Improve Anthocyanin and Zinc Concentration in Purple Rice by Nitrogen and Zinc Fertilizer Application

Suchila Utasee, Sansanee Jamjod, Sittisavet Lordkaew, Chanakan Prom-U-Thai,

Review

Improve Anthocyanin and Zinc Concentration in Purple Rice by Nitrogen and Zinc Fertilizer Application

Suchila Utasee1, Sansanee Jamjod1, Sittisavet Lordkaew2, Chanakan Prom-U-Thai1,3

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Zinc (Zn) is an essential micronutrient for plant growth and development, and anthocyanin is a secondary metabolite compound generally produced under stress conditions; both have benefits to human health. Rice is a staple food crop for most of the world’s population, and purple rice is well known as a natural source of Zn and anthocyanins, but their stability depends upon many factors. This review focuses on the opportunity to increase Zn and anthocyanin compounds in purple rice grains via Zn and nitrogen (N) management during cultivation. Variation in grain Zn concentration and anthocyanin compounds is found among purple rice varieties, thus presenting a challenge for breeding programs aiming at high grain Zn and anthocyanin contents. Genetic engineering has successfully achieved a high-efficiency vector system comprising two regulatory genes and six structural anthocyanin-related genes driven by endosperm-specific promoters to engineer purple endosperm rice that can provide new high-anthocyanin varieties. Grain Zn and anthocyanin concentrations in rice can also be affected by environmental factors during cultivation, e.g., light, temperature, soil salinity and nutrient (fertilizer) management. Applying N and Zn fertilizer is found to influence the physiological mechanisms of Zn absorption, uptake, transport and remobilization to promote grain Zn accumulation in rice, while N application can improve anthocyanin synthesis by promoting its biosynthesis pathway via the use of phenylalanine as a precursor. In summary, there is an opportunity to improve both grain Zn and anthocyanin in purple rice by appropriate management of Zn and N fertilizers during cultivation for specific varieties.

purple rice; anthocyanin; secondary metabolite; zinc deficiency; fertilizer

Zinc (Zn) is a micronutrient that plays an important role in plant growth and reproduction (Natasha et al, 2022). It is a major component and activator of several enzymes involved in the metabolic activities of plant growth, and as such is involved in improving crop yield and quality (Khampuang et al, 2021). Zn deficiency adversely reduces yield and deteriorates crop quality (Fongfon et al, 2021a; Bana et al, 2022). Regarding human health, a high grain Zn concentration in rice is a benefit for consumers for combatting Zn deficiency and preventing associated health problems such as cancer (Li et al, 2021), type I and type II diabetes (Chen et al, 2000), hair and memory loss, skin problems, weakening of body muscles, infertility in men and pneumonia in children (Li et al, 2021). The prevalence of dietary Zn deficiency in humans is often associated with Zn deficient soils and crops, especially in areas where Zn deficiency is common (Roohani et al, 2013). Anthocyanins are secondary metabolites that function in the protective mechanisms for plants under stress conditions (Enaru et al, 2021; Yamuangmorn and Prom-U-Thai, 2021). Anthocyanin in rice is also reported to possess many functional properties,relevant to human health, including antioxidant activity, protection of endothelial cells, prevention of heart and cardiovascular disease, anti-cancer activity, control of diabetes and ameliorating eyesight (Xia et al, 2021; Feng et al, 2022). Improving grain Zn and anthocyanin contents in rice crops is a promising avenue for decreasing the risk of several serious diseases among rice consumers; this would be especially effective in purple rice, in which anthocyanin is dominant. The agronomical management by soil and foliar application of Zn and N successfully increases grain yield and Zn accumulation and protectsagainst the chemical degradation of anthocyanins in purple rice crops (Farooq et al, 2018; Yamuangmorn et al, 2020; Fongfon et al, 2021a).

Purple rice with pigmented grains is referred to as black rice in the area where very dark pigment is common, and the color is formed by deposits of several compounds located in the pericarp layer, seed coat or aleurone layer. Purple rice has long been a unique and traditional food and medicinal plant in many cultures (Rerkasem et al, 2015), as a natural source of Zn and anthocyanin (Fongfon et al, 2021a; Jaksomsak et al, 2021; Yamuangmorn and Prom-U-Thai, 2021). Purple rice contains secondary compounds such as anthocyanins, flavones, tannins, phenolic acids, sterols, tocols, γ-oryzanols, amino acids and essential oils, among which anthocyanins account for more than 95% (Min et al, 2009; Bhat et al, 2020). China is recognized as the richest in purple rice resources (62%), followed by Sri Lanka (8.6%), Indonesia (7.2%), India (5.1%), the Philippines (4.3%), Bangladesh (4.1%), and lesser numbers in Malaysia, Thailand and Myanmar (Xia et al, 2021). The market for purple rice on both the domestic and international fronts is expanding due to the increasing demand among health-conscious consumers (Yammuangmorn and Prom-U-Thai, 2021). In Thailand, the major exports of purple rice are to China, Singapore, USA, Europe and Australia. The export volume of Thai purple rice has increased, particularly among the Association of Southeast Asian Nation members in relation to the per capita income due to greater health consciousness (Yammuangmorn and Prom-U-Thai, 2021). Currently, the standard classification of purple rice in global markets is similar to that for non-colored rice, mostly involving grain appearance as the main criterion, e.g., size and uniformity of color (Yammuangmorn and Prom-U-Thai, 2021). Thus, the functional properties will be applied to distinguish the quality of rice grains, including grain nutrients (e.g., Zn and Fe) and bioactive compounds (anthocyanins) that will determine the market value. Therefore, increasing grain Zn and anthocyanin concentration in purple rice not only benefits to consumer health but also promotes the price for farmers.

