Toxin ?-ODAP Accumulation And Allometric Relationship In Grass Pea(Lathyrus Sativus L.) Under Drought Stress

Grass pea (Lathyrus sativus L.) is considered an excellent legume crop as food and forage with a high protein content of up to 25% in seeds. It has a broad spectrum of resistances to adverse stresses such as drought, cold, waterlogging and insect pest and so on. These features lead it to become an important crop germplasm resource to cope with food and ecosystem security under global climate change. But the exploration and utilization on it is largely limited due to the presence of one non-amino acid, ?-N-oxalyl-L-?, ?-diaminopropionic acid (?-ODAP) in seeds and tissues, which is a neurotoxin that causes irreversible lathyrism in humans and animals after long-term excessive consumption. Since 1960s, a large number of efforts have been paid on the selection and breeding programs of low-or zero-toxin grass pea cultivars and the relevant tillage management technologies, however, the practical progresses achieved was restricted. For the solution to this obstacle, a fundamental scientific issue is how toxin ?-ODAP biosynthesis and allocation, individual growth and yield formation as well as the related ecological mechanism would be under drought stress. A large number of experimental results were found to be not mutually supportive and even contrary, which leads to failure of result incorporation. It is so far in a low progress for the fundamental scientific question mentioned above.On the basis of previous studies, we summarized the above fundamental scientific question into the following several relatively separated but closely linked issues:1) the characteristics of toxin biosynthesis and transportation in grass pea under drought stress; 2) the characteristics of individual growth and biomass allocation in grass pea under drought stress; 3) the relationship between ?-ODAP accumulation and growth plasticity and its related ecological mechanism under drought stress. In this thesis, three aspects of experimental researches have been conducted in the combination of growth environment-controlled experiments and field experiments using the grass pea genotypes with various toxin levels and yield potentials as testing materials.1) Pot-culture experiments with different water treatments and exogenous hormone applications were conducted using one local genotype of grass pea to reveal the characteristics of spatial-temporal distribution of toxin ?-ODAP, water use and yield formation. The data was further analyzed to develop the coupling relationship between toxin ?-ODAP accumulation and biomass accumulation and thereafter elucidate its regulatory mechanism.2) Extending experimental researches were conducted using pot-culture experiments with various water supply gradients. Seven genotypes of grass pea were used to determine the characteristics of spatial and temporal distribution of toxin, and further reveal the relationship between toxin biosynthesis and biomass accumulation and its related eco-physiological mechanism at reproductive growth period.3) Further extending and validating experiments were designed using 55 genotypes of grass pea with different toxin levels and yield potentials over two growing seasons under the field conditions. The experiments were aimed to quantitatively assess the responses of toxin P-ODAP accumulation and growth plasticity to the interactions between genotype and environment, and reveal its ecological mechanism. In those experiments mentioned above, various water supply regimes were designed to determine the parameters of toxin biosynthesis and distribution in different organs, plant hormone level, individual growth, biomass allocation, water use and yield formation and so on. Interdisciplinary researches and validating experiments were systematically conducted at the physiological level, individual level and population level. The major results were presented as follows.1. Pot-culture trials with varies water supplies and exogenous hormone application using one genotype throughout the whole growth period1mg plant growth regulator-24-epibrassinolide (EBL) and 10?mol drought response hormone-abscisic acid (ABA) were applied to pots with soil as root drenches in grass pea at the seedling stage in three water treatments [adequate water supply,80% field capacity (FC); moderate drought stress,50% FC; severe drought stress,35% FC]. The concentration and amount of P-ODAP in leaves and pods, ABA and proline concentration in leaves, and biomass accumulation from the seedling stage to maturity, and the grain yield and yield components at maturity were tracked at three water regimes.1) Leaf ?