With the development of the economy, the demand for high-nutrient nut-based complementary foods is increasing daily. Nuts of T. grandis are considered an important source of nutrients and bioactive ingredients and are frequently employed as food supplements [18]. T. grandis trees are widely cultivated in China’s subtropical mountainous areas, where the soil is very barren [26]. Although a large amount of chemical fertilizers has been applied, the effective fertilizer utilization rate is meager [27]. The excessive use of chemical fertilizers causes massive damage to the environment [28]. More importantly, chemical fertilizers are quickly fixed in the soil and converted into an insoluble form [29]. Previous studies have reported the remarkable effects of foliar spray application on the quality of crops. However, the understanding of the impact of foliar fertilizers on the cultivation of T. grandis trees is still limited [30].
As an essential element, Ca2+ is the third most abundant in plants [31]. Clear evidence confirmed the significance of Ca2+ in various biological processes, such as signal transduction, secondary metabolism, growth, and development [32]. In our study, CSA, a newly developed chelated calcium fertilizer, was applied to provide soluble Ca2+ to T. grandis trees. Proteins from T. grandis nuts are valuable nutritional sources in the food industry [33]. T. grandis kernels are enriched in unsaturated fatty acids, which are essential bioactive components for human health [34]. Analysis of quality parameters showed that CSA significantly up-regulated the contents of total protein and unsaturated fatty acids, suggesting the positive effects of CSA on the quality of T. grandis nuts.
Recently, integrated transcriptomic and metabolomic analyses revealed the regulatory mechanism underlying the secondary metabolism of T. grandis. A multi-omic analysis revealed the responses of terpenoid- and flavonoid-biosynthesis pathways to nanoplastic pollutants [35]. An integrated omic analysis investigated the involvement of WRKY21 in the regulation of age-induced amino acid biosynthesis in T. grandis nuts [19]. By referring to the newly published genome of T. grandis, our multi-omic analysis detected 47,064 transcripts, providing sufficient genetic information for uncovering the role of CSA in the activation of the phenolic acid biosynthesis pathway of T. grandis nuts.
KEGG enrichment analysis revealed that most of the CSA-responsive genes were enriched in ‘secondary metabolism’, ‘energy metabolism’, and ‘hormone signaling’, indicating that CSA has a comprehensive impact on T. grandis nuts. In plants, calcium sensors might participate in the regulation of secondary metabolite biosynthesis, such as stilbenes, phenolic precursors, and hormones [36]. Our data showed that most of the secondary metabolism-related DEGs were up-regulated by CSA, suggesting an active role of CSA in the secondary metabolism of T. grandis nuts. A previous study reported that starch and sucrose metabolism participated in the modulation of seed development in T. grandis [37]. Interestingly, most starch and sucrose metabolism-related genes were down-regulated by CSA. Thus, CSA treatment may modulate the processes involved in seed development in T. grandis by inhibiting energy metabolism. In T. grandis, phytohormones play an essential role in regulating the biosynthesis of squalene and β-sitosterol during the post-ripening process [38]. In our study, the zeatin biosynthesis pathway was inhibited by CSA, indicating an influence of CSA on the hormone signaling pathway.
In plants, flavonoids are classical phenolics containing a 15-C skeleton [39]. Although most of the phenolic acid biosynthesis-related genes have been identified in T. grandis, the impacts of CSA on the regulation of phenolic acid biosynthesis are largely unknown [40]. Recently, a number of TFs involved in the regulation of phenolic acid biosynthesis have been recognized in various plants. For example, several Salvia miltiorrhiza TFs, such as SPL7, MYB1, and NPR1-TGA2/NPR4 modules, are involved in phenolic acid biosynthesis [41–43]. In our study, two members of the AP2/ERF family, AP2-1 and AP2-3, were identified as regulators of phenolic acid biosynthesis in T. grandis. In S. miltiorrhiza, overexpression of the two AP2/ERF family TFs, ERF1L1, and ERF115, markedly elevated tanshinone production by regulating the expression of DXR, PAL3, 4CL5, TAT3, and RAS4, respectively [44, 45]. We identified HCT as a new downstream target of the AP2 subfamily members in T. grandis. The HCT enzyme, a significant regulator of phenolic acid biosynthesis, has been extensively investigated as a prominent member of the BAHD acyltransferase family [46]. In T. grandis, AP2-1/AP2-3 participated in phenolic acid biosynthesis by inducing HCT expression.
The roles of exogenous Ca2+ in the regulation of phenolic accumulation have been well-investigated in various plants. For example, exogenous Ca2+ regulates phenolic metabolism and physiological responses of wheat seedlings to UV-B radiation [47]. CaCl2 remarkably elevated phenolic acid contents by up-regulating the activities of C4H, PAL, and F5H [48]. The application of CSA could induce a higher direct entry of Ca2+ through the leaf vascular system [49]. We revealed a new regulatory module, Ca2+-AP2-HCT, that participated in the regulation of phenolic acid biosynthesis in T. grandis. Our data provides a theoretical basis for the application of Ca2+ fertilizer for the management of daily fertilizer application in T. grandis trees.