Functional analyses of chitinases in the moss Physcomitrella patens: chitin oligosaccharide-induced gene expression and enzymatic characterization

Figures & data

Table 1. Chitinase and CERK candidate genes in P. patens.

Gene	Length of predicted amino acid sequence from cDNA	Chitinase classification	GH family	Name	Transcript*
EDQ53501	ORF, 301; SP: 19; MP: 282	I	19	PpChi-Ia	+
EDQ64021	ORF, 323; SP: 26; MP: 297	I	19	PpChi-Ib	+
EDQ80360	–	I	19	PpChi-Ic	N.D.**
EDQ53624	ORF, 280; SP: 23; MP: 257	II	19	PpChi-IIa	+
EDQ53657	–	II	19	PpChi-IIb	N.D.**
EDQ64064	ORF, 292; SP: 19; MP: 273	II	19	PpChi-IIc	+
EDQ60220	ORF, 288; SP: 32; MP: 256	IV	19	PpChi-IV	+
EDQ72654	–	II-L	19	PpChi-II-L	N.D.**
EDQ51077	–	V	18	PpChi-Va	N.D.**
EDQ63241	ORF, 369; SP: 30; MP: 339	V	18	PpChi-Vb	+
	Length of predicted ORF	Extracellular domains	Intracellular domain
EDQ51621	637	LysM×3	Tyrosine kinase	PpCERK-a	+
EDQ67338	658	LysM×3	Tyrosine kinase	PpCERK-b	+

Notes: ORF, SP, and MP, indicate open reading frame, signal peptide, and mature protein, respectively.

^* Transcripts of chitinase and CERK candidate genes were determined by RT-qPCR using total RNA from samples at various stage or (GlcNAc)₆ treated samples.

^** N.D. indicates not detected.

Fig. 1. Schematic representation of primary structure of chitinases from P. patens.

Note: I–V and C with the loop regions indicate the name of each loop.

Fig. 2. Expression analysis of chitinase genes by RT-qPCR.

Notes: Expression profiles of each chitinase gene after 100 mM (GlcNAc)n (n = 6, 4, 2) treatment. (A) PpChi-Ia, PpChi-Ib, PpChi-IIa, PpChi-IIc, PpChi-IV and PpChi-Vb. (B) PpCERK-a and PpCERK-b. ●, (GlcNAc)₆; ○, (GlcNAc)₄; ▲, (GlcNAc)₂. Error bars represent the standard deviation from three replicated experiments.

Fig. 3. SDS–PAGE of recombinant PpChis.

Notes: Purified recombinant proteins were analyzed by SDS–PAGE. Lane 1, PpChi-Ia; lane 2, PpChi-IV; lane 3, PpChi-Vb. The gel was stained with Coomassie brilliant blue R250.

Fig. 4. Effect of pH and temperature on the chitinase activity.

Notes: (A) The effect of pH on chitinase activity was examined after incubation at 37 °C for 15 min. (B) The effect of temperature on chitinase activity was examined after incubation for 15 min. (C) The effect of pH on stability was examined by measuring residual activity after incubation at various pH at 37 °C for 24 h. (D) The effect of temperature on stability was examined by measuring residual activity after incubation in 0.1 M sodium phosphate buffer (pH 7.0) for 1 h. ●, PpChi-Ia; ○, PpChi-IV; ▲, PpChi-Vb.

Fig. 5. Time-courses of the PpChi-Ia and -IV catalyzed reaction toward (GlcNAc)_6–4. Time-course of the reaction products of (GlcNAc)_6–4 by PpChi-Ia (A, B, and C) and PpChi-IV (D, E, and F).

Notes: The reaction was conducted in 5 mM sodium acetate buffer (pH 5.0) at 25 °C. Concentrations of the enzyme and the substrate were 2 μM and 4 mM, respectively. □, (GlcNAc)₆; ■, (GlcNAc)₅; △, (GlcNAc)₄; ▲, (GlcNAc)₃; ○, (GlcNAc)₂; ●, GlcNAc. The dashed lines indicate the substrates.

Fig. 6. Time courses of (GlcNAc)_6–4 hydrolysis and transglycosylation catalyzed by PpChi-Vb.

Notes: Time-courses of the reaction products of PpChi-Vb with (GlcNAc)₆ (A), (GlcNAc)₅ (B), or (GlcNAc)₄ (C). D, E, and F indicate enlarged view of the vertical axis of A, B, and C, respectively. The reaction was conducted in 5 mM sodium acetate buffer (pH 4.0) at 25 °C. Concentrations of the enzyme and the substrate were 1 μM and 1.3 mM, respectively. ◇, (GlcNAc)₈; ◆, (GlcNAc)₇; □, (GlcNAc)₆; ■, (GlcNAc)₅; △, (GlcNAc)₄; ▲, (GlcNAc)₃; ○(GlcNAc)₂; ●, GlcNAc. (G) Reaction model of hydrolysis and transglycosylation from (GlcNAc)₆. The dashed lines indicate the substrates.

Fig. 7. Antifungal activity of PpChi against T. viride. Samples to be tested were placed in wells in 10 μL of distilled water.

Notes: (A) Blank (distilled water); (B) PpChi-Ia (300 pmol); (C) PpChi-IV (300 pmol); (D) PpChi-Vb (300 pmol).