This review focuses on several topics, including Zn deficiency in plant and human, grain Zn concentration in purple rice, anthocyanin in plants and its benefit for human health, grain anthocyanin concentration in purple rice, the environmental factors affecting anthocyanin compounds in purple rice, and the opportunity to improve grain Zn concentration and anthocyanin in purple rice by Zn and N fertilizer application. This review will be useful for efforts to remediate the low nutrient levels in purple rice by a common strategy of fertilizer management in order to improve both productivity and grain quality for the benefit of farmers and consumers.

Zn deficiency in plants and humans

In plants, Zn is well known as an essential micro- nutrient involved in many physiological functions, including protein and carbohydrate synthesis (Yadavi et al, 2014). Zn is an indispensable micronutrient for crop growth, an important component of carbonic anhydrase, and a stimulator of aldolase that is involved in carbon metabolism (Broadley et al, 2007). It is also an integral component of biomolecules, such as lipids and proteins, and a co-factor of auxins, and therefore,it plays an important role in plant nucleic acid metabolism (Mengel et al, 2001). In addition, mineral supply to a developing cereal grain takes place either by direct uptake from the soil or by remobilization of stored minerals in leaves. For estimating grain quality, nutrient density per unit of grain dry weight is important (Marles, 2017). Therefore, the application of Zn has been demonstrated to be beneficial in improving crop yield and quality (Chattha et al, 2017; Hossain et al, 2019;Das et al, 2020). However, Zn deficiency reduces yield, and deteriorates crop quality, resulting in poor accumulation of Zn in rice grains and poor human nutrition (Mousavi et al, 2007; de Valen?a et al, 2017; Liu et al, 2017). Zn deficiency can have severe biochemical and physiological consequences for plants, resulting in chlorosis and stunted growth as well as reduced yield and lowered grain micronutrient content when leaf Zn concentrations fall below 15 mg/kg, but this depends on the inherent sensitivity of the species and on the variety of growing conditions (Stanton et al, 2021). In general, there are three steps that limit the critical rate or barrier for effective Zn deposition in grains: the root barrier, the root-to-shoot barrier and the Zn loading barrier into grains (Swamy et al, 2016). Thus, Zn application not only improves productivity in crops but also increases grain quality in many ways, especially the involvement of Zn in plant nucleic acid metabolism and reactive oxygen species as related to anthocyanin synthesis in plants. Therefore, Zn application should be examined among the purple rice varieties.

In humans, Zn deficiency is a common occurrence among the world’s population together with deficiencies of iron (Fe) and vitamin A, particularly in regions where rice is consumed as the main staple food, as it contains a very low concentration of Zn compared with other cereal grains (Prom-U-Thai et al, 2020). Rice is one of the major food crops that has been raised in flagship programs such as HarvestPlus to achieve the micronutrient-enriched food for the target population (Bouis and Saltzman, 2017). It is estimated that one-third of the world population (around two billion people) suffers from mild Zn deficiency, and over 450000 children die each year due to such a deficiency (Harding et al, 2018). Zn is an essential micronutrient for all living organisms, as it performs both catalytic and structural functions in a wide variety of proteins; approximately one-tenth of the proteome (about 3000 human proteins) binds Zn (Andreini et al, 2006; Maret, 2012). Zn possesses therapeutic benefits for several chronic diseases in humans such as atherosclerosis, several malignancies, autoimmune diseases, Alzheimer’s disease and other neurodegenerative disorders, cancer, diabetes, depression, aging, Wilson’s disease and COVID-19-related liver injury (Gammoh and Rink, 2017; Choi et al, 2018; Coni et al, 2021). Thus, it is important for Zn deficiency to be fully corrected. Supplementation of Zn is potentially beneficial for managing the nutritional status as well as providing management of these diseases, and Zn may be used as an adjunct therapy (Tapiero and Tew, 2003; Chasapis et al, 2020). The individual Zn status of viruses is associated with respiratory tract infections, and most of the risk groups described for COVID-19 are associated with Zn deficiency (Wessels et al, 2020). This details how Zn is essential to preserve natural tissue barriers such as the respiratory epithelium, preventing pathogen entry and ensuring a balanced function of the immune system and the redox system. Deficiency can be added to the factors predisposing individuals to infection and detrimental progression of COVID-19, meaning that Zn is beneficial for most of the population, especially those with suboptimal Zn status (Wessels et al, 2020). Therefore, improving the concentration of Zn in rice grains would assist to increase Zn intake among consumers. Biofortification by fertilizer management for high grain Zn rice has been successfully developed, most effectively through the soil and foliar Zn application (Boonchuay et al, 2013; Prom-U-Thai et al, 2020). However, applying Zn fertilizer to boost grain Zn accumulation has been rarely investigated in purple rice varieties in relation to grain anthocyanin concentration. The double benefits of rice grain would greatly assist to promote consumer health.

