ABSTRACT
ABSTRACT
Deployment of the root system is highly sensitive to the levels and spatial distribution of nutrients like nitrogen. However, the genetic determinants of these sensing and deployment mechanisms are still poorly understood. Previously, using system approaches based on temporal changes in root transcriptome in relation to low nitrogen (LN), we have been able to identify a module that activates root production in poplar in response to LN conditions. Here, using comparative, gene ontology and expression analyses, we provide further evidence that the genes in this module are indeed involved in regulation of root development under LN. Better understanding of these modules will enable approaches for breeding for better nitrogen use efficiency through development of a more sensitive and plastic root system.
Addendum
In our recent paper, using time-series transcriptomic data, we have constructed a genetic network that encompasses ∼50% of the differentially-regulated-by-LN root transcriptome.1 The network displayed a 3-level organization and was centered on 11 genes, which we called superhubs, because they were exclusively connected to only hub genes.1 The eleven superhubs were further classified into 3 groups (A, B and C) based on the temporal changes in their expression in response to LN.1 We transgenically manipulated each of the 11 superhubs and found that 3 affect lateral root (LR) development under LN; the first encodes an F-box protein with homology to the Arabidopsis Hawaiian Skirt (HWS) and the 2 other genes encode transcription factors with homology to NAC1 and RAP2.11. As a result, we named the poplar genes PtaHWS, PtaNAC1, and PtaRAP2.111,2 Interestingly, these 3 genes comprised Group C which is characterized by a pattern of initial low expression that increases over time in response to LN.1,2
To better understand the regulatory context of the 3 superhubs, here we further analyzed the putative functions of the connected hubs and the enrichment of Gene Ontologies (GO) of the genes connected to each hub (Table 1). Similar analyses have been already reported for the PtaNAC1 superhub.2,3 Here we focused on the module associated with PtaHWS and PtaRAP2.11. Consistent with involvement in root development, orthologs for many of the hubs connected to PtaHWS and PtaRAP2.11 have been previously shown to be associated with root growth and development. Several have been found to be positive regulators of various aspects of root growth (ExpansinA17/EXPA17,4 Root Hair Defective 3/RHD3,5 Small Auxin Up RNA41/SAUR416) or to be regulated by auxin (SAUR71, SAUR55, SAUR417), the main hormone involved in LR development. Specifically, EXPA17 has been shown to be up-regulated by LBD18 and promote lateral root formation in Arabidopsis.4 RHD3 was initially isolated in Arabidopsis genetic screens of mutant defective in root hair development.5 Overexpression of SAUR41 in Arabidopsis has been shown to promote auxin-related phenotype including lateral root development.6 We further hypothesized that if these hub genes are indeed downstream of the 3 superhubs in the cascade leading to LR growth, their expression would be perturbed in the transgenic plants overexpressing the 2 superhubs. We therefore studied the expression of these genes in the transgenic roots. Consistently, the expression of EXPA17, RHD3 and the 3 SAUR poplar orthologous genes were significantly higher in the transgenics overexpressing PtaHWS or PtaRAP2.1 and this effect was specific for only LN conditions (Fig. 2).
