Based on our previous reports, the current results provide further evidence that CmANR1 has positive effects on root development in chrysanthemum. The CmANR1-transgenic plants have more robust root systems, such as longer and a greater number of LRs, ARs and root hairs, as well as a larger root system surface and volume, in comparison to WT plants (Fig. 1). We have observed and measured the root growth and developmental parameters of several periods of both transgenic and WT chrysanthemum (10-day-old tissue culture, 20-day-old tissue culture, and 40-day-old hydroponic culture), and it seemed that CmANR1 could respond quickly to the higher concentration of nitrate (10 mM, under the culture condition) and its positive role in root development was obvious at the initial stage of growth, which might continue into the later growth and development in transgenic plants. On the other hand, in this study, we confirmed that CmANR1-transgenic plants grew much higher, were greener, had better photosynthetic performance than those of WT plants under the tissue-cultured conditions (Fig. 6). As CmANR1-overexpression in the transgenic plants was triggered by a 35S promoter in our study, therefore, we could not determine whether the growth advantages of the transgenic plants on the shoot were due to the direct regulation of CmANR1 on other molecular players in shoots or due to the indirect stimulative effect of more nutrients and water uptake by the more extensive root system of transgenic plants. More work is required to determine the possible mechanism underlying the root-shoot interaction in chrysanthemum.
Previous studies have reported that ANR1 is a rapid nitrate-responsive and nitrate-dependent transcription factor gene [14, 28]. CmANR1 can act as a transcriptional activator of an auxin transport gene, thereby leading to auxin accumulation in roots during the nitrate signaling pathway in chrysanthemum . Meanwhile, the CmANR1-overexpression conferred more nitrate uptake and more nitrate-contained in the roots of the transgenic plants (Fig. 3a). Apart from being as a signaling molecule, nitrate has its basic nutritional role as the main nitrogen source for most land plants, and it will be taken up, translocated, assimilated and transitioned into other physiological compounds (such as amino acids) at the cellular level in the plants . In our root metabolomics assay, it was clear that there were significant increases in the metabolites that were involved in various amino acid metabolisms in transgenic plants, such as L-histidine, L-pyroglutamic acid, 4-guanidinobutyric acid, and L-phenylalanine (Table 1). These findings suggest that CmANR1 may have some influence on nitrate assimilation during root development under nitrate-rich conditions in chrysanthemum.
The TCA, well-known as an important central pathway connecting almost all metabolic pathways in plants, is responsible for the oxidation of respiratory substrates to drive ATP synthesis [38, 39]. Glycolysis, an oxidization process responsible for the conversion of glucose to pyruvic acid, is another ubiquitous cellular metabolism. Modifications of the expression of important kinases and mutases, such as hexokinase, pyruvate kinase, and phosphoglycerate mutase, could result in dramatic effects on photosynthetic metabolism . Notably, metatranscriptomics analysis of roots showed DEGs and metabolites related to amino sugar and nucleotide sugar metabolism were mostly enriched. The detailed expression level manifested by fold changes of the genes being involved in glycolysis and TCA cycle were clearly illustrated. The result shows that the expressions of genes encoding hexokinase (TR12735|C1-g1, 1.119), pyruvate kinase (TR99|C0-g3, -1.075), and phosphoglycerate mutase-like (TR15403|C0-g1, 1.585) were significantly altered in transgenic plants (Fig. 4c, d). Moreover, the significant changes in the content of some respiration substrates, such as citrate, L-malic acid, maltotriose, and D-mannose, suggest a possible energy enhancement in the roots of transgenic plants, which might be another explanation for their better root system development. In conclusion, this evidence hints that there is a link between sugar metabolism and root development that is mediated by CmANR1.
Moreover, there were some other interesting metabolites accumulated in transgenic plants. First, choline was one of the most abundant metabolites in both roots and shoots of transgenic plants. Choline, as the primary source for acetyl choline (ACH), is also related to glycine, serine and threonine metabolism, and synthesis of glycine betaine (GlyBet) in the chloroplast . ACH and GlyBet are essential for various important physiological processes and tolerance to abiotic stresses in plants [42, 43] .The increase in the content of choline gave some information on the possible amino acid transition enhancement and stresses-resistance improvement in transgenic plants, which provided a new idea for the future exploration. Second, rhoifolin, as one of the important flavonoids, has various significant biological activities, including anti-allergic, anti-inflammatory, antioxidant, antimicrobial, and anticancer effects . In our shoot metabolomics assay, the content of rhoifolin showed a significant increase in transgenic plants, suggesting the possible improvement of the pharmacological value of on transgenic plants. Also, some metabolites that were closely related to the biosynthesis of unsaturated fatty acids and fatty acids, such as alpha-Linolenic acid, (4Z, 7Z, 10Z, 13Z, 16Z, 19Z)-4, 7, 10, 13, 16, 19-Docosahexaenoic acid, and cis-9-Palmitoleic acid, were found significantly increased in shoots of transgenic plants, suggesting the improvement of cold and disease resistance in transgenic plants.
In conclusion, we now present a working model of CmANR1 on plant development (Fig. 8). In roots: CmANR1 senses the higher nitrate signal passed by the sensor NRT1.1, which is located on the plasma membrane in roots. Then, CmANR1 formed a homodimer with another CmANR1, and a heterodimer with AGL21, trans-activating downstream target genes, such as auxin-related genes , sugar metabolism genes and nitrate responsive genes, resulting in changes in the related physiological processes in plants. The final increases of auxin , AAs, metabolites related to glycolysis, and TCA cycle in roots (Table 1) promoted root system development in chrysanthemum. Moreover, TF expression analysis suggested some TFs, such as NLPs, LBDs, and other MADS-box TFs, were more likely to act together with CmANR1 on root system development in this background (Fig. 5). Therefore, it can be concluded that CmANR1 promoted root system development in chrysanthemum by combining the nitrate signaling pathway with auxin transport, nitrate assimilation, as well as glycolysis and the TCA cycle.
In shoots, the significant increases in shoot height, chlorophyll content, and three photosynthesis parameters (Pn, ETR, and ΦPSII) in transgenic plants indicated a more robust growth and better photosynthesis performance in the shoots of transgenic plants (Fig. 6). However, whether CmANR1 directly exerted some effects on shoot development could not yet be determined. At the minimum, overexpression of CmANR1 could trigger shoot development. Taken together, over-expressing CmANR1 could prompt both root and shoot development in chrysanthemum.