Nitrogenmetabolismgenesareresponsible forvarious N levels and auxin
Nitrogen and auxin significantly affect LRs development via N signaling, and regulate biosynthesis and transport of hormones such as ABA, GA and IAA. In previous studies, N-deficiency induced high affinity nitrate transporter NRT1.1, NRT2.1, NRT2.4 and NRT2.5 in roots of Arabidopsis plant. It was also reported that NRT2.1, NRT2.2, NRT2.4 and NRT2.5 could synergistically confer to plants ability to adapt to low N condition. NRT1 and NRT2 are not only for NO3- transportation but also for auxin transportation under low nitrogen condition. When plants are subjected to low nitrogen, NRT1 and NRT2 have high expression and induce auxin accumulation and transportation for regulating the LRs formation. Similarly, in this study, we found that two common DEGs (CSA011051; MSTRG.51865) NRT2.4 andNRT2.5 were identified from LRs under all four treatments. NRT2.4 and NRT2.5 were up-regulated and down-regulated in the LRs of seedlings under LN and LN + NPA treatments, respectively. And from the results of hormone determination, the N-deficiency could increase the content of auxin in lateral roots. Therefore, LN treatment could induce NRTs expression and thus increase auxin production and accumulation. And they could be regulated by nitrogen concentration and auxin treatment contributing to tea plant LRs formation.
Auxin and nitrogen signaling could control LR development. Tryptophan (Trp) aminotransferase of Arabidopsis1 (TAA1) is an important enzyme which plays important function in Trp transformation in to indole-3-pyruvic acid (IPyA), an auxin biosynthesis pathway (IPyApathway). Recent studies have shown that TAR regulates plant roots and shoots development: Tryptophan aminotransferase related genes (TAR1-4) were involved in IPyA pathway, while tryptophan aminotransferase related 2 (TAR2) gene was induced by N deficiency that can improve auxin biosynthesis in Arabidopsis, and increase IAA levels in LRs development[33, 34]. TAR2 is desired to accumulate auxin in LRs under low nitrogen condition. In the present study, one tryptophan aminotransferase related 2 gene (CSA015778) was down-regulated under HN + IBA treatment, indicating that LN treatment could induce TAR2 expression for auxin accumulation. However, under excessive exogenous auxin treatment, TAR2 would be down-regulated to balance LRs auxin concentration. The Tryptophan aminotransferase related 4 (TAR4) gene was up-regulated under low nitrogen condition in the shoot of Arabidopsis. Similar observation was present in this study as TAR4 (CSA001598) was up-regulated in LRs under LN treatment. Therefore, we speculate that TAR4 also participate in LRs development under low nitrogen condition. The auxin concentration increased as nitrogen level decreased in LRs of seedlings. In summary, the tea plant LRs formation could be induced by low nitrogen concentration via auxin biosynthesis and accumulation.
Arabidopsis has six AMT-type ammonium transporters including AMT1.1 to AMT1.5 and AMT2.1. Previous reports demonstrated that ammonium supply can induce the LRs initiation and branching in Arabidopsis, this may because that ammonium is complementary to nitrate in regulating LRs development, which happens in AMT-dependent manners. Ruan et al. (2016) reported that ammonia improves nitrate uptaking in tea roots, however, the present study revealed that AMT genes expression is diametrically opposite to NRT genes in LN and HN + IBA treatment. Among the six AMT-type ammonium transporters identified in the model plant Arabidopsis, AMT1.4 was expressed in the pollen, but AMT1.4 (CSA018499) was identified to express in tea plant roots in the present study. It was down-regulated in LRs under LN and HN + IBA treatments, and up-regulated in LN + NPA treatment. We concluded that AMT genes were down-regulated under low nitrogen condition and auxin treatment, and their expressions may inhibit LRs formation. In addition, the expression analysis of NRT, AMT. and NPF family genes revealed that the synergistic relationship of auxin and nitrogen signaling on LR development does exist. These genes contribute to N utilization efficiency exploration and provide gene reference for selection of high nitrogen-efficient varieties of tea plants. This can also contribute to the exploration of regulation of LRs formation through regulating nitrogen and auxin signal pathway.
