Metabolite profile and correlation in HP and LN
ANOVA analysis of the metabolites in the two maternal environments indicated that contents of 36 of 58 known metabolites were significantly changed between HP and LN maternal conditions. These metabolites included mostly amino acids such as serine, pyroglutamate and GABA and organic acids including TCA cycle intermediates such as galacterate, malate, succinate and malonate (Table 1 ).
In general Log2 ratios of HP:LN in the RIL population showed that most of the metabolites had a higher level in seeds from the HP environment as compared to the LN environment (Figure 3, Table 1 ).
The metabolite profiles of seeds from the two maternal environments showed that seeds grown in HP conditions contained a higher amount of metabolites, such as many sugars, amino acids and organic acids (Figure 3, 4 ). A high metabolite level was predominantly observed for GABA, sugars (maltose, fructose and glucose), organic acids including benzoate, salicylate, glycerate and also some TCA cycle intermediates such as galacterate, malate, malonate and succinate. The TCA cycle, including several catabolic reactions, plays a very critical role in energy metabolism in plants. In addition, it contributes in many other ways to the metabolome by being involved in a large number of metabolic networks (Araujo et al. 2012). In plants, it has been shown that amino acids such as leucine, iso-leucine and valine can be degraded into new products which can be used as precursors for the TCA cycle to provide additional energy for plants. The rate of the degradation of these compounds can be increased due to sub-optimal conditions such as abiotic stresses (Binder 2010).
Both HP and LN maternal environments could be sub-optimal environments for plant growth and seed development. Our results revealed that most of the amino acids were not significantly altered between the two maternal environments; however some of them such as GABA, pyroglutamate, glycine, leucine and aspartate showed significantly lower values in seeds grown in LN. This result was consistent with findings for tomato and Arabidopsis plants that reported a general decrease of amino acid levels under low nitrate conditions (Tschoep et al. 2009; Urbanczyk-Wochniak & Fernie 2004). GABA is one of the amino acids which frequently shows higher levels under stress conditions (Michaeliet al. 2011; Renault et al. 2011; Shelp, Bown & McLean 1999). GABA is an amino compound which is produced via the so-called GABA shunt pathway which has a primary role in keeping a balance in central C/N metabolism (Bouche & Fromm 2004). It has been shown that GABA levels increased rapidly under stressed growth conditions. Thus, GABA is thought to be involved in the tolerance of plants to sub-optimal environments (Fait et al. 2008; Kinnersley & Turano 2000; Renault et al. 2011). In our study we have observed that the GABA content was lower in seeds developed in LN conditions in comparison with those of the HP maternal condition. Our findings confirmed previous studies, where seeds also showed low amounts of GABA under LN maternal conditions (He et al. 2016); (Geshnizjani et al. 2019).
It has previously been reported that nitrate starvation resulted in a decrease in TCA cycle intermediates in tomato (Urbanczyk-Wochniak & Fernie 2004) and Arabidopsis (Tschoep et al. 2009) leaves. We also found that in comparison to the HP condition, seeds developed under LN possessed a lower amount of TCA cycle intermediates such as malate, succinate and malonate (Figure 4 ). This could be an indication of higher consumption of TCA cycle intermediates to produce more energy under LN to allow the plants to survive and continue growth.