capacities of grass pea (Lathyrus sativus L.) seeds in Valle Agricola district, Italy. Aust. J. Crop Sci. 2012, 6, 149−156. (8) Kumar, S.; Bejiga, G.; Ahmed, S.; Nakkoul, H.; Sarker, A. Genetic improvement of grass pea for low neurotoxin (β-ODAP) content. Food Chem. Toxicol. 2011, 49, 589−600.

AUTHOR INFORMATION

Corresponding Authors

*UWA Institute of Agriculture and Centre for Plant Genetics and Breeding, The University of Western Australia, M080, Perth, WA 6009, Australia. Tel: +61 418 286 487. E-mail: neil.turner@uwa. edu.au. *State Key Laboratory of Grassland Agroecosystems, Institute of Arid Agroecology, School of Life Sciences, Lanzhou University, Lanzhou 730000, Gansu Province, China. Tel: +86-9318914500. Fax: +86-931-8914500. E-mail: [email protected]. Funding

The research was funded by the Chinese Scholarships Council, Doctoral Fund of Ministry of Education of China (20110211110022), National Meteorological Industrial ProH

DOI: 10.1021/acs.jafc.5b01729 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry (9) Hillocks, R. J.; Maruthi, M. N. Grass pea (Lathyrus sativus): Is there a case for further crop improvement? Euphytica 2012, 186, 647−654. (10) Rao, S. L. N.; Adiga, P. R.; Sarma, P. S. The isolation and characterization of β-N-oxalyl-L-α, β-diaminopropionic acid: A neurotoxin from the seeds of Lathyrus sativus. Biochemistry 1964, 3, 432−436. (11) Spencer, P. S.; Ludolph, A.; Dwivedi, M. P.; Roy, D.; Hugon, J.; Schaumburg, H. H. Lathyrism: evidence for role of the neuroexcitatory amino acid BOAA. Lancet 1986, 328, 1066−1067. (12) Haimanot, R. T.; Kidane, Y.; Wuhib, E.; Kalissa, A.; Alemu, T.; Zein, Z. A.; Spencer, P. S. Lathyrism in rural northwestern Ethiopia: a highly prevalent neurotoxic disorder. Int. J. Epidemiol. 1990, 19, 664− 672. (13) Yan, Z. Y.; Spencer, P. S.; Li, Z. X.; Liang, Y. M.; Wang, Y. F.; Wang, C. Y.; Li, F. M. Lathyrus sativus (grass pea) and its neurotoxin ODAP. Phytochemistry 2006, 67, 107−121. (14) Lambein, F.; Kuo, Y. H. Lathyrism. Grain Legumes 2009, 54, 8−9. (15) Koh, H. L.; Lau, A. J.; Chan, E. C. Y. Hydrophilic interaction liquid chromatography with tandem mass spectrometry for the determination of underivatized dencichine (β-N-oxalyl-l-α, β-diamino propionic acid) in Panax medicinal plant species. Rapid Commun. Mass Spectrom. 2005, 19, 1237−1244. (16) Dahiya, B. S.; Jeswani, L. M. Estimation of genetic variances by full-sib and half-sib analysis in grass-pea. Indian J. Agr. Sci. 1974, 44, 829−832. (17) Dahiya, B. S.; Jeswani, L. M. Genotype X environment interactions for neurotoxic principle (BOAA) in grass pea. Indian J. Agr. Sci. 1975, 45, 437−439. (18) Ramanujam, K. L.; Sethi, K. L.; Rao, S. L. N. Stability of neurotoxin content in khesari. Indian J. Genet. Plant Breed. 1980, 40, 300−304. (19) Bell, E. A. Nonprotein amino acids of plants: significance in medicine, nutrition, and agriculture. J. Agric. Food Chem. 2003, 51, 2854−2865. (20) Campbell, C. G. Grass pea, Lathyrus sativus L. Promoting the conservation and use of underutilized and neglected crops. 18. Institute of Plant Genetic and Crop Plant Research; Gatersleben/International Plant Genetic Resources Institute: Rome, Italy. 1997. (21) Abd El-Moneim, A. M.; Van Dorrestein, B.; Baum, M.; Mulugeta, W. Improving the nutritional quality and yield potential of grasspea (Lathyrus sativus L.). Food Nutr. Bull. Suppl. 2000, 21, 493−496. (22) Grela, E. R.; Rybiński, W.; Klebaniuk, R.; Matras, J. Morphological characteristics of some accessions of grass pea (Lathyrus sativus L.) grown in Europe and nutritional traits of their seeds. Genet. Resour. Crop Evol. 2010, 57, 693−701. (23) Grela, E. R.; Rybiński, W.; Matras, J.; Sobolewska, S. Variability of phenotypic and morphological characteristics of some Lathyrus sativus L. and Lathyrus cicera L. accessions and nutritional traits of their seeds. Genet. Resour. Crop Evol. 2012, 59, 1687−1703. (24) Tavoletti, S.; Iommarini, L.; Mogliani, L. Agronomic, qualitative (β-ODAP) and molecular variability in grasspea populations of the Marche region (central Italy). Food Chem. Toxicol. 2011, 49, 601−606. (25) Hussain, M.; Chowdhury, B.; Haque, R.; Lambein, F. Effect of water stress, salinity, interaction of cations, stage of maturity of seeds and storage devices on the ODAP content of Lathyrus sativus. In Lathyrus and Lathyrism, a Decade of Progress; Haimanot, R. T., Lambein, F., Eds.; University of Ghent: Ghent, Belgium, 1997; pp 107−110. (26) Haque, R. M.; Kuo, Y. H.; Lambein, F.; Hussain, M. Effect of environmental factors on the biosynthesis of the neuro-excitatory amino acid beta-ODAP (beta-N-oxalyl-L-alpha, beta-diaminopropionic acid) in callus tissue of Lathyrus sativus. Food Chem. Toxicol. 2011, 49, 583− 588. (27) Jiao, C. J.; Xu, Q. L.; Wang, C. Y.; Li, F. M.; Li, Z. X.; Wang, Y. F. Accumulation pattern of toxin β-ODAP during lifespan and effect of nutrient elements on β-ODAP content in Lathyrus sativus seedlings. J. Agric. Sci. 2006, 144, 369−375. (28) Cocks, P.; Siddique, K. H. M.; Hanbury, C. Lathyrus - A new grain legume; No. 99/150; Rural Industries Research and Development Corporation: Kingston, Australia, 2000.