Grain Zn concentration in purple rice

Breeding rice varieties with high grain Zn concentration has been suggested to be a sustainable, targeted, food-based and cost-effective approach to alleviate Zn deficiency among rice consumers (Swamy et al, 2016). To do this, a wide variety of grain Zn concentrations is required as a source of genetic material. However, there is limited information available on grain Zn concentration in the purple rice varieties compared with the non-colored rice that has been well documented (Table 1). The ranges of 13.5–58.4 mg/kg grain Zn concentration were reported for a large rice germplasm of 939 varieties from International Rice Research Institute (Welch and Graham, 1999). In Thailand, a similar range of 17.0–59.0 mg/kg was also reported among 127 varieties from several studies (Saenchai et al, 2012; Jaksomsak et al, 2015; Jamjod et al, 2017). Additionally, variation in grain Zn concentration was also observed among 40 and 159 rice varieties from Lao People’s Democratic Republic (Lao PDR) (Xiongsiyee et al, 2018) and India (Maganti et al, 2020), ranging from 15.2 to 24.1 mg/kg and from 14.5 to 35.3 mg/kg, respectively. Among those reports, some mentioned grain Zn concentration in purple rice varieties. Xiongsiyee et al (2018) documented that grain Zn concentration variesfrom 15.5 to 19.5 mg/kg among five purple and four red rice varieties from Lao PDR, while the variation ranges from 19.0 to 41.3 mg/kg among 17 purple rice varieties from Thailand (Fongfon et al, 2021b; Jaksomsak et al, 2021). These reports indicate a narrower range of grain Zn in the purple rice compared with the non-colored rice, but with a limited number of varieties reported from different germplasms. Thus, a breeding program will be a promising strategy to achieve high grain Zn in purple rice, as it has successfully developed among non-colored rice varieties.

Additionally, agronomical management practices such as fertilizer and water manipulation are suggested as one of the potential methods for promoting grain Zn concentration in rice crops, but limited information is available on how such factors affect grain Zn concentration in the purple rice varieties. A few studies have been initiated and have reported that environmental factors during cultivation, e.g., growing elevation (Rerkasem et al, 2015), water condition (Jaksomsak et al, 2021), and fertilizer management (Prom-U-Thai et al, 2020; Fongfon et al, 2021a) affect grain Zn concentration to different extent among the purple rice varieties, but without detailed explanations of the mechanisms. This will present an opportunity to investigate the mechanisms of specific purple rice varieties in response to agronomical factors on grain Zn concentration. Applying Zn fertilizer has been carried out through the application to seeds, soil and leaves via foliar preparations (Farooq et al, 2018). Zn seed preparation improves germination, basal setting and growth; however, seed treatment with high Zn concentration (2 g/kg) significantly inhibits seed germination and growth (Rehman and Farooq, 2016). In maize and wheat, soil application of Zn (12–15 kg/hm2) effectively increases grain yield, biomass and grain Zn concentration (Khan et al, 2007). Primary seed addition significantly increase grain Zn concentration and Zn utilization efficiency by 82.9% and 49.0%, respectively (Li et al, 2014). Foliar Zn application has been suggested as an appropriate tool due to the direct application of Zn to the leaves without affecting soil chemical properties, and foliar Zn (140 g/L) increases the starch content, yield and Zn content in maize (Leach and Hameleers, 2001). Therefore, biofortification via breeding programs and agronomical management would be a promising way to achieve high grain Zn in purple rice varieties.

Table 1.Range of zinc (Zn) concentration in non-colored rice grains from different germplasms.

IRRI, International Rice Research Institute; Lao PDR, Lao People’s Democratic Republic.

Unpolished rice (brown rice) derived from husking the seed coat (palea and lemma) of the unhusked grains.

Anthocyanin in plants and its benefits to human health

Anthocyanin compounds are a group of reddish to purple flavonoids that are found inside the vacuoles of cells in several different tissues in plants (Stintzing and Carle, 2004). Anthocyanin compounds are plant secondary metabolites synthesized by the flavonoid biosynthesis pathway using phenylalanine as the precursor (Tanaka et al, 2008; Zhao et al, 2021) (Fig. 1). In the first three steps, phenylalanine is converted to 4-coumaroyl-CoA via the activities of phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumaroyl:coenzyme A-ligase (4CL). Anthocyanins are then synthesized by the activities of chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS). Several researchershave proposed different roles of anthocyanins in plants. In general, anthocyanin accumulation has been found to be upregulated in the presence of biotic and abiotic stressors such as too high/low temperature, strong light intensity, UV-B radiation, drought, nutrient deficiencies, and bacterial or fungal infections (Steyn et al, 2002; Truong et al, 2018). Therefore, anthocyanins have been regarded as both a symptom and a coping mechanism of plants under stressful conditions, although some studies have also reported their involvement in the photosynthetic process (Lo Piccolo et al, 2020). In purple rice, anthocyanins are found in form of cyanidin-3-glucoside and peonidin-3-glucoside (Hu et al, 2003), malvidin, pelargonidin-3,5-diglucoside, cyanidin-3-glucoside, cyanidin-3,5-diglucoside (Zhang et al, 2006), cyanidin-3-glucoside and pelargonidin-3-glucoside (Yawadio et al, 2007). The concentrations of anthocyanins in purple rice vary significantly, while much higher concentrations of anthocyanins were detected as cyanidin 3--glucoside and peonidin 3--glucoside (Xia et al, 2021; Wisetkomolmat et al, 2022). The accumulation of anthocyanin in different plants partly depends on genetic and environmental factors (temperature, UV light, pH, oxygen levels, hormones, osmotic stress, and nutrient status), and the interaction effects between these factors (Akhter et al, 2019; Lo Piccolo et al, 2020; Ling et al, 2021). Additionally, the harvesting process and postharvest conditions have also been reported to impact the anthocyanin concentration in purple rice grains (Rao et al, 2018). Thus, the appropriate growing conditions, environmental management during crop cultivation and postharvest management for the specific rice variety are required for maintaining high anthocyanin content in purple rice.

