Impact of COշ enrichment and its interaction with nitrogen on growth, physiology and secondary metabolites of Labisia pumila benth

The demand for Labisia pumila (Kacip Fatimah) keeps increasing annually as this plant is believed to have high phytoestrogen activity that widely used in neutraceutical industries. However the plant is relatively difficult slow to grow, and takes about 16 to 36 months before it is ready for use in medicinal preparation. Carbon dioxide (CO2) enrichment technique is known to be able to enhance slow growing plants and also to improve and alter plant bioactive compounds. Enriching L. pumila with elevated CO2 may extend these benefit. Therefore, a project was conducted with the objectives ; (i)to determine and establish the most suitable microclimate for CO2 enrichment of L. pumila under growth house based on ventilation per floor rate area and shading levels (ii) to investigate the effects of different CO2 concentration on the growth, leaf gas exchange and secondary metabolites of three varieties of L. pumila (iii) to determine the interaction between CO2 and nitrogen levels on growth, leaf gas exchange,primary and secondary metabolites of L. pumila and (iv) to establish and understand the biochemical regulation of secondary metabolites in L. pumila seedlings under interaction effects between CO2 and nitrogen levels. There were four experimen conducted to fulfill the objectives. In the first experiment , two levels of shade (0% ,and 90%) and five different ventilation to floor area (v/f) ratio (0 = close; 0.375,=open-up; 0.450 = open-doors; 0.750 = open-bottom; 1.35 = open-all) were conducted to determine the best combination of these factors for establishment of the suitable microclimate for L. pumila under growth house for CO2 enrichment. Result showed that shade+open-all give the best microclimate inside the growth chamber. Analysis of variance revealed that means of temperature and vapor pressure deficit (VPD) were significantly (P≤0.01) influenced by v/f ratio. Mean relative humidity (RH) inside the chamber was significantly (P≤0.01) enhanced by the shade used. Under shade+open-all, the stomatal conductance (gs) of L. pumila was found to be very sensitive to VPD. At VPD, temperature and RH of 1.41 kpa, 330C and 68% respectively, gs of L. pumila started to decrease from 1100 am onwards suggesting that CO2 enrichment should be carried out before this time to optimize benefit from CO2 exposure. In the second experiment, three varieties of L. pumila seedlings (alata,pumila and lanceolata) were exposed to three levels of CO2 (400, 800,1200 μmol mol-1). In this experiment, the manipulation of CO2 enrichment on L. pumila seedling seemed to be able to reduce the nursery period through growth enhancement, that was solely contributed by CO2 enrichment. Neither varietal differences nor its interaction effects with CO2 were observed. Increasing CO2 from 400 to 1200 μmol mol-1 had significantly improved growth, net photosynthesis (A), water use efficiency, maximum efficiency of photosystem II, carbohydrate, total phenolics and total flavonoids. However, chlorophyll content, specific leaf area, and net assimilation rate were found to decrease by end of the experiment. From this experiment, it was found that the production of secondary metabolites was negatively correlated with chlorophyll content implying that nitrogen levels might be playing an important role in the production of secondary metabolites in L. pumila. In the third experiment, three varieties of L. pumila seedlings were exposed to four levels of nitrogen (0, 90, 180 ,270 kg N ha-1) under 1200 μmol mol-1 CO2 in a split plot design. Absolute control at 400 μmol mol-1 CO2 and 180 kg N ha-1 (Standard) were also included. Under CO2 enrichment, application of low nitrogen (0 and 90 kg N ha-1) managed to enhance the production of secondary metabolites significantly regardless of differences in varieties. As levels of nitrogen increased, plant growth, photosynthesis, photosynthesis nitrogen use efficiency (PNUE) and chlorophyll content were enhanced. However, carbohydrate, total phenolics and flavonoids were reduced with increase in nitrogen fertilization. Increase in the production of secondary metabolites was found to be positively correlated with increase in the carbon to nitrogen ratio (C/N) and PNUE. In the last experiment, two CO2 levels (400 and 1200 μmol mol-1)and three nitrogen levels (0, 90, 270 kg N ha-1) were used to establish the mechanism of enhancement of secondary metabolites under varied CO2 levels and nitrogen fertilization. It was found that the highest production of secondary metabolites was obtained at 1200 μmol mol-1 + 0 kg N ha-1 and lowest at 400 μmol mol-1 + 270 kg N ha-1. The interaction effect between CO2 and nitrogen was found to influence the production of secondary metabolites, fructose, PAL activity and protein in L. pumila. Generally, these findings support the protein competition model that suggest the increase in production of secondary metabolites under high CO2 and low nitrogen levels was due to increased availability of phenylalanine (precursor for phenolics and flavonoids) due low sink ratio which increases the availability for production of secondary metabolites. From the project, it can be concluded that CO2 enrichment onto L. pumila was able to enhance growth and medicinal properties especially at low nitrogen fertilization.

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