Variation of the Impact Aroma Compound, 2-Acetyl-1-Pyrroline, Content in Thai Fragrant Rice Plants and its Enhanced Accumulation by Soil Nutritional Elements

This study investigated the accumulation characteristics of 2-acetyl-1-pyrroline (2AP) in the Thai Jasmine rice cultivar, Khao Dawk Mali 105 (KDML 105) under different soil types and addition of soil nutritional elements. There was significant variation in the 2AP contents of rice leaves, not only among different growth stages, but also among different leaf positions at each growth stage. The highest 2AP content was seen in the third leaves at booting stage (25.10±0.90 μg g), whilst for flag leaves it was seen at mature grain stage (8.00±0.72 μg g). During the ripening stage, plants in sandy soil acquired higher 2AP content in flag leaves, spikelets, and seeds than in clay soil, with the highest 2AP content in flag leaves at 25.00±0.70 μg g. The accumulation of 2AP in basal, middle, and upper branches of panicles showed no significant differences, whereas a steady decrease in 2AP content was detected in the panicle axis and branches. The effects of individual mineral elements (B, Cr, Cu, Mg, Mn, and Se) on 2AP content was different at each growth stage. B enhanced the 2AP content by more than two-fold in the leaves at the heading stage (63.84±4.56 μg g) compared to that of the control set, followed by Mn, Cu, and Se. Similar to the above results, the 2AP content was also heightened in grains. The extended knowledge gathered in this study will assist farmers who wish to improve the fragrance qualities of their rice varieties.


Introduction
Mineral elements in soil are known to have physiological functions in plants, acting as enzymatic activators, osmotic regulators, electron transporters, or constituents of organic/inorganic compounds (Hänsch & Mendel, 2009;Xie et al., 2004). However, the effect of mineral nutrients, other than Na and Cl, on rice plants, especially the effects on 2-Acetyl-1-pyrroline (2AP) content have not been widely investigated. The majority of research has focused on the effects of mineral elements on rice plant morphology and grain quality (Zeng et al., 2005), but little research has focused on the 2AP content in rice plant parts and grain.
Aroma compound 2AP is found in many plant species and is credited as one of the most important traits of aromatic rice. Thai jasmine rice variety, Khao Dawk Mali 105 (KDML 105), is a non-glutinous, fragrant rice variety, famous for its eating and cooking quality due to its high 2AP content. Multiple studies have been conducted on the gene responsible for 2AP in rice plants and research on environmental differences such as soil type, salinity, and water stress, have all been linked to the accumulation of 2AP in rice grains (Sarvestani, Pirdashti, Sanavy, & Balouchi, 2008;Thanachit, Jedrum, Anusontpor, & Wiriyakitn, 2014;Boontakham, Sookwong, Jongkaewwattana, Wangtueai, & Mahatheeranont, 2019). According to these reports, drought conditions and sandy soils are favourable for 2AP accumulation, although the predominant rice cultivation conditions are usually flooded clay soils (R. Singh, U. Singh, & Khush, 2000). Water stress treatment of KDML 105 after heading increases the amount of 2AP by 18% in leaves and 22% in grains (Boontakham et al., 2019). Another report described the correlation between 22 different locations possessing eight different soil textures in the northeastern part of Thailand and the 2AP content of KDML 105 rice grains. Sandy soil conditions and soil pH of 5.27 showed the highest 2AP content (Kong-ngern et al., 2011). The sandy soil texture is closely linked to water stress environments as the capacity for water retention is lower in sandy soils than that of clay soils. The effect of ripening is affected by temperature and geographical locations (Ishimaru et al., 2018;Pitiphunpong & Suwannaporn, 2009) and significantly affect 2AP content in grains and yield in rice plants. Saline conditions have also been extensively studied (Funsueb, Krongchai, Mahatheeranont, & Kittiwachana, 2016;Gay et al., 2010;Lutts, Kinet, & Bouharmont, 1996). In most reports, it is concluded that saline soil conditions increase the 2AP content in rice grains and improves the grain yield of aromatic rice at harvest. Furthermore, the effects of minerals, such as Zn, Fe, and La have been studied on the aroma content of brown rice and the proline content in leaves and panicles (Tang & Wu, 2006;Xiao et al., 2010;Mo et al., 2016). Zn and La promote higher 2AP concentrations in the rice panicles and grains due to increased proline concentrations and proline dehydrogenase activities. It can be hypothesised that the increase in aroma content of rice leaves at cultivation could increase the aroma content of rice grains.
The aim of this research was to enhance 2AP content in leaves and seeds of KDML 105 Thai rice. This was achieved by investigating the 2AP content in plants at different developmental stages, grown under different cultivation conditions, including different soil conditions and mineral element treatments. The 2AP content in rice seeds and leaves grown in sandy and clay soil were compared. The accumulation of 2AP in rice leaves at different leaf positions and panicle branches of rice plants was determined to observe the characteristics of 2AP accumulation. Moreover, the influence of nutritional elements commonly available in soil, such as Mg, B, Mn, Cu, Cr, Se, on the growth and variation in 2AP content in leaves and seeds was studied. These results will help to clarify the translocation characteristic of 2AP content in rice plants and expand the understanding of the true nature behind the development of 2AP in rice plants and environmental factors that influence the accumulation of 2AP. The extended knowledge gathered will be of assistance to farmers around the world who wish to improve the fragrant qualities of their rice varieties.

