Chestnut has been cultivated in China for more than 2700 years (Adua, 1999). It is an economically valuable plant, its wood used for timber, its leaf and flower utilized as raw material in the pharmaceutical and cosmetic industries, and its fruit peel a source of extracted tannin. Most of all, its fruit is a popular food resource with high nutritional value. The genus Castanea comprises 12 world chestnut species, including four cultivated species: Chinese chestnut (Castanea mollissima Blume), Japanese chestnut (Castanea crenata Sieb. et Zucc.), American chestnut (Castanea dentata [Marsh.] Borkh), and European chestnut (Castanea sativa Miller) (Jiang et al., 2019). Among these, Chinese chestnut and Japanese chestnut are the most commercially important (Tanaka and Kotobuki, 1992).

Chinese chestnut is commonly grown in China for its fruits. However, several insect pests can seriously damage the tree’s trunk, buds, and fruit, and reduce nut production and quality (Gokce et al., 2020). One such pest is the spruce spider mite (Oligonychus ununguis Jacobi), a typical polyphagous mite that consumes the chestnut’s leaves (Wang et al., 1989; Wang et al., 2010). In the main chestnut-producing region of China, the occurrence of spruce spider mite generally leads to a 15%–50% decline in production (Wang et al., 1991).

To date, several biological and chemical control measures have been taken to prevent this mite from attacking trees in China, including the use of natural enemy species and pesticides (Wang et al., 2010; Zhao and Fan, 2019). Nevertheless, because Chinese chestnut trees are tall and mostly distributed in mountains, it is inconvenient to spray pesticides. Moreover, it often results in the development of resistance to pesticides and environmental pollution, thereby slowing the movement toward green, pollution-free production of crops and restricting the development of the Chinese chestnut industry. In sum, the control measures for spruce spider mite prevention are currently expensive and poorly effective.

In previous work, we developed a number of foxtail millet (Setaria italica (L.) P. Beauv.) materials for interplanting with chestnut. They were early-maturing, shade-tolerant, could thrive beneath Chinese chestnut trees, realizing the high-efficient utilization of land resources under the trees. Surprisingly, we discovered that the leaves of these materials were attacked by spruce spider mites. This decreased the rate of bad chestnut nuts resulting from mite attack by 5%–15%, while damage to the millet was minimal (Li et al., 2022). Furthermore, these materials had a high content of crude protein (CP) in their leaves.

Plant–insect interactions are intricate and dynamic. To overcome insect attack, plants develop different defense mechanisms, including physical and chemical defenses (War et al., 2012; Belete, 2018). The first mechanical barrier to feeding is formed by structural features on the leave surface, such as a waxy cuticle, spines and thorns, trichomes, and thick and lignified cell walls. Secondary metabolites, including phenolics, flavonoids, tannins, and lectins, provide the next barriers of protection. Among chemical constituents, nutrients such as amino acids, vitamins, proteins, and carbohydrates have been also reported to affect the relationship between plants and insects (Li and Jin, 2005; Yang et al., 2014; Li and Li, 2018). In one study, nymphal density and the leaf damage index of grape leafhopper (Erythroneura apicalis Nawa) were negatively correlated with CP levels in grape leaf (Fan et al., 2008). Another study reported that total soluble protein content in cotton leaves was positively associated with the population densities of thrip (Thrips tabaci), whitefly (Bemesia tabaci), and jassid (Amrasca devastans), and these pests were more prone to attack cotton genotypes with high concentrations of such proteins (Rizwan et al., 2021). Hence, the protein content of host plant leaves may be related to the degree of insect infestation, which could explain the effect of the foxtail millet materials we bred in previous work. Consequently, it is important to mine genes related to CP accumulation in foxtail millet leaves and to elucidate their regulation mechanisms. This would be helpful for breeding of foxtail millet varieties with high concentrations of leaf CP, and provide an effective, economically, and environmentally safe control measure for spruce spider mite through interplanting.

In this study, we performed transcriptome analyses on four foxtail millet varieties with significantly different leaf CP levels to identify genes associated with leaf CP content. We identified and characterized differentially expressed genes (DEGs) and conducted gene enrichment analyses to investigate their possible functions. We also predicted differentially expressed transcription factor (TF) genes. Our results lay a foundation for clarifying the biological processes and regulation mechanisms underlying leaf CP accumulation, and provide potentially precious gene resources for the future development of foxtail millet varieties with high concentrations of leaf CP.

Below we provide a summary of our methods. More details can be found in the Supplementary Materials and Methods.