Published online:
22 July 2016Figure 1. Gene regulatory network (GRN) associated with the 3 superhubs. Superhubs are represented square whereas hub and other terminal genes are represented as circle and triangles. Hubs connected to only one superhub are presented in the same color as the corresponding superhub (purple for PtaNAC1, blue for PtaRAP2.11 and orange for PtaHWS). The hubs connected to all the 3 superhubs are presented as green triangle. The hubs connected to both PtaNAC1 and PtaRAP2.11 are presented as green circle whereas those connected to both PtaRAP2.11 and PtaHWS are presented as yellow circles. Abbreviations for the hub genes are provided in Table 1. The gene regulatory network is constructed based on previously-described transcription profiling or co-expression data of poplar roots grown under normal and LN conditions using the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE)1,2. The the expression profiles of 9,198 differentially expressed genes (DEGs) of the 6 time-point data as well as an additional list of 424 differentially expressed transcription factors were used as input for the ARACNE.2 Rank product was used for identifying DEGs.1
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kpsb20/2016/kpsb20.v011.i08/15592324.2016.1214792/20161222/images/medium/kpsb_a_1214792_f0001_oc.gif"})
Figure 1. Gene regulatory network (GRN) associated with the 3 superhubs. Superhubs are represented square whereas hub and other terminal genes are represented as circle and triangles. Hubs connected to only one superhub are presented in the same color as the corresponding superhub (purple for PtaNAC1, blue for PtaRAP2.11 and orange for PtaHWS). The hubs connected to all the 3 superhubs are presented as green triangle. The hubs connected to both PtaNAC1 and PtaRAP2.11 are presented as green circle whereas those connected to both PtaRAP2.11 and PtaHWS are presented as yellow circles. Abbreviations for the hub genes are provided in Table 1. The gene regulatory network is constructed based on previously-described transcription profiling or co-expression data of poplar roots grown under normal and LN conditions using the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE)1,2. The the expression profiles of 9,198 differentially expressed genes (DEGs) of the 6 time-point data as well as an additional list of 424 differentially expressed transcription factors were used as input for the ARACNE.2 Rank product was used for identifying DEGs.1
Published online:
22 July 2016Figure 2. Effect of PtaRAP2.11 and PtaHWS overexpression on the expression of connected hubs under normal and LN conditions. Expression was analyzed in root of WT-717, oe-PtaRAP2.11 and oe-PtaHWS plants after 40 d under control and LN. Primers used for the above hub genes are provided in Table S1. oe = overexpression using Camv35S promoter. Values show mean ± standard error of the mean (SEM) (n = 3). Asterisk represent significant difference between WT-717 and transgenic for a given treatment (P < 0.05) as determined by Student's t-test.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/kpsb20/2016/kpsb20.v011.i08/15592324.2016.1214792/20161222/images/medium/kpsb_a_1214792_f0002_b.gif"})
Figure 2. Effect of PtaRAP2.11 and PtaHWS overexpression on the expression of connected hubs under normal and LN conditions. Expression was analyzed in root of WT-717, oe-PtaRAP2.11 and oe-PtaHWS plants after 40 d under control and LN. Primers used for the above hub genes are provided in Table S1. oe = overexpression using Camv35S promoter. Values show mean ± standard error of the mean (SEM) (n = 3). Asterisk represent significant difference between WT-717 and transgenic for a given treatment (P < 0.05) as determined by Student's t-test.
Table 1. Significantly enriched GO-term associated with the genes terminally connected to hubs associated with PtaHWS and PtaRAP2.11 as shown in Fig. 1. The number of genes connected to each hub is shown in the parenthesis. The agriGO tool (http://bioinfo.cau.edu.cn/agriGO/) was used to perform the enrichment analysis using SEA (Singular Enrichment Analysis) coupled with available background data of Populus trichocarpa genome data (V 3.0). Only the top 5 most significantly enriched GO-terms in each regulatory module associated with each hub gene are shown. The GO-term associated with hub genes connected to PtaNAC1 has previously been reported (Wei et al., 2013, Fig. 5).
In contrast, RALF1 has been shown to be a negative regulator of lateral root formation, cell elongation as well as cell expansion-related genes.8,9 The expression of the poplar RALF1 ortholog was decreased in the transgenics, again under LN conditions (Fig. 2).
In addition to root development, other hubs were associated with other processes like nitrogen assimilation (NIA210) and C/N metabolism (PhosphoenolPyruvate Carboxylase Kinase 1/PPCK111), suggesting of a broader regulatory context of the module and/or coordination of root development with other processes.