Plant hormone metabolism and signaling pathways
The plant hormone, auxin is critical for plant growth and development processes, and plays its regulatory role mainly by inducing expression of early auxin response genes including Aux/IAA, GH3 and SAUR. GH3 can combine free auxin with disparate acid; therefore, overexpression of GH3 would result in expression of severe auxin-deficient phenotypes[38, 39]. In the present study, one GH3 gene was up-regulated under LN treatment but downregulated under LN + NPA treatment, while the HN + IBA treatment regulated GH3genes. In addition, Aux/IAA proteins play the role of transcriptional repressors by heterodimerizing with auxin response factor (ARF) transcription factors, while ARF family acts as key regulator of root development[40, 41]. At low auxin concentration, Aux/IAA proteins could restrain transcriptional activation of ARF proteins; thereby, preventing response genes transcription of auxin. But higher auxin concentration could induce ARF genes expression and then promote LRs development. Under LN treatment, the Aux/IAA proteins genes (CSA031541, MSTRG.7473) were down-regulated, while auxin response factor 2 (ARF2) gene (CSA011327) was up-regulated. This; therefore, shows that in LN condition, the LRs’ auxin concentration would be increased, and then inhibit Aux/IAA genes expression and improve ARF genes expression to induce LRs development. Moreover, under LN + NPA treatment, the ARF2 gene (CSA011327) was down-regulated, while ARF2 gene (CSA012843) was up-regulated under HN + IBA treatment. This; therefore, indicates that low nitrogen and auxin treatments could improve ARFs expression and enhance LRs formation.
Cytokinin is important for plants proliferation, plants cell division, secondary metabolism, and regulation of plants shoot and roots development. Other researchers have shown that some nitrogen signals are substituted by cytokinins as local and long-distance signal, and; thus, various genes were regulated by these plant hormones, including metabolism, growth development and nutrient absorption. High cytokinin content is reported to improve shoot development while high auxin content enhances root formation. Therefore, there exist an important signaling pathway among nitrogen, auxin and cytokinin in the regulation of plant development. The present study identified DEGs involved in cytokinin metabolism. Under LN treatment, one adenylate isopentenyltransferase (CAS006753) gene which induce cytokinin biosynthesis was down-regulated, while two cytokinin dehydrogenase genes which inhibit cytokinin biosynthesis were up-regulated. Under LN + NPA treatment, one cytokinin dehydrogenase gene (CAS011288) was down-regulated, but expressed in LN treatment. Under HN + IBA treatment, cytokinin hydroxylases gene (CAS017731) which induce cytokinin biosynthesis was down-regulated, while 7 cytokinin dehydrogenase genes were up-regulated. RP-UPLC technique revealed that cytokinin concentration increased with increasing nitrogen concentration and decreased with exogenous auxin treatment. It suggests that LN treatment and auxin treatment could inhibit cytokinin biosynthesis, while high auxin condition induces LRs formation in tea plants.
Ethylene is connected to plant’s physiological and morphological responses to nitrogen deficiency, and nitrate transporters NRT1.1 and NRT2.1are also sensitive to ethylene[45, 46]. Under low external nitrate concentration, NRT2.1 induces and promotes ethylene biosynthesis and signaling activity. Auxin and ethylene signaling pathways show specific regulation of plants growth and development, such as root elongation and root hair formation. Studies also suggest that ethylene might stimulate localized auxin biosynthesis. In the present study, ethylene biosynthesis varied with different levels of nitrogen treatment. Under LN, ethylene biosynthesis genes were up-regulated, but down-regulated under HN treatment. Under LN + NPA treatment, 3 ethylene biosynthesis genes were down-regulated while 14 genes were up-regulated under HN + IBA treatment. This; therefore, suggests that LN treatment improves ethylene biosynthesis; thereby, promoting auxin response genes expression and LRs formation, while HN treatment down-regulates ethylene biosynthesis; thus, inhibits LRs formation. Comparison between HN + IBA and LN + NPA treatments revealed that auxin could promote ethylene biosynthesis and stimulate LRs formation. These results; therefore, clearly indicate that auxin and nitrogen could regulate tea plant LRs formation through ethylene biosynthesis pathway.
Transcription factors (TFs) can control the expression of stress resistance genes. Many TF families such as NAC, MYB, MADS-box and WRKY have been explored, and these families can regulate cell division and expansion, lateral root development and secondary cell wall biosynthesis. Several TFs have been expressed in plants exposed to N-deficient situations[52, 53].
R2R3-MYB is reported to be the most abundant MYB protein. MYBs46/83 is speculated to be the prime regulator of secondary cell wall biosynthesis, while AtMYB58 specifically activate lignin biosynthesis, as regulated by AtMYB46. Therefore, MYBs expression would thicken the secondary cell wall and inhabit cells division and elongation[55, 56]. In the present study, the P-type R2R3 MYB protein (MYB83) homologous gene was down-regulated under LN and HN + IBA treatment, but up-regulated with NPA treatment. This; therefore, indicates that LN treatment could inhibit MYB genes expression and restrain secondary cell wall biosynthesis, and; thus, regulate roots development. Similar inference can be made with auxin treatment. This also confirms the hypothesis that accumulation of auxin can be promoted under low nitrogen conditions in LRs of tea plant.