(29) Yang, H. M.; Zhang, X. Y.; Wang, G. X. Relationships between stomatal character, photosynthetic character and seed chemical composition in grass pea at different water availabilities. J. Agric. Sci. 2004, 142, 675−681. (30) Gusmao, M.; Siddique, K. H. M.; Flower, K.; Nesbitt, H.; Veneklaas, E. J. Water deficit during the reproductive period of grass pea (Lathyrus sativus L.) reduced grain yield but maintained seed size. J. Agron. Crop Sci. 2012, 198, 430−441. (31) Kong, H.; Palta, J. A.; Siddique, K. H. M.; Stefanova, K.; Xiong, Y. C.; Turner, N. C. Photosynthesis is reduced, and seeds fail to set and fill at similar soil water contents in grass pea (Lathyrus sativus L.) subjected to terminal drought. J. Agron. Crop Sci. 2014, n/a. (32) Addis, G.; Narayan, R. K. J. Developmental variation of the neurotoxin, β-N-oxalyl-l-α, β-diamino propionic acid (ODAP), in Lathyrus sativus. Ann. Bot. 1994, 74, 209−215. (33) Kuo, Y. H.; Khan, J. K.; Lambein, F. Biosynthesis of the neurotoxin β-ODAP in developing pods of Lathyrus sativus. Phytochemistry 1994, 35, 911−913. (34) FAO-UNESCO. Soil map of the world: Revised legend. World Soil Resources Report; No. 60; FAO: Rome, 1988. (35) Wang, F.; Chen, X.; Chen, Q.; Qin, X. C.; Li, Z. X. Determination of neurotoxin 3-N-oxalyl-2, 3-diaminopropionic acid and non-protein amino acids in Lathyrus sativus by precolumn derivatization with 1fluoro-2, 4-dinitrobenzene. J. Chromatogr. A 2000, 883, 113−118. (36) Yan, Z. Y.; Jiao, C. J.; Wang, Y. P.; Li, F. M.; Liang, Y. M.; Li, Z. X. A method for the simultaneous determination of β-ODAP, α-ODAP, homoarginine and polyamines in Lathyrus sativus by liquid chromatography using a new extraction procedure. Anal. Chim. Acta 2005, 534, 199−205. (37) Falster, D. S.; Warton, D. I.; Wright, I. J. SMATR: Standardised major axis tests and routines, version 2.0, 2006. http://www.bio.mq.edu. au/ecology/SMATR. (38) Dahiya, B. S. Seed morphology as an indicator of low neurotoxin in Lathyrus sativus L. Qual. Plant. - Plant Foods Hum. Nutr. 1976, 25, 391−394. (39) Roy, D. N.; Rao, K. V. Physicochemical values in different varieties of Lathyrus sativus and their interrelationships. J. Agric. Food Chem. 1978, 26, 687−689. (40) Gresta, F.; Rocco, C.; Lombardo, G. M.; Avola, G.; Ruberto, G. Agronomic characterization and α- and β-ODAP determination through the adoption of new analytical strategies (HPLC-ELSD and NMR) of ten Sicilian accessions of grass pea. J. Agric. Food Chem. 2014, 62, 2436− 2442. (41) Xiong, J.-L.; Kong, H.-Y.; Akram, N. A.; Bai, X.; Ashraf, M.; Tan, R.-Y.; Siddique, K. H. M.; Xiong, Y.-C.; Turner, N. C. 24-epibrassinolide increases growth, grain yield and β-ODAP production in seeds of wellwatered and moderately water-stressed grass pea. Plant Growth Regul. 2015, (accepted 27 May 2015), DOI: 10.1007/s10725-015-0087-1. (42) Xing, G. S.; Cui, K. R.; Li, J.; Wang, Y. F.; Li, Z. X. Water stress and accumulation of β-N-Oxalyl-L-α, β-diaminopropionic acid in grass pea (Lathyrus sativus). J. Agric. Food Chem. 2001, 49, 216−220.

I

DOI: 10.1021/acs.jafc.5b01729 J. Agric. Food Chem. XXXX, XXX, XXX−XXX