Fig. 1. Flavonoid biosynthesis in plant.

PAL, Phenylalanine ammonia lyase; C4H, Cinnamic acid 4-hydroxylase; 4CL, 4-Coumaric acid:coenzyme A ligase; CHS, Chalcone synthase; CHI, Chalcone isomerase; FNS, Flavone synthase; F3H, Flavanone 3- hydroxylase; FLS, Flavonol synthesis; DFR, Dihydroflavonol 4-reductase; ANS, Anthocyanin synthase; UFGT, UDP-glucose:flavonoid-3-- glucosyltransferase.

Anthocyanins in purple rice have a long history of being beneficial for human health. Traditional medicine in China uses purple rice to prevent several diseases including anemia and to improve blood circulation, kidney function and eyesight (Deng et al, 2013; Feng et al, 2022). Purple rice porridge is given to aid the recovery of invalids; one Chinese variety is known as ‘healing of broken bones’ (Rerkasem et al, 2015). Additionally, one of the purple rice varieties in China has an exceptionally rich history and is known as ‘Imperial rice’, which was restricted solely for the emperor’s consumption, while another variety known as ‘forbidden rice’ has been consumed in Asia for thousands of years and reserved solely for Chinese royalty (Oikawa et al, 2015; Kushwaha, 2016). In Thailand, claims of medicinal properties of purple rice known as ‘Khao Kam’ or ‘Khao Niew Dam’ include stopping bleeding after childbirth, reducing fever, curing skin diseases and halting diarrhea (Waewkum and Singthong, 2021). Several studies have confirmed the anti-clastogenic and anti-carcinogenic properties of Thai purple rice varieties (Insuan et al, 2017; Nilnumkhum et al, 2017; Chariyakornkul et al, 2021). The purple rice varieties are considered as efficacious medicines to cure various diseases as well as being sources of staple foods with a pleasant taste, fragrance and fluffiness, and their beneficial properties for health have been identified (Kushwaha, 2016; Krishnan et al, 2021). Thus, high levels of anthocyanins in purple rice have many health benefits for consumers.

Grain anthocyanin concentration in purple rice

Purple or black rice is not the only pigmented rice with a pericarp color that originated among the rice-growing countries; red pericarp is also well established in Asia’s traditional pharmacopeia (Fig. 2-A) (Ahuja et al, 2007; Oikawa et al, 2015; Feng et al, 2022). Variation in the bioactive compounds occurs between pericarp colors, in which higher concentrations of phenolic acids and flavonoids are detected in rice with a purple pericarp than in red or non-colored pericarp (Vichapong et al, 2010; Zhang et al, 2010; Pengkumsri et al, 2015).About 85% of the anthocyanin in whole-grain purple rice is located in the aleurone and pericarp layers (Bhat et al, 2020; Yamuangmorn and Prom-U- Thai, 2021). The bran fraction comprises of the pericarp and aleurone layers removed by the polishing process from unpolished (brown rice) to polished rice grains. Thus, this fraction has the potential for concentrated anthocyanin use. The variation of anthocyanin distribution among plant parts affects the intensity of pigmentation in the grain and the other plant parts, and consequently impacts the concentration of anthocyanin (Rerkasem et al, 2015;Jamjod et al, 2017).The potential for anthocyanin synthesis and accumulation could be one of the key reasons for the variation of anthocyanin compounds among the purple rice varieties (Sani et al, 2018). A wide variation of anthocyanin concentration has been reported among rice varieties in different germplasms (Table 2). Total anthocyanin concentration in the bran fraction among 25 purple rice varieties from the USA ranges from 0.7 to 17.3 mg/g (Chen et al, 2017), while the concentration ranges from 0 to 6.5 mg/g in 11 local Thai purple rice varieties (Wisetkomolmat et al, 2022). The concentration in the bran fraction may have to be carefully compared between the varieties and germplasms, as it can vary according to the degree of milling, grain morphological characteristics and even the milling machine used (Prom-U-Thai et al, 2007; Saenchai et al, 2012). In the unpolished rice (brown rice), Yodmanee et al (2011) evaluated the functional compounds in nine red and three black rice varieties from Thailand, China and Sri Lanka, and reported that the total phenolic content differs significantly between the varieties but not between the colors, and the highest anthocyanin concentration is observed in the Chinese black rice (1.4 mg/g). Additionally, anthocyanin content ranges from 0 to 6.5 mg/g among 15 varieties from China (Chen et al, 2012; Xia et al, 2021) and from0.025 to 4.000 mg/g among 25 varieties from Thailand (Yodmanee et al, 2011; Somsana et al, 2013; Jaksomsak et al, 2021). In Lao PDR, nine purple varieties have anthocyanin concentrations ranging from 0 to 2.200 mg/g(Xiongsiyee et al, 2018); a range of 0.112–1.032 mg/g has been foundamong 18 purple varieties from Indonesia (Fitri et al, 2021), and 0–6.456 mg/g has been reportedamong six varieties from Korea (Islam et al, 2022). Thus, the selection of rice varieties with high amounts of anthocyanin compounds can be used as a source of genetic material to facilitate breeding programs for high anthocyanin purple rice varieties.