Ontology analysis of the genes connected to the various hubs corresponded to variety of processes (Table 1). However, they again included many ontologies directly or indirectly linked to root development, like root growth, hormone signaling, nitrogen metabolism and gene expression (Table 1). The above comparative, expression and ontology analyses further support the role of the 3 superhubs and the associated network in LR proliferation in response to LN. Further characterization of this novel network will enable breeding approaches for increased nitrogen use efficiency in agricultural and bioenergy crops.
| Hubs | GO Ontology | Corrected p-values |
|---|---|---|
| Protein Kinase (PK) (59 Genes) | GO:0006468: protein amino acid phosphorylation | 2.10E-06 |
| GO:0016310: phosphorylation | 4.00E-06 | |
| GO:0006464: protein modification process | 4.20E-06 | |
| GO:0005975: carbohydrate metabolic process | 4.20E-06 | |
| GO:0016301: kinase activity | 5.30E-07 | |
| Small Auxin Up-Regulated 55 (SAUR55) (169 Genes) | GO:0030001: metal ion transport | 2.80E-05 |
| GO:0005975: carbohydrate metabolic process | 2.80E-05 | |
| GO:0009725: response to hormone stimulus | 8.60E-04 | |
| GO:0045892: negative regulation of transcription, DNA-dependent | 8.60E-04 | |
| GO:0000023: maltose metabolic process | 1.70E-03 | |
| Small and basic Intrinsic Protein 2.1 (SIP2.1) (89 Genes) | GO:0009607: response to biotic stimulus | 2.30E-03 |
| GO:0009414: response to water deprivation | 3.40E-03 | |
| GO:0006807: nitrogen compound metabolic process | 3.40E-03 | |
| GO:0043169: cation binding | 3.90E-03 | |
| GO:0010504: regulation of cell cycle arrest in response to nitrogen starvation | 3.90E-03 | |
| Root Hair Defective3 (RHD3) (114 Genes) | GO:0031326: regulation of cellular biosynthetic process | 6.70E-03 |
| GO:0010468: regulation of gene expression | 6.70E-03 | |
| GO:0010015: root morphogenesis | 6.70E-03 | |
| GO:0006807: nitrogen compound metabolic process | 6.70E-03 | |
| GO:0003700:transcription factor activity | 7.00E-03 | |
| Small Auxin Upregulated 71 (SAUR71) (64 Genes) | GO:0019222: regulation of metabolic process | 2.20E-04 |
| GO:0009733: response to auxin stimulus | 2.00E-04 | |
| GO:0048364: root development | 1.32E-03 | |
| GO:0051171: regulation of nitrogen compound metabolic process | 2.20E-03 | |
| GO:0006350: transcription | 2.20E-03 | |
| Small Auxin Up-Regulated 41 (SAUR41) (113 Genes) | GO:0031326: regulation of cellular biosynthetic process | 2.50E-05 |
| GO:0006807: nitrogen compound metabolic process | 3.00E-05 | |
| GO:0010468: regulation of gene expression | 5.20E-05 | |
| GO:0019219: regulation of nucleic acid metabolic process | 2.00E-05 | |
| GO:0009734: auxin mediated signaling pathway | 1.40E-04 | |
| Rapid Alkalinization Factor 1 (RALF1) (93 Genes) | GO:0005262: calcium channel activity | 6.70E-04 |
| GO:0051716: cellular response to stimulus | 5.90E-04 | |
| GO:0048528: lateral root development | 4.10E-04 | |
| GO:0009791: post-embryonic development | 3.30E-03 | |
| GO:0051171: regulation of nitrogen compound metabolic process | 3.90E-03 | |
| Protein Kinase Like (PKL) (93 Genes) | GO:0003824: catalytic activity | 1.20E-04 |
| GO:0051252: regulation of RNA metabolic process | 7.10E-04 | |
| GO:0031326: regulation of cellular biosynthetic process | 6.10E-03 | |
| GO:0051171: regulation of nitrogen compound metabolic process | 6.30E-03 | |
| GO:0006355: regulation of transcription, DNA-dependent | 6.60E-03 | |
| Serine/Threonine/Tyrosine Kinase 17 (STY17) (85 Genes) | GO:0009534: chloroplast thylakoid | 2.00E-04 |