-ODAP concentration gradually decreased and P-ODAP amount firstly increased and then decreased from the seedling (30 DAS) to podding stage (90 DAS), pod ?-ODAP concentration and amount increased from flowering (70 DAS) to podding stage, and then decreased from podding stage to maturity (100 DAS) under sufficient water condition (80% FC) and moderated drought stress (50% FC). From seedling to podding stage, leaf ?-ODAP concentration at 35% FC had similar trends as at 80% FC and 50% FC, but pod ?-ODAP concentration and amount had slightly increase under severe drought stress (35% FC) from flowering stage to maturity. Drought stress significantly increased the ?-ODAP concentration and decreased P-ODAP amount in leaves, pods, and mature seeds.2) Exogenously applied with EBL and ABA showed that the ?-ODAP and growth in grass pea were significantly affected by EBL instead of ABA. The EBL applied as a root drench significantly increased the biomass in different organs and P-ODAP amount in leaves and pods under sufficient water condition and moderate drought stress, but it was not mediated by changing the concentration of ABA and proline in leaves. The final aboveground biomass, grain yield, the concentration and amount of grain P-ODAP, and water use efficiency for grain yield and aboveground biomass were increased by EBL at 80% FC and 50% FC in grass pea. EBL did not have a significant effect on above traits under severe drought stress (35% FC). ABA had no significant effect on the accumulation of biomass and final grain yield. The combination of EBL and ABA had the similar effect to that of EBL alone.3) Drought stress significantly changed the allometric relationships between organ biomass or allocation and total biomass, although exogenous EBL and ABA did not significantly affect it. Different water supply significantly affected the slopes between stem, pod or root biomass and total biomass, and had no effect on the slopes between the leaf and total biomass. Drought stress increased the root biomass and root biomass allocation, decreased the stem and leaf biomass, and their biomass allocation at the early growth period; water shortage reduced the leaf, root and pod biomass, and their allocation at the late growth period. There was an allometric relationship instead of an isometric relationship between underground and aboveground biomass (??1).4) Drought stress significantly increased the absolute value of the slopes of the allometric equations between ?-ODAP amount and organ biomass or total biomass during growth, although exogenous hormone did not significantly affect it. Leaf ?-ODAP amount and leaf biomass, and pod P-ODAP amount and pod biomass had isometric relationships (?=1), but ?-ODAP amount and total biomass was an allometric relationship (??1) at 80% FC. In addition, the allometric index (?, slope) of leaf ?-ODAP amount and total biomass at 80% FC was 2/3, drought stress significantly decreased it, which was -3/4 at 50% FC and -5/4 at 35% FC.2. Pot-culture trais with various water treatments using seven grass pea genotypes during the reproductive growth periodThe changes in concentration and amount of ?-ODAP and biomass of leaves and pods from the flowering stage to maturity, and grain yield and P-ODAP concentration and amount in the seed at maturity in seven grass pea genotypes with different seed P-ODAP concentrations grown in pots with soil were also measured at the same three water levels.5) The ?-ODAP concentration and amount in leaves decreased from flowering (54 DAS) to podding stage (75 DAS), ?-ODAP concentration and amount in pods also decreased from podding stage to maturity (103 DAS) in three water treatments. Water shortage significantly increased the P-ODAP concentration and decreased the ?-ODAP amount in leaves, pods, and seeds in all genotypes, expect IF 1310. In addition, drought stress increased the reduction of leaf P-ODAP amount from flowering to podding stage, and the average of the reduction of seven grass pea genotypes was 3.6 mg plant-1 at 80% FC,5.9 mg plant-1 at 50% FC, and 6.5 mg plant-1 at 35% FC. Drought stress also increased the reduction of pod ?