Table 2.Anthocyanin (cyanidin-3-O-glucoside) concentration in bran fraction and unpolished rice among purple rice varieties from different germplasms.

aBran fraction derived from pericarp and aleurone layers removed by the polishing process from unpolished (brown rice) to polished rice grains.bUnpolished rice (brown rice) derived from husking the seed coat (palea and lemma) of the unhusked grains.

In general, purple rice is well recognized in the form of glutinous endosperm type and it is commonly used in traditional food as a dessert(Appa Rao et al, 2006). The non-glutinous type has been bred, and this type gains more attention due to consumer preference and health issues (Yamuangmorn et al, 2018),and no significant difference in bioactive compounds (i.e., anthocyanin compounds) has been found between the two groups of the endosperm types, but the degree of anthocyanin content and antioxidant capacity losses depend on the cooking method applied and the rice variety. Thus, the appropriate cooking method is necessary to maintain the contents of bioactive compounds in purple rice. In both the endosperm types, the purple color may appear only in the grain along with the normally green leaves and the other shoot parts, or it may appear in both the grain and leaves, and the purple color may vary in shading (Fig. 2-B) (Yamuangmorn et al, 2018; Xia et al, 2021). Besides adjusting the process to reduce the loss of anthocyanins, genetic modification has been carried out to enhance the compounds in purple rice.

Fig. 2. Characteristics of rice plants and grains of color rice and non-pericarp color rice.

A, Grains of color rice and non-pericarp color rice. From left to right are purple color (Kum Doi Saket), red color (Hom Mali Deang), unpolished and polished rice (Khao Dowk Mali 105).B, Plants of color rice (left) and non-pericarp color rice (right).C, Grains of two purple rice varieties.

Study on purple rice has been performed in China, and there are currently more than 50 genetically improved varieties grown in China due to increases in consumption and utilization for health benefit purposes (Ito and Lacerda, 2019). The modification from white to purple rice grains occurs through both spontaneous and chemically induced mutation, where the pigmentation of grains is controlled by three different types of genes (,and). Thegene controls the purple pericarp, the purple color being dominant over the white color; thegene together with the absence of thegene produces a brown pericarp, while thegene does not produce any pigment by itself; and the red pericarp is produced when bothandgenes are crossed (Jeng et al, 2012). A previous study used a transgenic approach to reveal the function of anthocyanidin synthase (ANS), one of the four dioxygenases (DOX) of the flavonoid biosynthetic pathway in the synthesis of anthocyanidins from leucoanthocyanidins. ANS may act directly on different flavonoid substrates of DOX reactions inducing the expression ofafter treatment with methyl jasmonate (MeJA) and 2,6-dichloro-isonicotinic acid (DCINA), resulting in the accumulation of anthocyanin in rice (Reddy et al, 2017). The chemical treatment of transgenic plants increases the activities ofand, leading to high accumulation of anthocyanins in rice (Kawahigashi et al, 2007). Additionally, the Myc-type bHLH genefrom the purple rice variety Khum has been cloned and transformed into white rice Nipponbare and Taichung 65 using(Sakulsingharoj et al, 2014). The transgenic rice was found to up-regulate the expression levels of structural genes, both EBG (F3H) and LBG (DFR4, ANS) that were not regulated separately (van Long et al, 2011). Most recently, a high-efficiency vector system containing two regulatory genes and six structural anthocyanin-related genes driven by endosperm-specific promoters to engineer purple endosperm rice has been developed; this would provide a new rice germplasm resource and validate the successful use of a transgene stacking system for the biosynthesis pathways of functional compounds such as anthocyanin (Zhu et al, 2017). However, the acceptability of transgenic purple rice among consumers is unknown, especially when dealing with a worldwide health-conscious population. However, while the breeding and transgenic rice programs for high grain anthocyanin in purple rice are ongoing, other strategies such as biofortification are also required.

Environmental factors affecting anthocyanin compounds in purple rice

Many studies have examined the environmental factors influencing anthocyanin compound accumulation in purple rice. Somsana et al (2013) evaluated the total genetic variation of eight upland rice varieties based on the stability of anthocyanins and found high variation among varieties due to the environment, contributing 43% of the total variation. Anthocyanin synthesis can be induced by environmental conditions that are critical in rice production and may also affect the yield of purple rice (van Long et al, 2011; Anami et al, 2020).

