Rice (Oryza sativa L.) stands as one of the most important crops worldwide, providing nutrients and energy for more than half of the world’s population (Zhou et al., 2002). Plants are sessile and exposed to various environmental stresses, which are undoubtedly critical factors affecting crop production (Chakraborty and Newton, 2011; Deutsch et al., 2018). To cope with environmental stimuli, plants have involved a vast array of defense mechanisms, one of which is the generation of an enormous metabolite arsenal (Wang et al., 2019). Understanding the genetic basis of natural variation of the metabolome in major crops like rice is essential for the quality, reliability, and sustainability of the food supply (Chen et al., 2014). Moreover, the study of abundant metabolites in rice plants contributes considerably to basic rice research and breeding practices (Kang et al., 2019; Mutuku et al., 2019).

The adaptation of plants to harsh environments results in specialized metabolites that assertively enable plants to interact with abiotic and biotic stressors (Zhou et al., 2023). Among these specialized metabolites, the amine-conjugated phenolic compounds, hydroxycinnamic acid amides (HCAAs), have drawn a lot of attention in plant development and defense processes (Bouchereau et al., 1999; Campos et al., 2014; Cho and Lee, 2015; Roumani et al., 2020; Wang et al., 2020a, 2023a). It is formed through the condensation reaction between CoA esters of hydroxycinnamic acids and aliphatic, di/poly-amines, or aromatic monoamines. Accordingly, HCAAs can be broadly categorized into basic and neutral groups. The basic group is hydrophilic and ionizable, comprising aliphatic amines such as putrescine and spermidine. In contrast, the neutral group lacks free amino groups, is water insoluble, and includes aromatic amines such as tyramine, octopamine, and tryptamine (Leonard et al., 2022). It is generally proposed that HCAAs are synthesized in the cytosol and transported into the cell wall, where they undergo peroxidative polymerization mostly upon wounding or biotic attack (Facchini et al., 2002; Hagel and Facchini, 2005; Kang and Back, 2006; Lee et al., 2007). These compounds are widely distributed across plant species and play critical roles in plant physiology, including structural reinforcement, abiotic/biotic stress response, and signaling pathways (Muroi et al., 2009; Macoy et al., 2015; Li et al., 2018; Vogt, 2018; Roumani et al., 2021).

A growing number of studies demonstrate that a wide range of HCAAs accumulated in rice plays a vital role as defensive compounds exhibiting both antimicrobial and anti-herbivore activities (Park et al., 2014; Alamgir et al., 2016; Fang et al., 2022). Beyond their protective functions, HCAAs also contribute to the nutritional value of rice, especially in whole-grain varieties. The antioxidant properties of these compounds enhance the quality of rice by reducing oxidative stress during storage and processing, thereby maintaining the rice’s nutritional integrity. Additionally, rice grains enriched with hydroxycinnamic acid derivatives can offer health benefits when consumed, due to their bioactive properties (Călinoiu and Vodnar, 2018; Leonard et al., 2022; González-Rodríguez and García-Lara, 2024). Both in-vivo and in-vitro assays have demonstrated the potent antioxidant, anti-diabetic, anti-inflammatory, anti-melanogenic, and cytotoxic properties of HCAAs (Leonard et al., 2022). In traditional medicine, HCAAs, known for their antioxidant properties, have been used to treat skin and digestive disorders and even as anti-cancer agents (Liu et al., 2022; Khawula et al., 2023; González-Rodríguez and García-Lara, 2024). However, HCAAs may play vital roles in rice nutrition, stress tolerance, and growth regulation beyond current understanding, warranting further research.

A recent review has summarized current knowledge regarding HCAAs in maize, including its role in stress responses and its potential for conferring health benefits to human beings (González-Rodríguez and García-Lara, 2024). Both maize and rice are important crops in the grass family, and some key metabolites, including flavonoids and HCAAs, played vital roles in the domestication of maize and rice. However, flavonoids had higher variation in maize than in rice, and more abundant HCAAs existed in rice leaf than maize leaves, indicating that there is interspecific metabolic divergence between these two crops (Deng et al., 2020). Rice has been regarded as a model monocot plant for genomics study due to the smallest genome size among the domesticated cereals, high-efficiency transformation technology, and rich germplasm resources (Jiang et al., 2012; Li Y. et al., 2018). The development of metabolic genome-wide associated studies (mGWAS) provides a useful tool for gene-to-metabolite analysis to understand how rice defends against microorganisms, insects, and other threats through metabolic regulation. This article seeks to underscore the significance of HCAAs in rice development and defense mechanisms. This study aims to provide a comprehensive overview of the major groups of HCAAs, delving into their biosynthesis, chemical structures, distribution, and diverse functions, with a particular emphasis on their contribution to stress tolerance. Additionally, the existing challenges in rice HCAA research and promising opportunities for future investigation would also be discussed.

