N transfer occurred in the E. urophylla × E. grandis/ D. odorifera intercropping system
Effective utilization of nutrients is one of the two major advantages in legume/non-legume intercropping systems . This process mainly depends on the ability of the root to acquire external resources for plant survival in different environments and adapt to external disturbances , which increases the abundance of N metabolic proteins, such as glutamate dehydrogenase (GDH) and glutamine synthetase nodule isozyme (GS), by the rhizosphere effect . In this study, our results showed that interspecific rhizosphere effects significantly improved N uptake and promoted the development of E. urophylla × E. grandis roots. This finding was consistent with previous studies, indicating that planting NFT might be an attractive option for maintaining the N fertility of soils with Eucalyptus [4, 37]. Nevertheless, the effect on D. odorifera in the intercropping systems was limited, possibly because the root exudates of E. urophylla × E. grandis had an allelopathic effect on D. odorifera. In addition, it may also be caused by N transfer, which was consistent with previous studies [3, 15]. In our study, N transfer occurred from D. odorifera to E. urophylla × E. grandis by 14.61%, which was equal to enhancement 150.62 mg N to E. urophylla × E. grandis (Fig. 2). The transfer N was the key N resource for E. urophylla × E. grandis and increased the biomass by 45.09%, i.e., increased N content significantly improved seedling physiological performance by increasing plant growth and nutrient storage reserves for subsequent root growth .
Different stress responses between the two varieties
Comparative proteomics are frequently used to investigate stress-responsive mechanisms in plants . Intercropping may be more effective at decreasing diseases and enhancing disease suppression than monocropping [35, 40]. Our proteomic study indicated that most stress- and defense-related proteins increased in E. urophylla × E. grandis roots in the monocropping system to response oxidative stress, with significant differences in the abundance of proteins related to a series of biological functional processes, including response to biotic stimulus, defense response, response to stress, response to stimulus, etc. (Fig. 5), which was consistent with previous study, i.e., introduction NFT increased the anti-pressure ability of Eucalyptus . The root character and the molecular structure were altered due to the different microbial community composition in Eucalyptus/NFT plantations . In our study, the ecological advantage of E. urophylla × E. grandis was increased by mixture, and biological resistance was altered by regulating the ratio of oxidase and pathogenic proteins, which affected the defense proteins or pathological proteins. The E. urophylla × E. grandis abundance of the major allergen, peroxidase and peroxiredoxin proteins was significantly increased in the intercropping system. The peroxidase 15-like (id: TRINITY_DN22413) of D. odorifera was up-regulated, but peroxidase 4-like (id: TRINITY_DN34591) and nodulin-13-like isoform X1 (id: TRINITY_DN40711) were down-regulated in the intercropping system (Table S2). Peroxidase up-regulated by rhizosphere effects not only prevented active injury but also degraded auxin (probable indole-3-acetic acid-amido synthetase GH3.1 IAA) and reactive oxygen species (ROS) . These changes further promoted the hormone system to rebalance and regulate the formation of adventitious roots and lateral roots to adapt to environmental stresses . Peroxidases such as major allergen, peroxidase, peroxidoredoxins and chloroplastic peroxiredoxin Q were highly abundant in intercropping E. urophylla × E. grandis, indicating that the stress response of plants to external stress was improved by the intercropping system. However, peroxidase 4-like (TRINITY_DN34591) and nodulin-13-like isoform X1 (TRINITY_DN40711) were down-regulated in intercropping D. odorifera, which may also indicate restricting effects on D. odorifera in the intercropping system. Additionaly, when plants were placed in a stressful environment, ascorbic acid was required to regulate the H2O2 content in plants to improve their stress resistance . While the nucleobase-ascorbate transporter (A0A059CYL1) of E. urophylla × E. grandis showed a higher abundance after monocropping.