Light

Light intensity and duration are major environmental factors affecting the synthesis of primary and secondary compounds in plant pigments, e.g., chlorophyll and anthocyanin (Chen et al, 2016). Moreover, light is a significant stimulus for plant growth, and light intensity is positively correlated with the level of phenolic compounds such as anthocyanin (Jiang et al, 2016). However, in rice, no such experiment has been conducted to observe the effect of light quality on the synthesis of these compounds. For example, an experiment concerning the effect of ample sunlight on the fruit peel of blood oranges and purple pomelo conducted in an orchard demonstrated that the amount of anthocyanin in the orange fruit peel is reduced by 20-fold in bagged oranges compared to oranges under natural light, and the analysis showed that the promoter of, a key activator of anthocyanin biosynthesis, is light-inducible (Huang et al, 2019). Another study highlighted the effect of irradiance and light intensity on anthocyanin production inand found that light exposure positively regulates the accumulation of anthocyanins, whereas shade or a dark environment represses anthocyanin accumulation. A higher accumulation of anthocyanins can be observed with moderate light intensity (301–600 lx), where the cultures exposed to 10 d of continuous darkness show the lowest pigment content, while the cultures exposed to 10 d of continuous irradiance show the highest pigment content (Chan et al, 2010). Similar synergistic effects of low temperature and light have been demonstrated in grapes, where anthocyanin-related genes are upregulated independently under both conditions (Azuma, 2018).

Temperature

Temperature is another environmental factor influencing grain anthocyanin synthesis in plants. Anthocyanin biosynthesis is affected by temperature, and a suitable temperature increases anthocyanin synthesis while extremely high or low temperatures reduce the accumulation of anthocyanin and induce degradation of pigments (Shaked-Sachray et al, 2002; Niu et al, 2017). Inhibition of the expression of anthocyanin activator genes or enhancement of the expression of the repressor genes has been observed when plants are exposed to high temperatures, leading to decreased anthocyanin accumulation (Rowan et al, 2009; Wang et al, 2010). In the rice caryopsis, high temperature (≥ 35 oC), especially during grain filling, has an inhibitory effect on anthocyanin accumulation, whereas low temperatures of 22 oC to 27 oC enhance anthocyanin biosynthesis. High temperature induces alterations in the expression of CHS, F3H, DFR and ANS genes in rice and hinders anthocyanin synthesis, resulting in decreased accumulation of anthocyanin in the rice caryopsis(Zaidi et al, 2019; Mackon et al, 2021; Khantarate et al, 2022). A daily mean temperature of about 32oC compared to 22oC can result in a reduction of more than 20% in the amount of anthocyanin during grain filling in purple rice (Zaidi et al, 2019). Additionally, Kim et al (2007) reported that the expression levels of CHS, F3H, DFR, ANS and AN5 of the black rice variety Heugjinju were 200- to 500-fold lower for plants grown under 21 oC compared to those grown under 27oC. Thus, the growing altitude may impact the anthocyanin content and color density of purple rice; an appropriate altitude for a variety can enhance the accumulation of anthocyanin (Kim et al, 2007). A black rice variety G60 increases its anthocyanin content by about two- fold when grown at an altitude of 1360 m compared to when grown at an altitude of 76 m. Consequently, the color of the caryopsis is darker at high altitudes compared to at low altitudes due to the temperature difference between the two altitudes (Kushwaha et al, 2016). In addition, the response of anthocyanin synthesis among purple rice varieties grown at different elevations was found to depend on the rice variety. Some purple varieties have higher grain anthocyanin concentrations at lower elevations, while some varieties responded in the opposite direction (Rerkasem et al, 2015). Thermal stress was reported to enhance peroxidase enzyme activity and contributes to a high level of H2O2, resulting in the degradation of anthocyanin (Takahama et al, 2004). Thus, under high temperatures (≥ 35oC), the application of peroxidase inhibitors could assist to maintain metabolic activities and sustain anthocyanin accumulation in purple rice; this should be an interesting topic for further investigation. The variation in response to temperature among purple rice varieties at different growing elevations should also be considered.

Salinity or salt stress

Salinity or salt stress is among the most severe environmental stresses that cause the accumulation of damaging reactive oxygen species (ROS) in plants and reduce productivity (Kashif et al, 2020a, b). Anthocyanin accumulation is a protective mechanism for plants under stress conditions (Gould et al, 2004). Daiponmak et al (2010) studied the anthocyanin cyanidin-3-glucoside content and antioxidant activity of Thai rice varieties under salinity stress and found that increased total phenolic content, antioxidant activity and anthocyanin cyanidin-3-glucoside content in rice seedlings act as a protective mechanism against salinity stress. This ability is related to antioxidant activity, lipid peroxidation, total phenolic content and anthocyanin cyanidin-3-glucoside content. The anthocyanin concentration is decreased by about 25% when rice plants are exposed to a concentration of 150 mmol/L NaCl for 6 to 8 d (Chunthaburee et al, 2016). The expression patterns of the genes,,andare influenced during salt stress and are correlated with the anthocyanin variation in the leaves; the higher the total anthocyanin in leaves (deep purple leaf color), the less the plant is affected, indicating that higher anthocyanin in the leaf can significantly reduce salt stress in a plant (Chunthaburee et al, 2016). However, various degrees of salt stress affecting anthocyanin synthesis in purple rice has not yet been reported among different rice varieties; this would be very useful information for the selection of rice varieties concerning the potential of anthocyanin synthesis and productivity when grown under salt stress conditions.

