Flavonoid Accumulation Behavior in Response to the Abiotic Stress: Can a Uniform Mechanism Be Illustrated for All Plants?

This review concentrates on two aspects of how total flavonoid content and individual flavonoid compounds change with the perception of environmental stress and the subse quent changes in those metabolites after post-harvest conditions are of the main points of the study. Hereby, along with this study, the flavonoid synthesis or their accumulation with their importance in plants and then in humans is briefly described. According to the literature cited herein, it seems that a universal mechanism concerned with flavonoid accumulation in response to the abiotic stress factors cannot be illustrated. Flavonoid accumulation exhibits different reactions to the different stressors. Flavonoid accumula tion behavior not only varies depending on the developmental stage, species and even cultivars of the same species but also post-harvest processes.


Introduction
Phenolic compounds are secondary metabolites derived from pentose phosphate, shikimate and phenylpropanoid pathways in plants [1], and a wide range of functions including participation in the regulation of growth and developmental processes and interactions with biotic and abiotic environmental stimuli have been attributed to the those phytochemicals [2]. Of environmental conditions and their stimuli. Once plants cannot tolerate or overcome the unfavorable environmental conditions, plant growth and development are likely adversely influenced and subsequently significant loss of crop yields [7,8]. Along with the stress conditions, plant behavior may change with respect to the secondary metabolite synthesis, production, secretion and storage when subjected to the abiotic stress factors [9]. Some secondary metabolite synthesis, enzyme activities and soluble substance accumulation were positively influenced by abiotic stress conditions. These are considered as consequences of plant adaptive strategies concerned with establishment of some changes allowing to the plant to sustain its life under ever-changing conditions. Many results concerned with total flavonoid and their individual compounds in response to the different stressors. For total flavonoid content, increases were determined [2, 10-14, 16, 17, 19, 20] whereas decreases were found [15,18,21] under different stress conditions. Based on the literature review, we cannot deduce and explain the flavonoid accumulation or their compound profile using one simple sentence. The stress effect is compound specific. A uniform mechanism for compound profile variation cannot be illustrated [22]. Furthermore, the flavonoid accumulation is likely dependent stress factors, frequency, duration and timing.  1.2. Is it adaptive strategy to sacrifice the primary metabolites through increases in secondary metabolite production against stress conditions or high efficiency use of secondary metabolite biosynthesis pathway?
As previously mentioned, pentose phosphate, shikimate and phenylpropanoid pathways are of the three pathways in plants, which are responsible for biosynthesis of phenolic compounds [1]. Shikimic acid is a key intermediate in the synthesis of both aromatic amino acids and phenylpropanoids, and oxidative pentose phosphate pathway is of the precursors for the biosynthesis of aromatic amino acids, lignin and flavonoids [23]. Regulation and expression of the genes on the pathways have been well elucidated, but the pathway compartmentation is not yet known [24]. Some of the synthesis-associated genes and enzymes involved in phenolics biosynthesis were characterized in Arabidopsis (Arabidopsis thaliana), maize (Zea mays) and petunia (Petunia hybrid). Also Fragaria spp. has been studied for their genes and enzymes ( [25][26][27][28][29][30]; cited by [24]). In order to determine which pathway is preferred for biosynthesis secondary metabolites under abiotic stress factors, the expression of protein or enzymes associated with synthesis of secondary metabolites in the pathways should be determined and then compared with the control group-not stressed group. Determination of the long or short distance metabolic pathways or high or low energy cost pathways in response to the stress is also great concern to understand plant behavior and signaling.
Stressors bring about quantitative and qualitative changes in plant metabolites. Of those, in general, biosynthesis of proteins in the plant leaves is suppressed, triggering the changes at gene expression levels and subsequently the synthesis of new proteins. For the lipid content and composition, the disturbances concerned with fatty acid composition, especially changes in fatty acid carbon chains. The variations in the lipid composition influence membrane lipids and transport functions of membranes. Furthermore, accumulation of the compatible solutes is of the responses against drought, high temperature or high salinity, maintaining the osmotic adjustment and turgor regulation [31].
Plant secondary metabolites have been considered or often referred to as metabolites which are not fundamentals for sustainability of basic plant life processes. However, the crucial and wide range roles of secondary metabolites have been understood. The accumulation of phenylpropanoids increased in response to the environmental stress including pathogen attack, UV-radiation, high light, nutrient deficiency etc. According to Bryant et al. [32] hypothesis, an exchange occurs between carbon and biomass production or formation of defensive secondary metabolites, proposing that secondary metabolites are involved in protective processes of plants in response to stressors. For example, phenyl amide formation and accumulation of anthocyanin and polyamines have been reported as a response to the environmental stresses [33,34].