-ODAP amount from podding stage to maturity, and the average of reduction of seven grass pea genotypes was 16.0 mg plant-1 at 80% FC,24.9 mg plant-1 at 50% FC, and 31.2 mg plant-1 at 35% FC.6) There were significant positive relationships between the seed ?-ODAP concentration or amount at maturity and the sum of leaf and pod ?-ODAP amount at the podding stage. It existed no significant differences in the slopes of equations between seed P-ODAP concentration at maturity and the sum of leaf and pod ?-ODAP amount at podding stage among the three water regimes. The slopes of equations between seed ?-ODAP amount at maturity and the sum of leaf and pod ?-ODAP amount at podding stage for 80% FC and 50% FC were significantly higher than 35% FC.7) After fitting the allometric relationship between ?-ODAP amount and corresponding organ biomass or total biomass, we found that drought stress did not significantly affect the slopes of the allometric equation for leaf, but significantly increased the intercept between leaf ?-ODAP amount and leaf biomass, decreased the intercept between leaf ?-ODAP amount and total biomass; drought stress significantly increased the absolute value of the slopes for pod.3. Field trails using fifty-five grass pea genotypes with different toxin levels over two growing seasons with different rainfall amount at maturityIn addition, fifty-four grass pea genotypes from International center for agricultural research in the dry areas (ICARDA) and one native genotype were planted in a field located in a semi-arid area of the Loess Plateau in 2012 and 2013 to detect the genotype by environment interactions on the accumulation of biomass and grain ?-ODAP at maturity.8) The rainfall significantly affected the grain yield and harvest index (HI). Large differences existed among different grass pea genotypes for biomass traits. The average of grain DW was 2.58 g plant-1 (1.27-4.98 g plant-1) in 2012 and 4.40 g plant-1 in 2013 (2.20-7.20 g plant-1), and the average of aboveground biomass was 4.38 g plant-1 (2.02-10.46 g plant-1) in 2012 and 8.09 g plant-1 in 2013 (4.03-14.11 g plant-1). The grain DW increased by 70.5%, the aboveground DW increased by 82.6% and HI decreased by 7.1% in 2013 compared to 2012.9) The rainfall significantly affected the grain ?-ODAP concentration and amount. The grain ?-ODAP concentration and amount were different among the fifty-five genotypes. The average of grain P-ODAP concentration was 1.74% DW (1.04% DW-2.91% DW) in 2012 and 1.17% DW (0.54% DW-1.89% DW) in 2013. The average of grain ?-ODAP amount was 44.5 mg plant-1 (17.6 mg plant-1-93.1 mg plant-1) in 2012 and 51.1 mg plant-1 (23.5 mg plant-1-98.0 mg plant-1) in 2013. The grain ?-ODAP concentration decreased by 31.6%, but the grain ?-ODAP amount increased by 11.2% in 2013 compared to 2012. The greater amount of rainfall in 2013 significantly increased the P-ODAP amount, but decreased the P-ODAP concentration in the grain, as the greater grain yield diluted the toxin. There were no significant differences for the slopes of the allometric equation for grain P-ODAP amount and grain DW between 2012 and 2013, but the intercept in 2013 was significantly lower than in 2012.10) The slopes of allometric equations between reproductive (R, pod DW) organ biomass and vegetative (V, sum of leaf DW and pod DW) organ biomass in 2012 and 2013 were close to 1.0, and the slope in 2013 (0.85) was significantly lower than in 2012 (1.06).The study combined the mathematical statistics and allometric analysis to analyze the effect of drought stress on the accumulation of P-ODAP and growth plasticity in grass pea by conducting three experiments from one grass pea genotype throughout the whole growth period to seven grass pea genotypes during the reproductive growth stage to fifty-five grass pea genotypes at maturity. This series of experiments were mutual authentication and complement each other, and jointly revealed that drought stress significantly affected the distribution of P-ODAP during growth, the special allometric relationship between leaf P-ODAP amount and total biomass, and biomass allocation and vegetative-reproductive output in grass pea. In this study, we firstly systematic investigated the ?-ODAP accumulation and growth plasticity and its related ecological mechanism. These results not only provide the theoretical potential for breeding low-or zero-toxin grass pea cultivars, but also provide data support for enriching and developing allometric theory in the plant.