Chemicals
The standard compound 2AP was synthesized following two steps: hydrogenation and oxidation. In the first step, 2-acetylpyrrole was hydrogenated using 5% rhodium on activated alumina as the catalyst. After the maximum amount of the hydrogenation product had been obtained, the rhodium catalyst was removed and solvent was evaporated to give 2-(1-hydroxyethyl)pyrrolidine, a pale yellow viscous product. The oxidation reaction in the second step employed silver carbonate on Celite as a catalyst. The purified 2AP was yielded after separation on a packed column of a gas chromatograph and collected in a 3-mm o.d. Pyrex tube. This tube was then sealed under an N 2 atmosphere and kept at -20 °C. The purity and chemical structure of the synthetic 2AP were confirmed by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). The compound 2,6-dimethylpyridine (2,6-DMP) was used as internal standard. The basic fertilizer consisted of ammonium nitrate, potassium nitrate, and potassium dihydrogen orthophosphate. The individual mineral element included B (as B-amino acid complex), Se (as L-selenomethionine), Mn (as Mn amino acid chelate), Cu (as Cu amino acid chelate), Cr (as Cr amino acid chelate) and Mg (equivalent to Mg(OH) 2 ).

Cultivation Conditions
The non-glutinous, fragrant rice cultivar KDML 105 was grown at the experimental field of Chiang Mai Rice Research Centre, Sanpatong, Chiang Mai, Thailand. The growth period was from August to early December 2017 and 2018, with the average daily temperature ranging from 19 °C to 32 °C. KDML 105 rice seeds were soaked and kept in the dark for 24 h for germination and nursed in soil trays for two weeks before being transported to growing pots and experimental fields. Sampling was carried out at the same time of day (9-10 am) and the samples were stored at -20 °C one day before analysis.

Cultivation of KDML 105 Rice in Sandy Soil for Collection of Leaves and Panicles at Different Positions
Rice seedlings were transported to the experimental field (40 m × 20 m; in length and width respectively) and grown in sandy soil. Leaves were collected at random from various locations in the field during nine developmental stages including seedling, tillering, panicle initiation, booting, heading, milk grain, soft dough grain, hard dough grain, and mature grain. Panicles were carefully collected at booting, heading, milk grain, soft grain, hard dough, and mature grain stages. To investigate the variation in 2AP content in different leaf positions, samples were collected at the flag leaf (uppermost leaf below the panicle) and the second and third leaf position of rice plants. The primary panicles at flag leaf position were collected and the seeds of the panicle were separated evenly as the basal, middle and upper branches, and the panicle axis were analysed independently from the seeds. The characteristics of 2AP from leaves to grain was thus determined.

Cultivation of KDML 105 Rice in Different Soil Types
Rice seedlings were transplanted into 10-inch experimental plant pots (n = 3) filled with clay or sandy soil. Rice flag leaves and panicles were collected at heading, milk grain, soft dough grain, hard dough grain, and mature grain stages. The comparison of growth in sandy and clay soil was carried out to clarify research already established by other authors. The characteristics of 2AP from leaves to grains was also clarified in these experiments.

Cultivation of KDML105 Rice With Addition of Mineral Elements
To obtain the appropriate concentration of mineral elements, a preliminary study on KDML 105 plant developmental growth with the application of different concentrations of ten mineral elements was carried out. The mineral elements used were Ca, Mg, B, Zn, Mn, Cu, Mo, Fe, Cr, and Se. Four concentrations of individual elements were prepared following the minimum and maximum range reported in the literature (Schwarz, 1995). Rice seedlings were transplanted into 10-inch plant pots (n = 3) filled with clean sand and placed into a larger 12-inch pot filled with nutrient solution. The plants were supplied with a basic fertiliser solution (nitrogen (N), phosphorus (P), Potassium (K)), prepared by modifying the solution of Hoagland and Arnon (Jones, 2014). Individual elements were supplied along with NPK basic fertiliser solution twice a week over a period of 12 weeks. The experiment was conducted in open-air conditions with natural sunlight at the experimental field of the Chiang Mai Rice Research Center, Sanpatong.
From the preliminary experiment, six mineral elements applied at their most appropriate concentrations (Table 1) to obtain the largest grain yield and growth were selected for the investigation of individual mineral elements. Rice plants were transplanted into 10 and 12-inch plant pots (n = 3) containing sand and nutrient solution. Basic fertiliser NPK were supplied with individual elements twice a week for 12 weeks. Rice leaves and seeds were collected at four developmental stages for the quantification of 2AP content.