NAC transcription factors are important for plant growth as they could regulate plants cell division, lateral root development and secondary cell wall biosynthesis[57, 58]. A few NAC genes have been identified as key and effective regulation factor in auxin signaling pathway which directly affect LRs development[59, 60]. In the present study, 7 NAC DEGs were expressed under LN treatment,; 43 NAC DEGs were expressed under HN + IBA treatment and most of them were up-regulated. Similarly, DEGs of NAC also up-regulated under LN + NPA treatment. Consistent with previous reports, the present study; therefore, observed that NAC genes could be induced by nitrogen treatment and auxin treatment to regulate tea plants LRs formation; however, the detail signaling pathway still needs further exploration.
MADS-box TFs control plants root, flower and fruit development[61, 62]. Previous study has shown that AGL21 is induced by N-deprivation, and auxin also promotes AGL21, while AGL21 proteins interact with ANR1 (AGL44) to mediate LRs development[63, 64]. In the present study, 12 MADS-box protein genes were down-regulated under LN treatment. Under HN + IBA treatment, there are 19 DEGs. Also, AGL genes were up-regulated under LN + NPA treatment. Thus, it is tempting to suggest that in tea plant, the MADS-box protein genes expression might differ from that of Arabidopsis.
WRKY transcription factors could be involved in various plant developmental processes, such as biological and abiotic stresses, and seed germination and dormancy. WRKY TF is a major player in plant's innate immune system. Beet cyst nematode is reported to regulate WRKY TF genes expression to enhance roots development in Arabidopsis. In a previous study, WRKY TF families were induced under N-deficient condition. In the current study, WRKY TF family genes were induced by various levels of nitrogen treatments (Table S2). This reveals that WRKY TF family genes could be significantly induced by auxin signaling, and; thus, take part in nitrogen metabolism under various nitrogen conditions. It can also be deduced that TFs play important roles in nitrogen and auxin network in tea plant LRs formation. It is; therefore, important to explore the involvement of WRKY TF family gene via nitrogen and auxin signaling pathway in LRs formation in tea plant.
KEGG analysis revealed a significant change in glutathione metabolism in all treatments. GSH-dependent developmental pathway induces and sustains cell division during root development, and regulates auxin transport and evolution[67, 68]. Glutathione also acts as thiol/disulfide buffer. It can regulate the balance between GSH (reduced form) and GSSG (oxidized form) by GSH oxidation through reactive oxygen species, and GSSG reduction through glutathione reductase68. Exogenous GSSG could not induce roots in normal conditions but promote root development under auxin treatment; therefore, auxin and GSSG interaction would regulate plants roots development. It is reported that the reduction ratio of GSH/GSSG inhibits lateral roots in the presence of auxin.Glutathione S-transferases (GSTs) transforms GSH to GSSG, while glutathione reductase (GR) induces the reduction of GSSG into GSH. In the present study, 8 GSTs genes were up-regulated and 12 GST genes were down-regulated with LN treatment. Under HN condition, 14 GSTs genes were down-regulated, while 5 GSTs genes were up-regulated. IBA treatment up-regulated 5 glutathione reductase genes and 65 GSTs genes. Finally, 37 GSTs genes were up-regulated under NPA treatment. Nitrogen and auxin treatments had many DEGs in glutathione metabolism, both of them could affect GSH/GSSG ratio to regulate LRs formation in tea plant; however, the specific adjustment mechanism is still vague and requires further research.
F-box proteins are important ingredient of proteasome pathway and participate in cellular functions, such as auxin receptor (TIR1), which mediates transcriptional response to auxin in a F-box protein. Many researches have shown that adventitious roots formation accompanies soluble and insoluble carbohydrates accumulation, and the At1g23390 is shown to be related to such metabolism[73, 74]. When tea plants were treatment with IBA, a F-box/kelch gene similar to AT1g23390 was identified and this suggests a complex regulatory network during adventitious roots development. In the current study, AT1g23390 (CAS036587) was up-regulated by HN + IBA treatment, and its homogenous gene was down-regulated by LN + NPA treatment but up-regulated by LN treatment. It; therefore, depicts that this gene could be induced by nitrogen treatment, and its function was similar with auxin treatment in root development. It also indicates that LN treatment could induce auxin production in tea plant LRs. Hence, the putative role of this gene might be to regulate LRs formation through N and auxin signaling pathway in tea plants.