In E. urophylla × E. grandis roots, glutathione S-transferase (GST) (id: A0A059AX10) and the homolog glutathione S-transferase U25 (A0A058ZUA9) were increased after intercropping (Table S1). GST, which catalyzes the conjugation of glutathione to electrophilic substrates, plays a crucial role in cell detoxification and stress tolerance in plants , increasing the toxin removal through increased enzyme levels . High contents of gibberellins can ameliorate plant diseases, and gibberellin-related protein expression increased in intercropping E. urophylla × E. grandis roots (A0A059A3Y5) but was down-regulated in D. odorifera (TRINITY_DN35143) at our site. Thus, Eucalyptus can improve the adaptability of the stress response when mixed with an NFT, but the NFT showed inhibition. In addition, thioredoxin is involved in plant stress resistance by regulating stress tolerance, and it was up-regulated in E. urophylla × E. grandis roots (A0A059BPT5). Notably, the number of different stress response proteins in D. odorifera was much lower than that in E. urophylla × E. grandis, which indicated that D. odorifera was weakly sensitive to environmental stresses. The thaumatin-like protein abundance was increased in the two species, suggesting that it could significantly improve the antifungal activity in the intercropping system. Alcohol dehydrogenase was up-regulated when plants were under stress and was increased in monoculture E. urophylla × E. grandis (A0A058ZYN2). Importantly, the SOD enzyme, which eliminates the production of harmful substances in metabolism, showed a nonsignificant difference of 1.5-fold in the roots of the two plants. Nevertheless, the above evidence still showed that Eucalyptus resistance was improved in the intercropping system, and the resistance of D. odorifera was hindered. Jasmonic acid (JA) signaling may play an important role in the self-protective responses against opportunistic damage . Our data showed that there was a high level of JA in the intercropping D. odorifera roots, result in increasing the D. odorifera stress resistance through rhizosphere interactions.
Overall, the increased levels of stress-responsive proteins in both species indicated that the intercropping systems provided plants with stronger stress resistance than monoculture systems [33–34], i.e., the advantages of the interaction of E. urophylla × E. grandis in intercropping systems may improve the ecological adaptation compared to monoculture.
N metabolism in the molecular structure represents a major consequence N transfer
The abundance of N metabolized proteins changed in the two species via root interactions, with a low protein abundance of GDH and GS in monocropped E. urophylla × E. grandis, but with a high abundance in monocropped D. odorifera. GS and GDH are key enzymes in the N assimilation and metabolism pathways , and a previous study showed that GS and GDH in transgenic plants improved plant growth and productivity . Our result as well showed that rhizosphere effects promoted N assimilation in E. urophylla × E. grandis roots and productivity but restricted D. odorifera. The amino acid metabolic proteins were found at higher levels in monocropped E. urophylla × E. grandis roots. Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase A (A0A059A9F1) and aspartyl protease AED3 isoform X2 (A0A059AD87) (Table S1) showed higher accumulation in E. urophylla × E. grandis roots in monocropping treatment than that in the intercropping treatment, and the two proteins play a leading role in organelle activity, especially during plant senescence and programmed cell death (PCD) [46–47]. Meanwhile, these proteins also participate in abiotic stress responses . Aminotransferase promotes the decomposition of amino acids and the synthesis of new amino acids through the activity of amino acids, which mediates the level of N metabolism. Our study found higher levels of these proteins in the intercropping system of the two species, i.e., alanine-glyoxylate aminotransferase (A0A059A1E9) and alanine aminotransferase (A0A059AS13) (Table S1) of E. urophylla × E. grandis and acetylornithine aminotransferase (TRINITY_DN44984) (Table S2), putative branched-chain-amino-acid aminotransferase 7 isoform X1 (TRINITY_DN45090), and tryptophan aminotransferase-related protein 4-like (TRINITY_DN39873) of D. odorifera were up-regulated.
In addition, proteins related to N compound transport, which promote the synthesis and transport of N in plants, were only found at a higher abundance in intercropping of E. urophylla × E. grandis but were not found in D. odorifera roots. We believe that the higher abundance proteins of N transport is beneficial to the synthesis and absorption of N for E. urophylla × E. grandis. Most importantly, through the analysis of KEGG pathway, the larger proteins of synthesized functional were found than the metabolic proteome in E. urophylla × E. grandis, while the opposite result was found in D. odorifera, which may be a key signal for the N transfer from D. odorifera to E. urophylla × E. grandis. These data showed that in addition to increased Eucalyptus N assimilation through the intercropping system, as well as N transfer were occurred from the N2-fixing tree to Eucalyptus, as shown in previous studies [3, 15].