HCAAs, also known as phenolamides or phenylamides, are widely distributed types of secondary metabolites in plants and considered as one of the main classes of phenylpropanoid metabolites, along with lignin and flavonoids (Herrmann and Nagel, 1989; Bassard et al., 2010; Dong and Lin, 2021). As illustrated in Figure 1 , HCAAs are chemical conjugates of various hydroxycinnamic acid esters like coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA with aromatic amines like tyramine and tryptamine or polyamines including putrescine, spermidine, and spermine (Edreva et al., 2007; Tanabe et al., 2016). The conjugation connects two different classes of plant protective compounds, spanning the continuum from primary to secondary metabolites, and is catalyzed by those inducible hydroxycinnamoyl transferases (Edreva et al., 2007; Bassard et al., 2010; Macoy et al., 2015). In rice, several genes encoding these transferases have been identified: Os09g37200 encoding a putrescine hydroxycinnamoyl acyltransferase (OsPHT), Os04g56910 encoding an agmatine hydroxycinnamoyl acyltransferase (OsAHT2), and Os12g27220 encoding a spermidine hydroxycinnamoyl acyltransferase (OsSHT) associated with the natural variation in levels of HCAAs were identified based on mGWAS (Chen et al., 2014). Further mGWAS in rice leaf and in-vivo metabolic analysis of the transgenic plants identified Os12g27220 and Os12g27254 as two OsSHT genes that might underlie the natural variation of levels of spermidine-conjugated HCAAs in a collection of rice germplasms (Dong et al., 2015). Further molecular and biochemical studies identified another four genes coding tryptamine/tyramine hydroxycinnamoyl transferases including OsTHT1/2 and OsTBT1/2 (Peng et al., 2016). In rice, the levels of aliphatic p-coumaroyl putrescine and feruloyl putrescine increase markedly in plants attacked by herbivores and pathogens (Alamgir et al., 2016; Morimoto et al., 2018), and two tandem-duplicated genes encoding putrescine hydroxycinnamoyl acyltransferases were found to be involved in causing the accumulation of HCAAs and the enhancement of HCAAs-dependent resistance against the fungal pathogen Magnaporthe oryzae (Fang et al., 2021). Taken together, there are 10 different hydroxycinnamoyl transferases, including OsAHT1/2, OsPHT3/4, OsSHT1/2, OsTBT1/2, and OsTHT1/2, summarized in Table 1 , and they can transfer acetyl donors to agmatine, putrescine, spermidine, tryptamine, and tyramine receptors in rice plants, respectively. This process results in the formation of five types of HCAAs in rice, including hydroxycinnamoyl agmatine, hydroxycinnamoyl putrescine, hydroxycinnamoyl spermidine, hydroxycinnamoyl tryptamine, and hydroxycinnamoyl tyramine (Dong et al., 2015; Peng et al., 2016).

The majority of hydroxycinnamoyl transferases are members of the BAHD acyltransferase family, and they are able to acylate a variety of metabolites and participate in secondary metabolic reactions (D’Auria, 2006; Dong et al., 2015; Fang et al., 2021). All proteins in this family typically share two conserved motifs, including HXXXD and DFGWG. The HXXXD motif, central to each enzyme, is crucial for catalysis and strictly conserved in BAHD acyltransferases. The DFGWG motif is located near the C-terminal of the protein and might play an important role in the catalytic process and binding of CoA (Xu et al., 2023). The genes of this family are mostly classified into five categories, among which the N-hydroxycinnamoyl transferase utilizing branched-chain amines as receptors belongs to Clade V (D’Auria, 2006). In rice, OsSHT1/2 and OsPHT1/2 have been identified as members of Clade V ( Table 1 ), whereas OsAHT1/2, OsPHT3/4, OsTBT1/2, and OsTHT1/2 belong to Clade IV (Peng et al., 2016). OsAHT1/2, OsPHT3/4, OsSHT1/2, OsTBT1/2, and OsTHT1/2 can utilize various CoA thioesters, such as feruloyl-CoA, caffeoyl-CoA, sinapoyl-CoA, and coumaroyl-CoA, as acyl donors to catalyze mono-, di-, and tri-acylation reactions with receptor aromatic amines or polyamines, thereby generating a structurally diverse array of HCAA compounds. HCAAs could possess a variety of metabolic, chemical, and functional capabilities due to the large number of possible combinations among the parent compounds (Edreva et al., 2007; Zeiss et al., 2021). Acylation significantly enhances the structural diversity, stability, and bioavailability of HCAAs, thereby contributing to their multiple biological functions in plant growth and development.