Over accumulation of flavonoid versus reactive oxygen species? Non-enzymatic antioxidant system but any relations with the enzymatic antioxidant system (SOD, CAT, APX)?
Flavonoids are secondary metabolites synthesized by general phenylpropanoid pathway in plants [35]. They have been considered as a secondary (non-enzymatic) reactive oxygen spe-cies scavenging system in plants and humans [36]. Flavonoids exhibit direct scavenging of reactive oxygen species [36] one of the ways scavenging reactive oxygen species, flavonoids can easily donate hydrogen atom. Thus, while reactive oxygen species are inactivated by flavonoids, flavonoids return to phenoxyl radical [37]. Flavonoids phenoxyl radical can react with other free radicals and then acquiring a stable quinone structure [38]. The other way of scavenging reactive oxygen species, flavonoids return to phenoxyl radical by donating hydrogen atom at the first step. At the second step, phenoxyl radical scavenge other high reactive radical (R • ) by radical-radical termination. Flavonoid phenoxyl radical is highly stabile radical due to presence of a resonance structure redistributed the unpaired electron on the aromatic core [39] (Figure 3).

The possible protective defense roles of flavonoids in response to UV light have been documented in many studies but what happens if the flavonoid and other pigments cannot completely block the sunlight transmission?
UV light from sunlight is primarily required to perform photosynthesis as basic function and developmental process such as de-etiolation, phototropism and flowering of the plants [40,41]. But interestingly, the UV light causes damage to DNA, protein and cell membranes of the plants, because, as sessile organisms, plants are more exposed to the UV-light. Subsequently, normal growth and development of plants are retarded [41]. Short-wavelength UV light is grouped into three categories. Of those, UV-A (315-400 nm) directly reaches the earth's surface, and UV-B (280-315 nm) and UV-C (100-280 nm) are blocked by the ozone layer. However, a small quantity of UV-B reaches the earth's surface because of ozone layer depletion and subsequently causes DNA damages [42].  UV-B has highest energy of UV light that reaches the earth surface [43]. Although the high level of UV-B causes damage to biomolecules, low level of UV-B regulates morphology, development, phycology and biochemical compositions [44]. While long wavelengths UV lightinduced regulation is provided with photoreceptors including phototropins, neochromes, phytochromes, rhodopsins and cryptochromes [45] (Figure 5), UV-B-induced regulation is provided with UV RESISTANCE LOCUS 8 (UVR8) receptor protein [46]. UVR8 directly absorbs UV-B radiation and induces the transcription of flavonoids biosynthesis genes by orchestrating UV protective gene expression responses [47]. Upregulation of CHS genes transcription in response to the several stressors was reported to induce flavonoid biosynthesis [49]. UVR8 protein interacts with the WD40-repeat domain of COP1 after perception of UV-B light that is one of the stressors [50]. Consequently, UVR8-COP1 complex leads to activation of HY5 gene expression [47]. HY5 proteins as a transcription factor play an enhancing role for UV-B induced-CHS gene expression during seedling development by binding to a conserved G-box sequence [51,52]. Thus, flavonoids that accumulate in upper epidermis layer specially absorb a large amount of 280-340 nm wavelengths [53]. Thus, the flavonoids accumulated in upper epidermis layer protect the internal tissues of leaves and stems against UV-B. Since the synthesis of kaempferol is deficient in chalcone flavone isomerase mutant tt4 A. thaliana, the plant exhibits high sensitivity to UV light [54]. Along with the absorption of UV light with chromophore group of flavonoids, flavonoids may undergo a transformation. While flavonoids with aromatic chromophore absorb light in the 250 nm region of UV spectra, flavonoids contain carbonyl that are conjugated with the aromatic ring chromophore absorb light in the 350 nm region of UV spectra. The transformation of flavonoids after of UV light absorption in vitro conditions is illustrated in Figure 4 [55].
It is worthy to note that the flavonoids are not unique functional UV-blocker absorbing all UV-B irradiation but the other protective roles of flavonoids cannot be ignored in spite of deficient in absorption of all UV-B irradiation since UV-B induced increase in the quantity of flavonoids has been reported, suggesting that flavonoids may exhibit functions including signal molecules, antioxidant molecules, defensive compounds, allelochemicals [56] after UV-B exposure in plant.
When the UV light cannot be completely blocked via defense system apparatus of the plants, UV light reaches to DNA, resulting in formation of cyclobutene pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) on DNA [57,58]. There are two mechanisms repairing the photoproduct that can inhibit transcription and replication and induce mutations [48]: first one is photolyases enzyme, and the other one is nucleotide excision repair mechanism [60]. CPD Photolyase gene expression is regulated by a wide spectrum of light, including far-red, red and blue light [61]. But recent studies reported that photolyase gene expression was regulated by UVR8 receptor protein and the regulation mechanism remains poorly understood [62]. In order to exhibit DNA repair activity, photolyase enzyme needs UV-A light [63] but the light is not required for the activity of dark repair called nucleotide excision repair mechanism. Recently, photolyase and nucleotide excision repair mechanism in A. thaliana were well described [60].