| GO:0005975: carbohydrate metabolic process | 1.10E-03 | |
| GO:0009414: cellular response to stimulus | 4.70E-03 | |
| GO:0051716: response to light stimulus | 6.20E-03 | |
| GO:0048731: system development | 3.00E-02 |
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References
- Dash M, Yordanov YS, Georgieva T, Kumari S, Wei H, Busov V. A systems biology approach identifies new regulators of poplar root development under low nitrogen. Plant J 2015; 84:335-46; PMID:26315649; http://dx.doi.org/10.1111/tpj.13002 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Wei HR, Yordanov YS, Georgieva T, Li X, Busov V. Nitrogen deprivation promotes Populus root growth through global transcriptome reprogramming and activation of hierarchical genetic networks. New Phytol 2013; 200:483-97; PMID:23795675; http://dx.doi.org/10.1111/nph.12375 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Wei H, Yordanov YS, Kumari S, Georgieva T, Busov V. Genetic networks involved in poplar root response to low nitrogen. Plant Signal Behav 2013; 8:483-97; PMID:24300216; http://dx.doi.org/10.4161/psb.27211 [Taylor & Francis Online], [Google Scholar]
- Lee HW, Kim J. EXPANSINA17 up-regulated by LBD18/ASL20 promotes lateral root formation during the auxin response. Plant Cell Physiol 2013:pct105; PMID:23872272; http://dx.doi.org/10.1093/pcp/pct105 [Crossref], [Web of Science ®], [Google Scholar]
- Yuen CY, Sedbrook JC, Perrin RM, Carroll KL, Masson PH. Loss-of-function mutations of ROOT HAIR DEFECTIVE3 suppress root waving, skewing, and epidermal cell file rotation in Arabidopsis. Plant Physiol 2005; 138:701-14; PMID:15908600; http://dx.doi.org/10.1104/pp.105.059774 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Kong Y, Zhu Y, Gao C, She W, Lin W, Chen Y, Han N, Bian H, Zhu M, Wang J. Tissue-specific expression of SMALL AUXIN UP RNA41 differentially regulates cell expansion and root meristem patterning in Arabidopsis. Plant Cell Physiol 2013; 54:609-21; PMID: 23396598; http://dx.doi.org/10.1093/pcp/pct028 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Jain M, Tyagi AK, Khurana JP. Genome-wide analysis, evolutionary expansion, and expression of early auxin-responsive SAUR gene family in rice (Oryza sativa). Genomics 2006; 88:360-71; PMID:16707243; http://dx.doi.org/10.1016/j.ygeno.2006.04.008 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Bergonci T, Ribeiro B, Ceciliato PH, Guerrero-Abad JC, Silva-Filho MC, Moura DS. Arabidopsis thaliana RALF1 opposes brassinosteroid effects on root cell elongation and lateral root formation. J Exp Bot 2014; 65:2219-30; PMID:24620000; http://dx.doi.org/10.1093/jxb/eru099 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Bergonci T, Silva-Filho MC, Moura DS. Antagonistic relationship between AtRALF1and brassinosteroid regulates cellexpansion-related genes. Plant Signal Behav 2014; 9:e976146; PMID:25482784; http://dx.doi.org/10.4161/15592324.2014.976146 [Taylor & Francis Online], [Google Scholar]
- Caboche M, Rouzé P. Nitrate reductase: a target for molecular and cellular studies in higher plants. Trends Genetics 1990; 6:187-91; PMID:2196722; http://dx.doi.org/10.1016/0168-9525(90)90175-6 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Walker RP, Chen Z-H, Técsi LI, Famiani F, Lea PJ, Leegood RC. Phosphoenolpyruvate carboxykinase plays a role in interactions of carbon and nitrogen metabolism during grape seed development. Planta 1999; 210:9-18; PMID:10592027; http://dx.doi.org/10.1007/s004250050648 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]