Quantification of 2-Acetyl-1-Pyrroline in Leaves and Seeds of KDML 105 Rice
Rice samples were dried under ambient conditions to reach a moisture content of approximately 14%. Leaf samples (0.20 g) were ground with a blender (Y46; Moulinex, Paris, France) and placed into 20 mL headspace vials with 1.0 µl of 500 ppm and 2, 6-DMP added as an internal standard. The headspace vials were sealed immediately with polytetrafluoroethylene silicone (PTFE/silicone) septa and aluminum caps prior to analysis by static headspace-gas chromatography coupled with a nitrogen/phosphorus detector (SHS-GC-NPD).  Vol. 12,No. 6; of KDML 105 < 0.05) within in the same gr ith the additio ±1.18 µg g -1 ) 1 ), and the co booting stage 49.33±0.65 µg rences between ants grown wit 41 µg g -1 ), an n stage, the hig (5.93±0.46 µg n mature grain d by Se (2.61± ment with diffe < 0.05) within olumn in the 2020 rice n the owth on of was ontrol e, the g -1 ), n the h the nd Se ghest g g -1 ) n, the ±0.02 erent n the same jas.ccsenet. of diseases as well as boosted root development in rice plants, whilst calcium is believed to impact straw stiffness, seed, and grain formation (Kushwaha, 2016).

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Rice plants treated with Mg had the highest 2AP content at tillering, followed by Mn. In rice plants Mg is a component of chlorophyll and is involved in the translocation of other minerals. At tillering, Mg is taken up in higher concentrations than most other minerals and acts as an enzymatic activator (Marschner, 2012). The presence of Mg in rice plants is linked to high yield due to its involvement in chlorophyll synthesis. However, Mg showed no effect on 2AP content in rice leaves in other growth stages whilst Mn increased the 2AP content in all four stages of rice plant development.
At booting stage, Cu and Cr highly promoted 2AP formation, and the 2AP content was twice as high as that at the tillering stage. Cu is necessary at the seedling stage as it stimulates the absorption of water and nutrients from the soil (Kushwaha, 2016). The first report of Cr involvement in 2AP content is described in our study. Previous reports have suggested the association of Cr with the root systems of similar cereal plants, such as oats and corn, but according to our knowledge, its exact consequence on rice has not yet been studied (Samantaray, Rout, & Das, 1998). The role of Cr on 2AP production at booting stage suggested that it is an important mineral for the reproductive stage of rice plants as opposed to the vegetative or ripening stages.
At heading, the 2AP content in the leaves of rice plants treated with Cu, Cr, and Mg, decreased while those treated with B, Se, and Mn increased significantly. B stimulated a very high production of 2AP at the heading stage, double the amount at the booting stage. At the mature grain stage, grains of plants treated with B and Se had the highest amount of 2AP. B is associated with carbohydrate chemistry as well as pollen germination, pollen grain development, and seed production (Fageria, 2014). The involvement of B in seed production and grain development could have stimulated rice seeds and leaves to accumulate more 2AP than other mineral-treated rice plants. In addition, Se mitigates stress in plants because of its ability to induce the synthesis of S and N compounds, in addition to stimulating the activity of antioxidant enzymes and metabolites (Mikkelsen & Wan, 1990;Shalaby et al., 2017). In this study, the addition of Se helped to increase the 2AP content of rice leaves at heading and seeds at mature grain stage. This suggests Se as an important mineral for the ripening stages of rice plants.
In summary, our result showed that the addition of Mg resulted in an increase of 2AP content at the tillering stage, whilst other elements reduced 2AP content compared to that of the control. Cu showed importance at the booting stage whilst B at the heading stage. Although Mn addition did not produce plants with the highest amount of 2AP, its contribution was seen as significant through-out the cultivation period. Mn increased the 2AP content in all four stages of rice plant development. Overall, addition of B to rice plants significantly increased the 2AP content in the leaves and seeds of KDML 105 rice plants.

Conclusions
This study investigated the effect of some mineral elements on the 2AP content in KDML105 rice plants and yielded positive results in elucidating the accumulation and translocation characteristics of 2AP in rice plants.
The 2AP content varied among rice leaves, not only in the different growth stages, but also in different positions of the rice plant at each growth stage. Sandy soil facilitated higher 2AP content than that of clay soil. B treatments showed the highest amount of detected 2AP in both seeds and leaves of KDML 105 rice plants. There was a positive correlation between the amount of 2AP detected in rice leaves at the heading stage and seeds at the mature grain stage. Accordingly, mineral elements that stimulated 2AP production in the leaves at heading stage, as B did, had the most effect on the amount of 2AP that was accumulated in the rice seeds.