Proteins involved in protein metabolism cause by N transfer
Plants adapt to environmental stress by regulating protein metabolism, such as the synthesis of stress-related proteins and cold-shock proteins . There were 40 groups of proteins involved in intercropping E. urophylla × E. grandis roots. We identified 31 (77.5%) down-regulated and 9 (22.5%) up-regulated proteins, whereas 9 groups of down-regulated and 41 groups of up-regulated proteins were detected in D. odorifera roots. Ribosomes, the organelles that catalyze protein synthesis, consist of a small and a large subunit, and these subunits are composed of several RNA species and various structurally distinct proteins, which play a significant role in DNA repair, apoptosis, transcriptional regulation and translational regulation . Many of these proteins showed increased levels in monoculture E. urophylla × E. grandis roots but decreased levels D. odorifera. Moreover, casein kinase 1-like II regulatory subunit is involved in translation, and eukaryotic translation initiation factor isoform X1 is involved in the initiation phase of eukaryotic translation; both of these proteins were increased in the monoculture E. urophylla × E. grandis roots but down-regulated in the monoculture D. odorifera. In our investigation, we believe that the Eucalyptus and N2-fixing species showed an opposite trend in terms of protein metabolic function, which may also be consistent with our hypothesis that the two species show a decreasing trend. The results are highly consistent with previous results, i.e. the physiological metabolism of Eucalyptus was promoted but restricted the growth of N2-fixing species (see Fig. 1) since N transfer was from N2-fixing species to Eucalyptus [3, 15]. Glycine-rich RNA-binding protein (TRINITY_DN41532) was found in intercropping D. odorifera with a higher level than in monoculture,which can respond various plant stresses . The 70-kD heat shock (hsp70) proteins are encoded by a highly conserved multigene family whose proteins function in all major subcellular compartments of the cell, and numerous studies have elucidated the hsp70 chaperone functions under stress conditions and in protein metabolism . These proteins were up-regulated in monoculture E. urophylla × E. grandis roots, but all of these proteins were down-regulated in D. odorifera roots. Bedon et al. (2011) concluded that the protein abundance expression of Eucalyptus can be down-regulated under stress .
N transfer altered proteins involved in the cell wall and cytoskeleton metabolism of both species
The cell wall is an important structure that determines cell shape and acts as a critical barrier against pathogens . For example, expansive proteins are a group of cellular structural proteins that relax the noncovalent interaction between components of the plant cell wall and play a defensive role . These proteins were expressed at higher levels in intercropping E. urophylla × E. grandis roots than in the monoculture system, but the opposite results were observed in D. odorifera. Growing plant cell walls typically extend faster at low pH, and expansive protein activity increases in drought regions . Eucalyptus planting can result in soil acidification , as Eucalyptus consumes large quantities of water, thus increasing the abundance of the expansive proteins to promote defense adaptation. The plant cytoskeleton is a highly dynamic network of proteinaceous components consisting mainly of microtubules and microfilaments, which are involved in coordinating cell structure and signaling , and these components were up-regulated in D. odorifera roots. Sucrose synthase (SuSy) was found in D. odorifera roots (TRINITY_DN40013) and was down-regulated in the intercropping system; this protein is related to cellulose synthesis and increased thickness of cell walls. Gordon et al. (1999) suggested that sucrose metabolism regulates and controls SuSy expression in N2-fixing trees to alter N fixation efficiency . Some studies have also shown an improvement in the N fixation efficiency of N2-fixing species mixed with Eucalyptus relying on N transfer [3, 59]. Therefore, N transfer may directly alter proteins involved in cell wall and cytoskeletal metabolism at our site.
The glycolytic pathway and the TCA cycle was different of both species
The glycolytic pathway degrades sugars to pyruvate. The mitochondrial pyruvate carrier was less abundant in the intercropping of both varieties, which decreased the pyruvate carrier protein on the mitochondrial intima. Pyruvate kinase, the rate-limiting enzyme for transferring the high-energy phosphate from phosphoenol pyruvate to ADP and producing ATP, was found in the D. odorifera roots, and it was more abundant in intercropping compared to monocropping, possibly due to the Fe deficiency in intercropped D. odorifera. The TCA cycle, the key process of the energy cycle and ultimate metabolic pathway for nutrients, includes three key enzymes for citrate synthase (CS): isocitrate dehydrogenase (ITD) and ketoglutarate dehydrogenase alpha (KLDA). CS (A0A059AKY9, A0A059B6K2, A0A059BEH3, A0A059D8E5) was only found in the E. urophylla × E. grandis roots. ATP-citrate synthase alpha chain protein 3 (ACLA-3) (A0A059BEH3) abundance was down-regulated in the intercropping system. ACLA-1 (A0A059D8E5) was increased in the intercropping treatment, which reduced the synthetic accumulation of chlorophyll and carotene in plants to retard plant dwarfing and senescence. It is largely due to the increase N content by N transfer from aodorifera to E. urophylla × E. grandis. NADP-dependent malic enzymes catalyze the oxidation decarboxylation of malic acid pyruvate, participate in the glycolytic pathway and TCA cycle, and play a defensive role in plants . Our study found the NADP-dependent malic enzyme isoform X1 (A0A059BVK1) in the E. urophylla × E. grandis roots and was higher in intercropping than that in monoculture. Nevertheless, aconitate hydratase is the key enzyme responsible for the conversion of citrate into isocitrate in the TCA cycle, and enolase is the enzyme responsible for catalyzing the reversible dehydration of 2-phospho-D-glycerate into phosphoenolpyruvate as part of the glycolytic and gluconeogenesis pathways , but there were no significant difference in these two enzymes of both varieties between the intercropping and monocropping treatments.