2.1. Flavonoids and Phenolics Content (HPLC Analysis)
High performance liquid chromatography (HPLC) analysis of flavonoids is present in
Table 1. According to the data obtained (
Table 1), the concentration of the majority flavonoids (quercetin, rutin, catechin, epicatechin and naringenin) was increased in plants when grown under 310 μmol m
−2s
−1. Accumulation of the studied flavonoid components from the sink (leaves) to the source (rhizomes) increased under low light intensity. The analysis of flavonoid components using HPLC in leaves of ginger showed that quercetin possessed the highest concentration, followed by cathechin. The analysis of quercetin concentration under two light intensities indicated a higher concentration of quercetin in ginger leaves grown under 310 μmol m
−2s
−1 compared with plants grown under 790 μmol m
−2s
−1. In addition, quercetin decreased the photosynthesis rate through inhibition of the ATPase activity and electron transport rate in photosynthesis photosystems [
21].
Rutin in Malaysian ginger ranged from 0.173 and 0.451 mg/g dry weight. The highest concentration of rutin was recorded in rhizome of Halia Bentong exposed to low light; however, when light intensity increased, accumulation of rutin in rhizome decreased by about 31%. Rutin concentration in Halia Bentong leaves was low, especially when exposed to high light intensity. A moderate concentration of rutin was observed in Halia Bara rhizome under a high light level (0.324 mg/g dry weight). Rutin accumulation in Halia Bara, although still favored in the rhizomes more than the leaves, declined in concentration in both plant parts with increasing light intensity from 310 to 790 μmol m−2s−1.
Catechin and epicatechin are polyphenolic antioxidant plant secondary metabolites. The term catechin is also commonly used to refer to the related family of flavonoids and the subgroup flavanols. Catechin concentration in Halia Bara under low light intensity was higher in the leaves than rhizomes, which was also comparable to that contained in the rhizomes of Halia Bentong under low light. Meanwhile, epicatechin concentration was highest in leaves of both varieties under low light intensity (0.117–0.118 mg/g dry weight) although the amount was not clearly different from those obtained in the rhizome of Halia Bara under low or high light conditions. Generally, the concentration of epicatechin in Halia Bentong was lower than that found in Halia Bara, especially in rhizomes under high light intensity (0.078 mg/g dry weight).
Kaempferol is a rare flavonoid in plants. However, in the leaves and rhizomes of Halia Bara and Halia Bentong, it was detected in small concentrations (between 0.042 and 0.068 mg/g dry weight).
Naringenin is a flavonoid that is considered to have a bioactive effect on human health as an antioxidant, free radical scavenger, anti-inflammatory, carbohydrate metabolism promoter, and immune system modulator. It is the predominant flavanone in grapefruit and was found to have an inhibitory effect on carcinogens [
22]. Although a lack of information has been gathered about naringenin in ginger, from the present study, its concentration was low, ranging from 0.02 to 0.094 mg/g dry weight. Naringenin concentration in ginger was clearly affected by the differences in varieties, light intensity, and plant parts. Generally, Halia Bentong had a higher concentration than Halia Bara, with more accumulation found in the leaves than in the rhizomes, especially under low light condition.
Irradiance increases leaf area-based phenolics content, which is mainly accumulated in the epidermis [
23,
24]. Shui
et al. [
25] found that ecological factors influenced flavonoid concentration primarily during the young stage of
Ginkgo biloba development; and amongst the ecological factors studied, light and temperature had the greatest effects on flavonoid synthesis in
Ginkgo biloba.
Salicylic acid, belonging to plant phenolics group, is found in some plant species, and its highest levels are observed in the inflorescence of thermogenic plants and in spice herbs [
26]. According to
Table 1, salicylic acid was not detected in gingers grown under a light intensity of 310 μmol m
−2s
−1. A high content of salicylic acid (0.673 mg/g dry weight) was detected from Halia Bara leaf extract grown under 710 μmol m
−2s
−1 light intensity. The results of previous studies showed that salicylic acid is capable of enhancing plant growth and yield. Jeyakumar
et al. [
27] reported that salicylic acid was able to enhance the dry matter production in blackgram. Induction of photosynthesis rate and stomatal conductance by salicylic acid was provided in previous studies [
28].
Nevertheless, although the leaves’ flavonoid content is highly sensitive to biotic and abiotic control of PAL expression [
29], the results of Waterman
et al. [
30] and Mole
et al. [
31] showed parallel variations of phenolics and flavonoids under different irradiance levels. Contrary to our results, higher phenolics content was reported in the rhizomes of
Z. officinale rather than its leaves [
32]. In addition, the results of Chan
et al. [
13], reporting a high level of flavonoid components in ginger leaves compared to the rhizomes of
Z. officinale, supported our finding. The synthesis of isoflavones and some other flavonoids is induced when plants are infected or injured [
33,
34], or under low light and low nutrient condition [
22,
34]. The increase in soluble phenolics such as intermediates in lignin biosynthesis can reflect the typical anatomical change induced by stressors: An increase in cell wall endurance and the creation of physical barriers prevent walls against harmful actions [
35]. However, some plant products such as anthocyanin, cumarin and lignin are biosynthesized while phenolic compounds are being transformed into flavonoids [
36]. From
Table 2, it can be observed that Halia Bentong had a higher average increase in flavonoid components (26.1%) compared to Halia Bentong (19.5%) when the light intensity was decreased.
From
Table 1, it is apparent that total flavonoids (TF) and total phenolics (TP) accumulation and partitioning in the plant were significantly affected by the differing light intensities (
p ≤ 0.01). With decreasing TP in the leaves and rhizomes observed when decreasing light intensity from 790 to 310 μmol m
−2s
−1, TF content increased significantly in the leaves and rhizome of both varieties.