Nutrient

The essential nutrients for plant growth and development are important in plant production, and some minerals such as calcium, magnesium, iron, manganese and copper can form a complex with anthocyanin in plant cells (Sinilal et al, 2011). Improving Mg uptake in rice can form a metalloid co-pigment complex for stabilizing anthocyanin, increasing the concentration in different parts such as leaves and the pericarp by promoting the expression levels of,,,and(Tisarum et al, 2018). This agrees with the results of Sinilal et al (2011), which indicated that Mg2+acts as a co-enzyme regulator in anthocyanin biosynthesis and can sustain a high level of anthocyanin in the rice pericarp. Foliar application of phosphorus at 23.6% P2O5enhances the total anthocyanin concentration in apples by 75% (?tampar et al, 2015). Furthermore, N fertilization affects the yield of bioactive substances differently in each plant species; for example, increasing N application increases caffeine and theobromine contents in the leaves ofbut has no effect on phenolic or flavonoid concentrations (Palumbo et al, 2007). However, the mechanism of the effect of nutrient stress on anthocyanin synthesis is not well understood in purple rice. In other plants, the addition of nutrients can affect the anthocyanin concentration in the fruits; for example, applying N at 120 kg/hm2decreases the anthocyanin levels in grape skins (Soubeyrand et al, 2014). The application of 120 and 150 kg/hm2N increases the concentration of anthocyanin and the antioxidant properties in the colored potato (Michalska et al, 2016). N fertilizers applied to rice increase anthocyanins in the leaves and shoots but have no effect on the anthocyanin concentration in the grains (Yamuangmorn et al, 2018). In addition, Zn application to berries has a significant influence on gene expression in phenolic biosynthesis pathways (Tang et al, 2015) and is able to protect the color and chemical degradation of anthocyanins in purple corn (L.) (Luna-Vital et al, 2018). However, no information is available concerning the effect of Zn application on grain anthocyanin concentration among purple rice varieties, suggesting that this should be further investigated, as biofortification by applying Zn fertilizer is an ongoing international collaboration among rice-growing countries to increase grain Zn concentration for the benefit of human health.

Even though many environmental factors affect grain anthocyanin concentration in purple rice as indicated above, there is an opportunity to increase both grain Zn and anthocyanin compounds in purple rice for the maximum benefit to consumers (Fig. 3).

Improve anthocyanin and Zn concentrations in purple rice by N and Zn fertilizer application

N is an essential component of all proteins, and also an essential constituent of chlorophyll. A sufficient amount of available N in plants is required, making N a key factor in crop production (Nadeem et al, 2013). N facilitates improved fruit and seed production as well as rapid plant growth, and affects the quality of crops (Marschner, 2012). N deficiency results in common abiotic stress for plants and leads to changes both in primary and secondary metabolism. The accumulation of reactive oxygen species is the major deleterious effect of N deficiency (Ková?ik and Ba?kor, 2007; Giorgi et al, 2009). In rice crops, applying N fertilizer is a common practice among farmers in all rice-growing countries. Currently, 50% of cereal agricultural systems apply N fertilizer, approximately 100 million tons each year globally, especially in rice crops (Liu et al, 2016; Duncan et al, 2018). Applying N fertilizer not only increases the production of rice crops, but also enhances the accumulation of grain Zn that could benefit seedling growth and development as well as human nutrition (Boonchuay et al, 2013;Prom-U-Thai et al, 2020). Increasing grain N concentration in plant tissues by N fertilizer application is a key parameter for improving grain Zn concentration in rice crops, but the appropriate rates for each variety should be considered, as different responses can be observed among the varieties depending on yield potential and original grain Zn concentration (Kankulanach et al, 2021). Applying N results in higher tissue protein concentration together with higher P, Zn and Fe concentrations that influence nutrient absorption and productivity in wheat (Kutman et al, 2011; Liu et al, 2018). In addition, applying N fertilizers has indirect effects on grain Zn.For example, the soil acidifying effect of ammonium sulfate [(NH4)2SO4] results in increasing Zn availability, especially in alkaline soils (Alloway, 2008). At the molecular level, an abundance of transporter proteins are involved in Zn uptake, absorption, transport and remobilization in rice plants (Cakmak et al, 2010; Waters and Sankaran, 2011). Additionally, a number of transport proteins,such as Zn-regulated transporter/Fe-regulated transporter-like proteins, yellow stripe-like transporters and heavy metal ATPase family proteins, are involved in the root uptake, xylem loading and unloading, xylem-to-phloem exchange, phloem loading and unloading, and grain deposition of Zn or nicotianamine (NA)-chelated Zn (Palmer and Guerinot, 2009; Pedas et al, 2009; Nahar et al, 2020). The application of Zn induces the expression levels of N assimilation genes (,,and) and the activities of NR, NiR and GS in the shoots, but decreases the expression levels of,andin the roots. Therefore, the N and Zn supply promotes N and Zn assimilation in the rice shoots, thereby contributing to higher plant biomass and grain Zn accumulation. The application of N significantly increases the root-to-shoot translocation and distribution of Zn into the leaves by upregulating the expression levels of Zn transporter genes () in both the roots and shoots (Ji et al, 2021). Therefore, applying N should carefully consider the co-transport of Zn with N, a factor that is important for improving Zn concentration in rice by fertilizer management. In the future, it may be necessary to investigate the functional relationship between N and Zn by an intensive molecular research program, particularly when dealing with a specific purple rice variety.