In the natural environment, plants are constantly threatened by herbivorous insects. For Chinese chestnut, damage from spruce spider mite is a primary challenge restricting its output and quality, and generally leads to a serious commercial loss. In previous work, we developed several shade-tolerant foxtail millet varieties for interplanting with chestnut trees, and found that it improved the quality of chestnut fruits by attracting spruce spider mite to feed on their leaves. This strategy could become a new, effective, and environmentally friendly, pest-management option for chestnut tree.

Nutrients of host plants, such as proteins and carbohydrates, are essential sources of energy for the growth and development of herbivorous insects. CP content in leaves affects the lifespan, development duration, and egg laying outcomes of mites, and in certain plant species the degree of leaf damage by insects is higher for leaves with higher CP levels (Wu et al., 1995; Fan et al., 2008; Wei et al., 2009; Yang et al., 2014; Sonalkar, 2020). The foxtail millet varieties we previously bred for interplanting with chestnut tree all had high CP levels in leaves, which may be one major reason why they attracted the spider mites. In present study, four foxtail millet varieties with significant differences in leaf CP content were used to identify genes related to CP levels via transcriptome sequencing technology. We successfully identified 435 DEGs, including 337 upregulated DEGs and 98 downregulated DEGs in varieties with high CP concentrations. Our findings provide a precious resource for research on the mechanisms that control the accumulation of CP, and offer a chance to effectively control spruce spider mite outbreaks among chestnut trees.

Protein is an important product of nitrogen metabolism, and therefore CP levels are closely associated with nitrogen metabolism. For example, an increase in CP levels of vegetable leaves is correlated with an increase in N uptake (Mhlontlo et al., 2007). Most plants assimilate inorganic and organic forms of nitrogen, including nitrate and ammonium (Temple et al., 1998). Metabolism normally begins with nitrate being converted into nitrite in the presence of nitrate reductase, and then the nitrite is reduced to form ammonia by nitrite reductase. Following its formation, ammonia is assimilated by a number of pathways, including reductive amination and transpiration, to yield a variety of amino acids (Mokhele et al., 2012; Kishorekumar et al., 2020). In the current study, two DEGs, SETIT_014128 mg and SETIT_014124mg, were enriched in the nitrogen metabolism pathway and may code for carbonic anhydrase (CA). The reversible conversion of carbon dioxide to bicarbonate (CO₂+H₂O ⇔ HCO₃ ⁻ + H⁺) is catalyzed by this enzyme in the leaves of C₄ plants (Ludwig, 2016). Cyanate in plants, which is derived from endogens cyanide or comes from the environment, is decomposed into carbon dioxide and ammonia (CNO⁻ + HCO₃ ⁻+2H⁺ ⇔ NH₃+2CO₂), and produced ammonia enters into biosynthesis pathway of amino acids. This reaction is bicarbonate-dependent (Johnson and Anderson, 1987; Kozliak et al., 1995; Ebbs, 2004; Yu et al., 2019). Thus, SETIT_014128 mg and SETIT_014124 mg may promote nitrogen metabolism and subsequent biosynthesis of proteins by upregulating their expression level and the activity of CA. In addition, in the first step of C₄ photosynthesis, phosphoenolpyruvate carboxylase (PEPC) uses bicarbonate as a substrate, whose generation from carbon dioxide is catalyzed by CA (Ludwig, 2016); the decrease in CA accumulation and CA activity plays a considerable role in protein accumulation of PEPC (Chatterjee et al., 2021). In addition to having a significant impact on carbon and nitrogen metabolism, PEPC also influences protein synthesis by promoting the distribution of the carbon skeleton to amino acid production (Shinano et al., 2006). One study found that applying the nanobiomaterial ‘nanochitin whisker’ primarily facilitated the accumulation of nitrogen and translocation more than carbon metabolism and enhanced CP concentration in grain by stimulating PEPC activity and reducing the ratios of sucrose phosphate synthase (SPS) to PEPC in flag leaves and spikes of wheat during the anthesis and grain-filling stages (Cheng et al., 2019), revealing the role of PEPC in the coordination of carbon and nitrogen flow to amino acids and carbohydrates. These findings further support that SETIT_014128 mg and SETIT_014124 mg may be hub genes associated with leaf CP concentration in foxtail millet, and can be used to develop high-CP varieties by genetic manipulation; however, their detailed functions need to be further clarified.

