Analysis of Gene Regulatory Networks of Maize in Response to Nitrogen

Abstract

Nitrogen (N) fertilizer has a major influence on the yield and quality. Understanding and optimising the response of crop plants to nitrogen fertilizer usage is of central importance in enhancing food security and agricultural sustainability. In this study, the analysis of gene regulatory networks reveals multiple genes and biological processes in response to N. Two microarray studies have been used to infer components of the nitrogen-response network. Since they used different array technologies, a map linking the two probe sets to the maize B73 reference genome has been generated to allow comparison. Putative Arabidopsis homologues of maize genes were used to query the Biological General Repository for Interaction Datasets (BioGRID) network, which yielded the potential involvement of three transcription factors (TFs) (GLK5, MADS64 and bZIP108) and a Calcium-dependent protein kinase. An Artificial Neural Network was used to identify influential genes and retrieved bZIP108 and WRKY36 as significant TFs in both microarray studies, along with genes for Asparagine Synthetase, a dual-specific protein kinase and a protein phosphatase. The output from one study also suggested roles for microRNA (miRNA) 399b and Nin-like Protein 15 (NLP15). Co-expression-network analysis of TFs with closely related profiles to known Nitrate-responsive genes identified GLK5, GLK8 and NLP15 as candidate regulators of genes repressed under low Nitrogen conditions, while bZIP108 might play a role in gene activation.

Keywords: maize; network analysis; nitrogen response genes; regulatory network inference.

Figure 1

A Venn diagram of differential expressed genes and their common genes across two microarray datasets.

Figure 2

Functional categorization of differential expressed genes in common between two microarray datasets. The probe set sequences were mapped to the UniRef protein database and then subsequently to KEGG function categories, as described in the text. KEGG: Kyoto Encyclopedia of Genes and Genomes.

Figure 3

The known network of maize N response genes from Study 1. (A) The Biological General Repository for Interaction Datasets (BioGRID) network with maize N response genes: 108 genes with 97 interactions retrieved. Nodes in white represent genes only mapped in Arabidopsis; nodes in yellow represent genes with no expression difference; nodes in blue represent down-regulated genes; and nodes in red represent up-regulated genes. (B) The IPA network with N response genes. The grey nodes are from the input set of nine genes.

Figure 4

Expression patterns of three common genes selected from artificial neural network (ANN) across chips comparison. (A) Gene expression date from Study 1 [24], (B) Gene expression date from Study 2 [25]. Note: LN: low nitrogen; SN: sufficient nitrogen; Recovery: recovery from low N to SN.

Figure 5

Top 200 interactions from all genes by ANN analysis. (A) All genes network with Top 200 interactions in Study 1. Nodes are coloured according to gene function: amino acid related in pink; carbon related in green; nitrogen related in red; phosphate related in purple; regulator and transcription factors (TFs) in yellow; stress related in brown; and others in white. (B) Extracted subnetwork in Study 1. AtRL1 interacted with other TFs such as RING-H2, EREBP and cysteine proteinase. (C) All genes network with Top 200 interactions. The nodes highlighted by red circle represent the three common genes: ZmASN4, dual specific kinase and Ser/Thr protein phosphatase. (D) Extracted sub-network with two central nodes: NLP15 and miR-399b. NLP15 and GLK5 were significantly down-regulated TFs under low N treatment.

Figure 6

Top 200 interactions from TFs by ANN analysis (Study 1). All nodes are coloured based on fold change value (FC > 2): blue represents down-regulated, while red represents up-regulated. Edge with red colour indicates a positive interaction (stimulation), while edge with blue colour indicates a negative interaction (inhibition). The edge thickness based on Z-score value. (A) All TFs network with Top 200 interactions. Eighty-one TFs included 11 up-regulated and 13 down-regulated TFs. (B) Extracted subnetwork with GRMZM2G132628 as core hub surrounded with 30 positive interactions. (C) Extracted subnetwork with ZmGLK5 connected with four differentially expressed genes: ZmHB54, ZmbHLH121 and two TFs (GRMZM5G825312 and GRMZM5G868618).

Figure 7

Top 200 interactions from TFs by ANN analysis (Study 2). All nodes are coloured by expression fold change value (FC > 2), blue represents down-regulated, while red represents up-regulated. Edge with red colour indicates a positive interaction (stimulation), while edge with blue colour indicates a negative interaction (inhibition). The edge thickness based on Z-score value.

Figure 8

Sub-network of ZmASN4 interacted with its 13 neighbours. Note: CLC-a: chloride channel protein-a; EREBP: ethylene-responsive element binding protein; PEPC: phosphoenolpyruvate carboxylase; SUS: sucrose synthetase; TPS: terpene synthetase; ZmASN4: asparagine synthetase; ZmGLK5: G2-like 5 transcription factor; ZmGLK8: G2-like 8 transcription factor. Blue represents down-regulated gene.

Figure 9

Subnetwork associated with N response genes (A); and their expression pattern (B) in maize leaves. 12A: Subnetwork with 14 N response genes was implemented in Cytoscape with hierarchic layout; 12B: Expression pattern of these N response genes under low N, sufficient N and N recovery. Note: LN: low nitrogen; SN: sufficient nitrogen; recovery: recovery from low N to SN; AAP8: Amino acid permease 8; bHLH: basic helix-loop-helix protein; CLC-a: chloride channel protein-a; SUS: sucrose synthetase; UKL2: Uridine kinase-like protein 2; ZmASN4: asparagine synthetase; ZmGLK5: G2-like 5 transcription factor; ZmGLK8: G2-like 8 transcription factor; ZmNLP15: nodule-inception like protein.