Root ethylene signalling is involved in Miscanthus sinensis growth promotion by the bacterial endophyte Herbaspirillum frisingense GSF30(T)

Abstract

The bacterial endophyte Herbaspirillum frisingense GSF30(T) is a colonizer of several grasses grown in temperate climates, including the highly nitrogen-efficient perennial energy grass Miscanthus. Inoculation of Miscanthus sinensis seedlings with H. frisingense promoted root and shoot growth but had only a minor impact on nutrient concentrations. The bacterium affected the root architecture and increased fine-root structures. Although H. frisingense has the genetic requirements to fix nitrogen, only minor changes in nitrogen concentrations were observed. Herbaspirillum agglomerates were identified primarily in the root apoplast but also in the shoots. The short-term (3h) and long-term (3 weeks) transcriptomic responses of the plant to bacterial inoculation revealed that H. frisingense induced rapid changes in plant hormone signalling, most prominent in jasmonate signalling. Ethylene signalling pathways were also affected and persisted after 3 weeks in the root. Growth stimulation of the root by the ethylene precursor 1-aminocyclopropane 1-carboxylic acid was dose dependent and was affected by H. frisingense inoculation. Minor changes in the proteome were identified after 3 weeks. This study suggests that H. frisingense improves plant growth by modulating plant hormone signalling pathways and provides a framework to understand the beneficial effects of diazotrophic plant-growth-promoting bacteria, such as H. frisingense, on the biomass grass Miscanthus.

Keywords: Biomass; Miscanthus; diazotroph; endophyte; ethylene; plant-growth-promoting bacteria..

Fig. 1.

Growth promotion of M. sinensis by H. frisingense GSF30^T (Hf). The graphs show total biomass from 6-week-old plants. (A) Fresh weight [mg, ±standard error (SE)] of seedlings grown with (+Hf, dark grey bars) or without H. frisingense (–Hf, pale grey bars) under low (N100, 50 µM NH₄NO₃; 12 plants) or higher (N500, 250 µM NH₄NO₃; 13 plants) nitrogen levels. Different lower-case letters indicate a significant difference by ANOVA test (P < 0.05). (B) Fresh weight (% compared with –Hf, ±SE) following H. frisingense inoculation (n=11 independent experiments; total 223 plants +Hf and 259 plants –Hf) under N100 conditions. Different lower-case letters indicate a significant difference by ANOVA test (P < 0.001). (C) Dry weight (mg, ±SE) of 6-week-old seedlings, separated for roots and shoots, without or with H. frisingense inoculation under N100 conditions.

Fig. 2.

Green fluorescent spots in M. sinensis roots inoculated with GFP-labelled H. frisingense. Bacterial aggregates were visible in the intercellular apoplastic space. Bright field image (right) and fluorescence image (left). The red colour indicates residual background fluorescence from the cell wall. The inset shows a magnification of the bacterial colony aggregates. (This figure is available in colour at JXB online.)

Fig. 3.

Root morphology and fine root structural changes of M. sinensis following inoculation with H. frisingense. (A) Root length of different diameter classes without inoculation (triangles) and with inoculation (circles). Quantitative root parameters were deduced using a WinRHIZO system. (B) Seedling appearance without inoculation (left) and with inoculation (right). Note the increased root system. (This figure is available in colour at JXB online.)

Fig. 4.

Differential element concentrations in roots (A) and shoots (B) without (grey bars) and with (black bars) H. frisingense inoculation. Macroelements (left graphs) and micronutrients (right graphs) are given in concentrations [mg/g dry weight (DW)]. Asterisks indicate significant differences at the P < 0.05 level (one-way ANOVA).

Fig. 5.

Differential expression of M. sinensis transcript categories related to hormones and key metabolic functions in response to H. frisingense inoculation. Red indicates lower expression of gene categories compared with non-inoculated plants, while blue indicates higher expression. Non-significant differences (z-score <1.96) are shown in white. Colouring is according to the z-scores of the bin-wise Wilcoxon test. A z-score of ±1.96 represents a P value of 0.05. The plot was generated using PageMan.

Fig. 6.

Normalized expression of M. sinensis transcripts related to hormones in response to H. frisingense (qRT-PCR, log₂ scale). Note the different scale of jasmonate-regulated genes (BGAF-1, BGAF-2, and dirigent-like; left graph). Auxin-responsive genes were: auxin response factors ARF1 and ARF6, and the auxin-responsive IAA18 and IAA20 genes. Ethylene-related genes were ethylene insensitive 3 (EIN3), ethylene-resistant 1 (ETR1), the sugarcane ethylene receptor-like SCER1-like SCER1a and SCER1b, and the ethylene response factor SCERF1-like. A SCERF2-like gene (Cavalcante et al., 2007) was not identified in Miscanthus. 3h, 3h after inoculation; 3 w, 3 weeks after inoculation. Error bars: +SE of three (3h) or two (3 w) biological replicates. Qualitatively similar results were obtained from two further replicates.

Fig. 7.

Mechanistic scheme of the interaction of M. sinensis and H. frisingense. The beneficial effect of Herbaspirillum-induced root hormone signalling leads to higher root biomass, improved nutrient absorption, and, as a consequence, also increases the shoot biomass. Red arrows: up-regulated genes, blue arrow: down-regulated genes. JA, jasmonate; ER, ethylene receptor; ERF, ethylene response factor. (This figure is available in colour at JXB online.)