Constitutive HCAAs show natural variation and accumulation patterns in rice plant tissues/organs across developmental stages, supporting rice development and stress defense (Dong et al., 2015; Li Z. et al., 2018).

Extraction is a key step in the determination of chemical compounds in rice. Generally, a sample pre-treatment step is required before the extraction. For example, a drying process of rice samples is necessary to stabilize the samples, preventing the microbial spoilage and the hydrolytic rancidity (Chan et al., 2013). In addition, sterilization of rice samples can be conducted through washing with a 1% sodium hypochlorite solution (Mohd. Esa et al., 2013). Finally, the sample is generally ground to a fine powder in order to obtain a homogeneous material, which can be stored in a freezer for further analysis (Goufo et al., 2014). In some conditions, prior to extraction, lipids are removed from the sample by adding hexane (Zhou et al., 2014). Both basic and aromatic HCAAs form ester bonds to structural polymers of the cell wall (Edreva et al., 2007). Sometimes enzymatic hydrolysis with cellulase of rice samples is used to break the bonds between the phenolic compounds and the insoluble polymers of the cell walls to increase the total phenolic content (Wanyo et al., 2014; Sumczynski et al., 2017).

For the actual extraction phase, maceration is the most adopted procedure for the isolation of rice HCAA compounds. Polar solvents like methanol/water or ethanol/water mixtures are used. For instance, acetone/water (70/30, v/v) was used to extract phenolic compounds from rice, and a hydrolysis with acid or NaOH is performed to break the bound phenolics in specific cases (Alves et al., 2016). After impurities from the extract were removed using a filtration membrane, HCAAs in the rice samples were analyzed using chromatographic methods (Chen et al., 2013; Dong et al., 2015; Xue et al., 2024). The selected features of chromatographic methods for the analysis of HCAAs are similar to those of phenolic compounds, which have been well summarized (Ciulu et al., 2018). Therefore, the quali-quantitative profile of HCAAs in rice involves the separation, the identification, and the quantification of the extracted analytes, and HPLC is the most adopted analytical technique.

In rice, HCAAs are more abundant in leaves, which also show more metabolic interactions than seeds (Deng et al., 2020). Notably, HCAAs accumulate significantly in leaves under environmental stress (Gaquerel et al., 2014; Alamgir et al., 2016; Roumani et al., 2021). Although the general function of HCAAs in plant immunity has been well reviewed (Roumani et al., 2021; Zeiss et al., 2021; Liu et al., 2022), this review will mainly focus on the protective roles of HCAAs in the vital model crop rice, especially under various stresses ( Table 3 ).

HCAA metabolism is regulated by multiple mechanisms in response to varying developmental stages and environmental conditions (Roumani et al., 2021). Based on recent advances in rice HCAAs, we summarized potential regulatory mechanisms, including phytohormone signaling regulation, transcription factor regulation, and epigenetic regulation in rice under biotic attacks as examples ( Figure 2 ).

This review provides a thorough analysis of HCAAs in rice plants, with special attention to their varied distribution, biological roles, and biosynthetic mechanisms. HCAAs are crucial for plant defense, acting as a direct toxin to predators and pathogens and contributing to cell-wall cross-linking and reinforcement (Bassard et al., 2010). HCAAs in growing rice panicles and roots likely provide key protection against herbivores and pathogens, as flowering plants are more resistant to herbivory than young vegetative plants (Diezel et al., 2011). In contrast to constitutive HCAAs in flowers and roots, young rice leaves are frequently exposed to various stresses and have shown a strong response through enhanced accumulation of HCAAs (Alamgir et al., 2016; Tanabe et al., 2016). The molecular mechanisms of HCAA biosynthesis were intensively investigated in other plants such as Nicotiana tabacum (Figon et al., 2021) and Arabidopsis thaliana (Luo et al., 2009). For instance, the induction of HCAAs in response to herbivory was finely controlled by a dominant regulator, MYB8, in N. tabacum (Kaur et al., 2010). However, regulatory networks of HCAAs biosynthetic pathways in rice plants under various stresses remain to be further investigated.

