## 3 Discussion
### Role of Precursors in the High Expression of 2 AP in P. amaryllifolius  
_P. amaryllifolius_ is a potent producer of 2 AP. The _P. amaryllifolius_ leaf tissue showed the presence of 2 AP. Basmati rice has been reported to accumulate up to 1 ppm of 2 AP and in the highly scented rice variety Khao Dawk Mali-105 up to 3 ppm of 2 AP [33]. [PERSON], [PERSON], and [PERSON] [19] reported relatively higher 2 AP in the aerial parts of the rice plants when compared to the milled grains. Moreover, 2 AP has been reported to be synthesized throughout rice plants except in the roots [34]. Similar to the reports for aromatic rice, the reports for 2 AP indicate that it is synthesized in aerial parts of _P. amaryllifolius_[PERSON] The presence of 2 AP prompted us to analyze the precursors related to the 2 AP biosynthetic pathway. Proline has been identified as a precursor amino acid for the biosynthesis of 2 AP and higher concentrations have been reported to contribute to enhanced 2 AP levels. Several reports have demonstrated the role of proline in 2 AP pathway. Using tracer experiments with 15N-proline, [PERSON] et al[PERSON] [34] demonstrated that the nitrogen of 2 AP was derived from proline. [PERSON] et al. [17] reported enhanced production of 2 AP by feeding L-proline to the semi-differentiated callus culture of _P. amaryllifolius_. The maximum level of 22 mg/kg of 2 AP was synthesized when rice calli were treated with 1 mmol/1 of L-proline. [PERSON] et al[PERSON] [18] demonstrated that proline addition enhanced the aroma in callus cultures of aromatic rice, suggesting that proline was related to aroma synthesis. [PERSON] et al. [24] overexpressed the _P5 CS_ gene in scented rice in order to achieve enhanced accumulation of proline, P5C, and 2 AP. The leaf samples accumulated 8.07 \(\pm\) 0.09 umoles/g of free proline and a higher amount of P5 CS enzyme activity 67.8 \(\pm\) 2.83 with the presence of transcripts supporting the higher accumulation of 2 AP in _P. amaryllifolius_ leaves.  
In addition to proline, methylglyoxal has also been identified as another precursor for the biosynthesis of 2 AP. [PERSON] et al. [35] reported higher (1.9 umol/g) MG levels in aromatic than nonaromatic cultivars (1.2 umol/g) of soybean. MG was identified as the carbon source for 2 AP. Vegetable soybeans with higher levels of 2 AP were reported to contain higher amounts of MG demonstrating a direct relation between MG and 2 AP. They also demonstrated that an intermediate compound derived from proline reacts nonenzymatically with MG to produce 2 AP.  
In addition, the synthesis of GABA is regulated by _BADH2_. In aromatic rice varieties, since _BADH2_ is nonfunctional, low GABA synthesis takes place. However, the amount of GABA present in _P. amaryllifolius_ was considerably higher. The concentrations of GABA in various plant parts commonly range from 0.03 to 5.5 \(\upmu\)g/g fresh weight [22]. This study demonstrated higher levels of proline and methylglyoxal that might be responsible for the high 2 AP expression in _P. amaryllifolius_. BADH2 enzyme activity leads to an increased level of its substrate, GAB-ald, the immediate precursor of 2 AP [36]. Utilization of GAB-ald by converting it into GABA blocks 2 AP synthesis, whereas the enhancement of GAB-ald results in increased 2 AP synthesis.