The fate of the flavonoid-enriched crop plants through the food chain: terminal
Any direct and indirect biotic or abiotic stressors or their combinations at certain time or simultaneously influence the phytochemistry and subsequently the changes orchestrate the plant protection and plants' biological activities. Herewith, phytochemistry of a plant can be regarded as protective roles for plants against stressors and health-promoting properties for humans. The quality of the crop plants is a combination attributed to their composition and   contents that shape the commodity value for human consumption. Since humans are, in general, considered to be at the top of the food chain, the terminal of the flavonoid-enriched/poor crop plants would be the human, resulting the health standards.
Nothing stays the same as its former form and the changes are inevitable for all living and non-living things. Therefore, numerous studies-as listed in Table 1 but not limited in this chapter-have been performed in order to keep the stability or dynamic changes of flavonoid content and its compound. Of those studies, atmosphere conditions are of great interest for long-term storage and subsequently essential for keeping biological value of the crop. In the study reported on Allium cepa var. calonicum Backer) by [64] (see the detail in Table 1), the highest content after storage at conditions with gas composition of 5% CO 2 + 5% O 2 was achieved. Two major compound-quercetin 3,4′-di-O-glucoside and quercetin 4′-O-glucoside (spiraeoside)-exhibited an increase [64]. Effect of carbon dioxide-enriched atmosphere on total

Total flavonoid content Plant species Researchers
Different storage temperatures (0, 2, 4, and 6 + 2°C) + 5%, 10%, 20% or 0.03% CO 2 Total flavonoid content Phoenix dactylifera L. [54] Storage conditions were at 50, 25, 4, and −20°C Total flavonoid content Anemopsis californica [59] Freeze and thermal drying Total flavonoid content Oxycoccus palustris Pers. [60] Stability testing at different temperatures flavonoid content changes in Phoenix dactylifera L. fruit in response cold storage was tested and the fruits stored under low temperature conditions (0°C) or relatively high CO 2 concentration (20% CO 2 ) was reported not to exhibit any chilling or CO 2 injury symptoms. Modified conditions have been reported to extend not only the date storability and then fruit quality but also magnify the maintenance of fruit quality in response to the cold temperature storage [65].
Furthermore, the influence of different plant parts, developmental storage and storage durations (1, 2, 3 and 4 days) [66], different temperature and sampling days [67], different storage days [68], Light (photosynthetically active radiation (PAR) level and maturity [69] has been examined to indicate that the there is no constant stability or dynamics of flavonoid content and its compounds in quantity and quality.

Conclusion
As a conclusion, since plants are open systems and do not exist in a vacuum, they are continuously interacted with their biotic and non-biotic surroundings. Based on the literatures cited in the present chapter, a universal mechanism with respect to the accumulation behavior of flavonoid cannot be illustrated even the flavonoids commonly exhibit a tendency toward increase in response to the unfavorable conditions. Up to our best research, flavonoid accumulation behavior varies depending on the developmental stage, species and even cultivars of the same species. It also exhibits different reaction to the different stressors.
Beyond physiological aspect for the plants for their survival mechanism, plants are also sources for other living organisms. The quality and then biological efficacy of the flavonoid containing crops are great issue for human beings. According to the literature cited herein, the fate of the flavonoids containing herbal products including bulbs, leaves, fruits etc is influenced by the storage temperatures, storage time, modified storage conditions, cultivars, different parts and subunit parts of the plant, light and maturity.