Apart from their biological functions in plants, HCAAs exhibit a wide range of health-promoting properties. For example, N-(p-coumaroyl) serotonin has been found to have antimicrobial properties against pathogenic bacteria, as well as anti-inflammatory and antiatherogenic effects (Takii et al., 2003). Furthermore, their potential to positively impact gut health and lipid metabolism presents significant opportunities for improving human well-being (González-Rodríguez and García-Lara, 2024). Both sakuranetin and HCAAs, renowned as the unique phenolic phytoalexins in rice, have been reported to have antimicrobial properties (Cho and Lee, 2015). A recent study demonstrated that rice under attack by the phloem-feeding brown leafhopper (BPH) produces an antifungal flavonoid, sakuranetin, which targets the yeast-like beneficial endosymbionts of BPH. In addition, disrupting sakuranetin biosynthesis in rice increased the reproductive performance and nutrition of BPH, along with enhancing plant damage (Liu et al., 2023). Thus, we propose that HCAAs could influence the performance of biological organisms through modulating their symbionts and lipid metabolism. Currently, there are limited studies on the bioavailability and biotransformation of HCAAs, which could be hydrolyzed in vivo to produce the phenolic acid and amine moieties through the action of enzymes from the host and/or gut microbiota. For instance, agmatine can be freed from these HCAAs in vivo, and these compounds may contribute to health benefits partially through agmatine, an important factor in the longevity of its host (Laube and Bernstein, 2017). This indicates that agmatine-conjugated HCAAs may serve as agmatine carriers and display similar bioactivities. HCAAs are now seen more as metabolic intermediates than mere end products (Bassard et al., 2010). Therefore, HCAAs can be hydroxylated and methylated, stored as conjugates, and mobilized when needed.

Plants primed to accumulate high levels of HCAAs can launch a faster, stronger defense response to future infestations, which suggests a connection between preexisting and newly produced defense-related metabolites, indicating a state of readiness (Tugizimana et al., 2019). Plants store defensive secondary metabolites in specialized structures: water-soluble ones in vacuoles and lipophilic ones in latex, resin ducts, trichomes, and glandular hairs (Khare et al., 2020; Al-Khayri et al., 2023). Once plants are attacked by environmental stressors, these metabolites, including HCAAs, could release rapidly from the injured sites and defend against attackers. Epigenetic regulation in plants facilitates defense priming by introducing specific chemical modifications to DNA and histone within chromatin (Lämke and Bäurle, 2017; Jiang et al., 2020). Histone modification is one type of epigenetic regulation of gene expression that occurs during plant development and environmental responses (Dong and Lin, 2021). It has been found that MeJA or powdery mildew treatments upregulated the expression of JMJ705, an H3K27me3 demethylase, which reduced H3K27me3 levels from the four BAHD N-acyltransferases, including TBT1, TBT2, ACT, and PHT3, in the resistant qingke line (Xu et al., 2022). A recent study in our lab showed that the infestation of fall armyworm induced H3K9ac levels from OsSHT1/2, which enhanced the biosynthesis of spermidine-derived HCAAs in rice (Xue et al., 2024). Further research is crucial to identify additional histone markers and histone-modifying enzymes and to understand the mechanisms of epigenetic regulation in the HCAA-mediated defense network in plants.

Therefore, using HCAAs to fight herbivores and pathogens is cost-effective for plants and may have influenced plant evolution. Additionally, HCAAs have been widely reported to have health-promoting and pharmacological properties (Roumani et al., 2020; Wang et al., 2020a). Value addition in rice has caused great interest, as it will benefit food security and human health. For example, pigmented rice grains are important food resources for health, as they contain various nutrients and bioactive metabolites, including some HCAAs (Zhao et al., 2024). HCAAs can aid in breeding stress-resistant plants and enhancing human health, with efforts already underway to engineer HCAA production in rice grains (Park et al., 2009). Importantly, current studies on rice hydroxycinnamoyl transferase genes report no phenotypic changes from knocking out or overexpressing these genes (Fang et al., 2021; Shen et al., 2021). These results suggest overexpressing hydroxycinnamoyl transferase genes can boost rice resistance without harming agronomic traits, offering a new genetic resource for better crop resistance. This review aims to provide a foundation for further HCAA research in rice and other cereals and explore their potential in breeding superior varieties for sustainable agriculture.