In eukaryotes, the ubiquitin-proteasome system (UPS) is responsible for the majority of cytosolic protein breakdown, accomplished through the degradation of the substrate protein by ATP-dependent 26S proteasome after covalent attachment of many Ub molecules to the substrate protein (Ciechanover et al., 2000; Nandi et al., 2006). Binding of Ub to target proteins includes the following steps. First, the C-terminal Gly of Ub is activated and transferred to a carrier E2 protein (Ub-conjugating enzyme) by the Ub-activating enzyme E1. Then with the aid of E3 (a Ub-protein ligase enzyme), the E2 protein tags Ub to the target protein. Various E3 ligases within the UPS recognize substrates carrying diverse degradation signals, giving the system high specificity and selectivity. In this study, we identified two DEGs that encode Ub-conjugating enzyme E2 J2 (UBE2J2) and E3 Ub-protein ligase SIAH1, namely, SETIT_025290 mg and SETIT_004756mg, respectively. The UBE2J2 protein is a Ub-conjugating enzyme which, in association with Ub ligases including TEB4, PARKIN, CHIP, and cIAP1, promotes proteasomal degradation in mammals and responds to proteotoxic stress (Imai et al., 2002; Lenk et al., 2002; Kim et al., 2003; Wu et al., 2005; Choi et al., 2010; Claessen et al., 2010; Burr et al., 2011). Nevertheless, no UBE2J2 proteins have been characterized in plants to date. The seven in absentia (SINA)/seven in absentia homolog (SIAH) family proteins are E3 Ub-protein ligases with a RING domain and are highly conserved from plants to mammals (Polekhina et al., 2002). This superfamily was first characterized in Drosophila melanogaster and plays a crucial role in the regulation of development and proteasome-mediated protein degradation (Hu and Fearon Eric, 1999). To date, 18 SINA proteins homologous to the Drosophila SINA protein have been defined in Arabidopsis, among which SINAT5 targets the noapical meristem/cup-shaped cotyledon 1 (NAC1) TF and late elongated hypocotyl (LHY) for degradation and plays a role in various developmental processes in plants (Xie et al., 2002; Park et al., 2010). Therefore, SETIT_025290 mg and SETIT_004756 mg may be involved in specific ubiquitination and subsequent proteolysis of proteins, and have an impact on leaf CP content in foxtail millet by regulating protein levels and the stability of related proteins. However, their biological functions and regulation mechanisms remain to be elucidated in future work.

TFs regulate a variety of biological processes, such as plant metabolism, growth, and development, by binding to the promoters (or intragenic regions) of target genes (Kaufmann et al., 2018; Burgess et al., 2019). Many TFs have been found to control nitrogen assimilation. Recently, Wei et al. reported that overexpression of the dehydration-responsive element-binding protein 1C (OsDREB1C) gene in rice (Oryza sativa), a member of the AP2/ERF TF family, improved nitrogen content, nitrogen use efficiency (NUE), and protein abundance in leaves by elevating nitrogen uptake and transport activity (Wei et al., 2022). Furthermore, a few TFs affect protein synthesis in plants. For example, overexpressing the MYB family TF TaODORANT1 in common wheat (Triticum aestivum) and its homolog in Triticum urartu reduces the transcription levels of seed storage protein (SSP) genes and total SSP levels of mature grains, revealing an inhibition effect on SSP synthesis (Luo et al., 2021). OPAQUE 11 (O11) encodes a seed-specific bHLH TF in maize, and the protein levels of loss-of-function mutant o11 endosperm per kernel are significantly decreased compared to wild type (Feng et al., 2018), revealing regulatory role in protein accumulation. In addition, several NAC TFs have also been reported to regulate protein accumulation and content in grains of wheat, rice, and maize (Zea mays) (Uauy et al., 2006; Liang et al., 2014; Zhang et al., 2019; Wang et al., 2020). An endosperm-specific NAC TF, TaNAC019, modulates SSP accumulation in wheat seeds (Gao et al., 2021). It directly binds to the promoters of high-molecular-weight glutenin (HMW-GS) genes and activates their expression, and triple knock-out mutants of TaNAC019 homologs have lower gluten levels. In the current research, 40 DEGs were identified as TF genes, containing several members of the bHLH, MYB, NAC, and especially ERF gene families (Figure 5). It can be speculated that these genes may function as TFs and improve CP accumulation in leaves of foxtail millet by modulating nitrogen assimilation and protein synthesis. However, the detailed underlying molecular mechanisms involved require further research, and will be a focus of ours in the future.

Transcriptome analysis of genes in foxtail millet associated with CP accumulation in leaves was performed and 435 DEGs were identified. Several DEGs related to nitrogen metabolism and ubiquitin-mediated proteolysis pathways were characterized. We also predicted the TFs that participate in the control of CP levels. These findings provide a resource that clarifies the accumulation and control mechanisms of CP levels in leaves. This information can be used to develop foxtail millet varieties with high CP concentrations for interplanting with chestnut, to improve production of this important crop.