In this study, cinnamic acid was not detected in plants grown under 310 μmol m−2s−1 where instead high content of flavonoids was registered; but cinnamic acid was detected in ginger grown under 790 μmol m−2s−1 with low content of flavonoids.
These results suggest the ability of different light intensities to alter or modify both the concentration and profiling of phenolic components in ginger plants; although accumulation of phenolics components favored high light intensity, in contrast, low light intensity generally promoted the accumulation of flavonoids. Considering the intricacy of flavonoid biosynthesis and flavonoid metabolism processes, it is difficult to figure out linear relationship between flavonoids and their basic precursors.
2.2. Photosynthesis Rate, Stomata Conductance and Transpiration
The leaf net photosynthetic rate, stomata conductance and transpiration rate increased with increasing light intensity (
Table 3). According to the results in
Table 3, a high photosynthesis rate (12.25 μmol CO
2 m
−2s
−1) was obtained in Halia Bara varieties when grown under 790 μmol m
−2s
−1. Under low light intensity (310 μmol m
−2s
−1), the stomata conductance was low compared to plants grown under high light intensity (790 μmol m
−2s
−1). Stomata behavior and regulation are very important factors in the control of photosynthetic rate. The current study also showed that increasing light intensity from 310 to 790 μmol m
−2s
−1 increased the net photosynthetic rate significantly as well as the stomata conductance and transpiration rate. This suggests that the increase in photosynthetic rates resulted from increased CO
2 uptake activity at the chloroplast level, rather than simple increases in stomata opening (reduced resistance to CO
2 entry in the leaves). Either situation could lead to an increase in photosynthetic rate, however, when an increase in stomata opening is the primary cause of increased photosynthetic activity, an increase in internal carbon would be expected. Ajithkumar
et al. [
37] point out that photosynthetic rate, stomata conductance, transpiration rate, stomatal index, and stomatal frequency decreased significantly with increasing shade level. The results of the current study showed that increasing flavonoids in ginger decreased the photosynthesis rate significantly and
vice versa. A possible explanation is that flavonoids and shikimic acid could be part of the regulatory system controlling the flux of carbon into secondary compounds in plants [
38]. Consequently, decreasing the photosynthesis rate resulted in increased shikimic acid activity and flavonoid contents. The effect of shikimic acid inhibition on photosynthesis enzymes is supported by Dixon plots study [
39]. Further, competitive inhibition pattern on PEP carboxylase activities was also reported previously [
39]. Although previous studies on the relationship between photosynthesis and flavonoids content have been carried out, most of the results remain contradictory. A number of studies have found that increased photosynthesis and carbohydrate content in leaves and dry weight of leaves were not accompanied by increased flavonoid content and its synthesis speed [
39]. Stomata conductance, photosynthesis rate and transpiration also increased with increasing phenolic compounds [
40]. The mechanism involved in increased photosynthetic rates and leaf area is not known.
When photosynthesis occurs with the presence of light, flavonoid components are able to change the rate of electron transport and photophosphorylation, bringing about the change of ATP/NADPH ratio [
21]. In carbon metabolism reactions, they can shift the dynamic equilibrium of pentosephosphate reduction cycle to enhance the synthesis of main metabolites due to both the change in energy substrate intake and the interaction with enzymes of the cycle. Additionally, flavonoids exercise a feedback control over their own biosynthesis, although this phenomenon is not clearly understood [
41]. However, inhibitory effect of flavonoids on photosynthesis rate was reported in previous studies [
21]. As opposed to the shade leaves, the sun leaves typically exhibit high photosynthetic capacity [
42], therefore, they have high carbon input, which can exceed the demand for protein synthesis and stimulate phenolic synthesis [
43].
Ginger is a semi-shade loving plant that does not require high light intensity for its photosynthesis [
44]. Furthermore, the difference in photosynthesis rate under different light intensities may be related to the absence or presence of salicylic acid in ginger. Hence, further study is required to establish the effect of salicylic acid on photosynthesis rate in ginger.
2.3. Chlorophyll Content
Chlorophyll concentration significantly increased with decreasing light intensity from 790 to 310 μmol m
−2s
−1. A high concentration of chlorophyll was obtained in Halia Bara 273.5 μg mL
−1 grown under 310 μmol m
−2s
−1. Such an increase in chlorophyll content when grown under shade conditions was reported by Khan
et al. [
45] and Souza
et al. [
46]. Chl a:b ratio decreased with increasing light intensity (
Table 3). With increasing chlorophyll a + b content, TF increased but TP decreased in both varieties. This negative relationship between TP and chlorophyll a + b content was not significant. Competition between TP and chlorophyll synthesis was reported in previous studies [
46,
47]. The described competition between chlorophyll and phenolics in leaves fits well with the predictions of the Protein Competition Model (PCM), that is, that the total leaf mass-based polyphenols content is controlled by the competition between protein and polyphenol biosynthetic pathways and its metabolic regulation. This indicates an accumulation of dry matter that dilutes chlorophyll and polyphenols [
47]. The increased TF in Halia Bentong with increasing shading could be associated with significantly higher leaf chlorophyll and carotenoids contents under lower light levels. Michel
et al. [
17] concluded that TF production is related to plant pigments (chlorophyll and carotenoids), and in contrast to flavonoids, the xanthophyll cycle seem to be mainly relevant to protect photosynthesis against sudden increases in light intensity. Increasing chlorophyll content is usually followed by an increase in flavonoid content in plant [
47].