Hormone regulation of rhizome development in tall fescue (Festuca arundinacea) associated with proteomic changes controlling respiratory and amino acid metabolism

Abstract

Background and aims: Rhizomes are underground stems with meristematic tissues capable of generating shoots and roots. However, mechanisms controlling rhizome formation and growth are yet to be completely understood. The objectives of this study were to investigate whether rhizome development could be regulated by cytokinins (CKs) and gibberellic acids (GAs), and determine underlying mechanisms of regulation of rhizome formation and growth of tall fescue (Festuca arundinacea) by a CK or GA through proteomic and transcript analysis.

Methods: A rhizomatous genotype of tall fescue ('BR') plants were treated with 6-benzylaminopurine (BAP, a synthetic cytokinin) or GA3 in hydroponic culture in growth chambers. Furthermore, comparative proteomic analysis of two-dimensional electrophoresis and mass spectrometry were performed to investigate proteins and associated metabolic pathways imparting increased rhizome number by BAP and rhizome elongation by GA3 KEY RESULTS: BAP stimulated rhizome formation while GA3 promoted rhizome elongation. Proteomic analysis identified 76 differentially expressed proteins (DEPs) due to BAP treatment and 37 DEPs due to GA3 treatment. Cytokinin-related genes and cell division-related genes were upregulated in the rhizome node by BAP and gibberellin-related and cell growth-related genes in the rhizome by GA3 CONCLUSIONS: Most of the BAP- or GA-responsive DEPs were involved in respiratory metabolism and amino acid metabolism. Transcription analysis demonstrated that genes involved in hormone metabolism, signalling pathways, cell division and cell-wall loosening were upregulated by BAP or GA3 The CK and GA promoted rhizome formation and growth, respectively, by activating metabolic pathways that supply energy and amino acids to support cell division and expansion during rhizome initiation and elongation in tall fescue.

Keywords: Rhizome; hormone; protein; tall fescue; transcription.

Fig. 1.

Phenotypes of ‘BR’ tall fescue. (A) Rhizomatous phenotypes that had been grown in fritted clay for 3 months in the greenhouse. (B) A rhizome bud was observed 3 d after transplanting to the hydroponic system. (C) Rhizomatous phenotypes in hydroponics after 12 d in the growth chamber. (D) Average numbers of rhizomes per plant in hydroponics. Values are mean ± s.e. for 30 plants. (E) Average length of rhizomes in hydroponics. Values are mean ± s.e. for 30 rhizomes. Columns marked with different letters indicate significant differences among treatments based on the LSD value (P≤0·05); FW, fresh weight.

Fig. 2.

Effects of BAP on rhizomes of ‘BR’ tall fescue. (A) Average number of rhizomes per individual plant treated with different concentrations of BAP in hydroponics. (B) Endogenous iPA content in rhizome nodes of ‘BR’ tall fescue treated with 1 µm BAP. Columns marked with different lowercase letters indicate significant differences among treatments at a given day of sampling based on the LSD value (P≤0·05). Columns marked with different uppercase letters indicate significant differences among sampling time points for a given treatment based on the LSD value (P≤0·05); FW, fresh weight.

Fig. 3.

Two-dimensional SDS–PAGE gels of rhizome nodes with BAP treatment in ‘BR’ tall fescue. Differentially expressed protein spots are marked. There were at least four replicated gels for each treatment (P ≤ 0·05).

Fig. 4.

Cluster analysis and functional distribution of differentially expressed proteins of rhizome nodes with BAP treatment in ‘BR’ tall fescue. (A) Biological process and cellular components were analysed against the Arabidopsis database (http://bioinfo.cau.edu.cn/agriGO/analysis.php) (threshold −log10(P value) > 4). (B) Functional distribution was grouped using MapMan software (http://mapman.gabipd.org/web/guest/home).

Fig. 5.

Expression levels of genes in rhizome nodes of ‘BR’ tall fescue with BAP treatment. (A) Expression levels of genes responsible for CK metabolism and the signalling pathway in rhizome nodes with BAP treatment. (B) Expression levels of genes involved in cell division in crown nodes with BAP treatment. FaIPT, tRNA dimethylallyltransferase 2-like; FaCKX, cytokinin oxidase; FaHK1, histidine kinase 1; FaHK2, histidine kinase 2; FaRR1, response regulator 1; FaRR6, response regulator 6; FaCYCD, cyclin-D2-2; FaHis4, histone h4; FaPCNA, proliferating cell nuclear antigen; FaCDKB, cyclin-dependent kinase B1-1.

Fig. 6.

Effects of GA₃ on rhizomes of ‘BR’ tall fescue. (A) Rhizomatous phenotypes with GA₃ treatment in hydroponics after 12 d in the growth chamber. White arrows indicate rhizomes. (B) Average length of rhizomes with GA₃ treatment in hydroponics. Values are mean ± s.e. of 30 rhizomes. Vertical bars are LSD values (P ≤ 0·05) indicating significant differences among treatments at a given day. (C) Endogenous GA₄ content in rhizomes treated with 10 µm GA₃. Columns marked with different letters indicate significant differences among treatments based on the LSD value (P ≤ 0·05); FW, fresh weight.

Fig. 7.

Two-dimensional SDS–PAGE gels of tall fescue rhizomes with GA₃ treatment. Differentially expressed proteins are marked. There were least four repeat gels for each treatment (P≤0·05).

Fig. 8.

Cluster analysis and functional distribution of differentially expressed proteins of rhizomes with GA₃ treatment in ‘BR’ tall fescue. (A) Biological processes and cellular components were analysed against the Arabidopsis database (http://bioinfo.cau.edu.cn/agriGO/analysis.php) (threshold −log10(P value) > 4). (B) Functional distribution was grouped using MapMan software (http://mapman.gabipd.org/web/guest/home).

Fig. 9.

Expression level of genes in rhizomes of ‘BR’ tall fescue with GA₃ treatment. FaKO, ent-kaurene; FaGA20OX1, gibberellin 20-oxidase1; FaGA20OX2, gibberellin 20-oxidase2; FaEXPA5, EXPANSIN A5; FaEXPA11, EXPANSIN A11; FaEXPB2, EXPANSIN B2; FaEXPB11, EXPANSIN B11; FaXET1, xyloglucan endotransglycosylase1; FaXET2, xyloglucan endotransglycosylase 2.

Fig. 10.

Differentially expressed proteins in rhizome nodes involved in the respiratory metabolism pathway according to the KEGG pathway in ‘BR’ tall fescue. Red colour indicates proteins that were upregulated with BAP treatment compared with the control, and black colour indicates proteins that were downregulated with BAP treatment compared with the control.

Fig. 11.

A model for hormone-regulated rhizome development in tall fescue ‘BR’.