Fig. 3. Diagram of purple rice with high grain Zn and anthocyanin concentrations.

The combined application of N and Zn fertilizer improves yield and Zn concentration in the grains, Zn uptake and accumulation in purple rice varieties. Applying a combined N and Zn fertilizer to the soil during cultivation at the rates of 60 kg/hm2N and 50 kg/hm2ZnSO4increases grain yield and Zn concentration in purple rice varieties (Yamuangmorn et al, 2020; Fongfon et al, 2021a). However, limited information is available concerning a wider range of N and Zn application rates or for rice varieties under suitable management. At present, little is known concerning the mechanisms of translocation and distribution of N and Zn from plant tissues into the grain. In addition, priming seeds with a combined N and Zn solution enhances seedling growth performance, while grain yield and Zn concentration can be improved by foliar application of N and Zn fertilizers (Tuiwong et al, 2022). The interaction mechanisms between N and Zn involved Zn playing a major role in plants by acting as the cofactor for enzymes involved in N metabolism such as alcohol dehydrogenase (Castillo-González et al, 2018). The increase in seedling growth performance is due to positive interactions between N and Zn within the plant body and the increased activity of several enzymes that facilitate vegetative growth and photosynthesis (Aboutalebian et al, 2012). Wang et al (2017) showed that foliar application of combined N and Zn increases Zn concentration of wheat grain yield by 101%, and the molar ratio of grain phytic: Zn is decreased by 53.1% compared with the control. Applying soil Zn (0.2 mg/kg) and medium soil N (180 mg/kg) increasesgrain yield and Zn concentration in wheat (Erenoglu et al, 2011). Thus, the combined N and Zn fertilizers act synergistically to improve the productivity and grain Zn concentration, which can be a way to solve the problem of Zn deficiency among the population.

For grain anthocyanin concentration, N deficiency results in abiotic stress for plants and leads to changes both in primary and secondary metabolism due to the accumulation of reactive oxygen species. The N deficiency causes an increase in the biosynthesis of secondary metabolites based on carbon compounds, mainly phenolic compounds such as hydroxycinnamic acids, flavonoids and anthocyanins (Rubio-Wilhelmi et al, 2012; Guillén-Román et al, 2018). Applying N fertilizers can alter the quality of antioxidant compounds. In particular, N fertilizers can alter the antioxidant compounds in many plants (Nguyen et al, 2008; Amarowicz et al, 2020; Frías-Moreno et al, 2020). Anthocyanin synthesis in plant parts can be influenced by both the low and high N rates, but there are no available studies explaining the difference of responses. The use of Zn spraying is a useful and applicable method that can improve the colorless aril disorder and significantly improve pomegranate quality (Asadi et al, 2019). However, limited information is available on how Zn application affects anthocyanin compound synthesis and accumulation in purple rice varieties. In purple rice, the levels of some phenolic acids,such as p-coumaric acid and ferulic acid,are increased in plants with N deficiency (Chishak and Horiguchi, 1997). Grain yield and anthocyanin of some purple rice varieties increased simultaneously with increased N fertilizer rates, while foliar Zn application is suggested as an effective tool to increase grain Zn concentration among the varieties, although soil Zn application should be carefully considered, as it may reduce grain anthocyanin in some varieties (Fongfon et al, 2021a). Therefore, it would be interesting to investigate how the involvement of Zn in plant nucleic acid metabolism and reactive oxygen species is related to anthocyanin synthesis in plants among the purple rice varieties. This would assist to achieve high grain Zn and anthocyanin concentrations in specific purple rice varieties.

Prospects

Purple rice is a potential food source with high nutritional value. A current challenge is to improve grain anthocyanin and Zn concentrations in purple rice in order to benefit human health. The wide variation of grain Zn and anthocyanin concentrations among rice varieties provides an opportunity to facilitate breeding programs for high grain quality purple rice. Genetic engineering has succeeded in producing high anthocyanin purple rice varieties, but the acceptability among rice consumers is an issue that needs to be examined concerning long-term consumption. The biofortification strategy of applying N and Zn fertilizer would be a convenient pathway to improve both grain anthocyanin and Zn concentrations in purple rice varieties, although further investigation is required to better understand the interaction between rice variety and fertilizer management.

ACKNOWLEDGEMENTS

The financial support was partially provided by Chiang Mai University (Grant No. coe2565).We thank all the members of the Plant Nutrition Laboratory, Faculty of Agriculture, Chiang Mai University, Thailand for their help and support and Dr. Dale Taneyhill for English editing.

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Copyright ? 2022, China National Rice Research Institute. Hosting by Elsevier B V

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Peer review under responsibility of China National Rice Research Institute

http://dx.doi.org/

26 October 2021;

22 April 2022

Chanakan Prom-U-Thai (chanakan.p@cmu.ac.th)

(Managing Editor: Fang Hongmin)