Abstract

Media summary

Allelopathic activity, methyl jasmonate, DIMBOA, phenolic acid, maize

Introduction

The contribution of cyclic hydroxamic acids (e.g., 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, DIMBOA) to plant resistance for both insect and pathogen pests in Gramineae has been previously described (Frey et al. 1997; Sicker et al. 2000). In maize (Zea mays L.), the major benzoxazinoid is DIMBOA (Sicker et al. 2000), which had been identified as an important allelochemical providing maize resistance to insects, diseases, and the herbicide atrazine (Frey et al. 1997). Due to their muti-funtional phytotoxicity, the benzoxaziniods are involved in allelopathic interaction (Sicker et al. 2000). The biosynthesis of the DIMBOA has been elucidated in maize, totally nine genes including BX1-BX9 involved in the pathway (Frey et al. 1997, 2003). Recent studies have indicated that JA and MeJA functions as a key signal molecular in plant chemical defence (Kessler and Baldwin 2002). Treatment of plant with exogenous JA (or MeJA) can induce the increase of the amounts of DIMBOA derivatives (HDMBOA-Glc) in maize leaves (Oikawa et al. 2001). It seems possible that some JA-induced plant secondary compounds may serve as allelopathic agents. So far there is no direct evidence had been published to show this phenomenon. In the present study, we tested the inducible effects of MeJA on hydroxamic acid and phenolic acid content in maize, the related genes expression and allelopathic effects of maize to barnyardgrass and aimed to provide direct evidence by chemical analysis, gene expression and bioassay for the relationship between allelopathy and induced defense of plant.

Methods

Plant, soil and standard chemicals

A standard susceptible maize variety Yuenong No.9, was used in this study. The soil (0-20 cm), a clay loam, was collected from a paddy field in Guangzhou, China. The standard compounds of protocatechuric acid, p -hydroxy- benzoic acid, vanillic acid, caffeic acid, syringic acid, p -hydroxycinnamic acid and ferulic acid were purchased from ACROS Company. DIMBOA standard was isolated, purified and identified in our laboratory according to the method described by Larsen (2000).

Plant treatment

Seeds were germinated papers for 3 days and were transplanted to plastic pots (φ10 × 15 cm) with 3 seedlings per pot. Totally, 110 pots were arranged in a growth room at 25°C and L12:D12 photoperiod for about 10 days. Maize seedlings at V3 were used for treatment. MeJA (Sigma-aldrich company, St. Louis, MO) was dissolved in ethanol as a stock solution of 30%. MeJA were applied to the cotton ball, and transferred into a desiccator (6L) together with 3 pots of maize seedlings. Two treatment levels (40μl and 80μl) of MeJA (30%) were used in experiments, the final concentrations of MeJA were expressed as 2μl /L and 4μl/L. Same amount of ethanol was used to maize seedlings as controls. The treated desiccators were put in a growth chamber and the maize seedlings were cultivated for 48 h prior chemical analyses, total RNA extraction and bioassays.

Separation, extraction and determination of DIMBOA and phenolic acids

Separation, extraction and determination of DIMBOA and phenolic acids accorded to the method described by Larsen (2000).

Analysis of gene expression

Expression patterns of defense-related genes in different treated maize leaves and roots were analysed using reverse transcription- polymerase chain reaction (RT-PCR) according to the method described by Xu (2003). The gene-specific primers used in PCR were listed in Figure 2.

Bioassay of allelopathic effects of aqueous extract of MeJA-induced maize leaves

The second leaves were sampled from maize after treated for 48h. Ten milliliters of ddH₂O per gram of fresh sample was added to the samples. Fresh samples were ground to powder, kept at 4°C for 15 min and then centrifuged at 1500 rpm for 15 min. The supernatant was recovered and stored at 4°C until it was used as a crude aqueous extract. The concentrations of aqueous extract used for bioassays were 5% and 10%. Bioassay of the allelopathic effects of aqueous extract of maize leaves accorded to the method described by Zeng (2001).

Statistics

The data are expressed as the means ± the standard errors of the means. Analysis of variance (ANOVA) and unpaired t test was carried out using SAS8.1, and significant treatment differences were tested at 0.05 level by using Duncan’s multiple range test or t test.

Results

Induction of DIMBOA and its derivatives by treatment with MeJA

An accumulation of DIMBOA was observed in the second, third leaf and root of maize of MeJA treated at two concentrations after 48h (Figure 1 A). The induced effects of DIMBOA were significant compared with control except third leaf of 4 μl /L treatment. But, its derivative was only increased significantly in third leaf at 4 μl /L treatment and in root at 2μl /L (Figure 1 B). This indicated a transformation of DIMBOA and its derivatives in different tissues at different MeJA treatment concentration. The total peak areas of DIMBOA and its derivatives in all MeJA treatment tissues were significant larger than that of control (Figure 1 C). These findings suggest that the JA evoked hydroxamic acid biosynthesis in shoot and root of maize.

Figure 1. Amount of DIMBOA (A), peak area of its derivatives (B) and total peak area of hydroxamic acids in the 2nd, 3rd leaf and root of maize treated by different concentration of MeJA for 48h. The bars in the same category sharing the same letter are not significant different at P=0.05.

Induction of phenolic acid by treatment with MeJA

Protocatechuric acid, p-hydroxybenzoic acid and vanillic acid were three detectable phenolic acid in the leaf of maize. But, only protocatechuric acid and vanillic acid were in root. There was an unknown phenolic acid in root too; the retention time was about 15min. Table 1 shows that variations in the concentrations of various forms of phenolic acids existed among the different treatments. In particular, vanillic acid increased significantly both in roots and leaves after two MeJA treatments for 48h. Protocatechuric acid appeared in roots after treatment and was only significantly higher in the second leaf in MeJA treatment at 2μL/L level. The unknown compound in roots was significant higher in two MeJA treatments as compared with that of control. However, there was no significant difference in the concentrations of p-hydroxybenzoic acid between the control and MeJA treated leaves.

Table 1. Changes in Phenolic Acid content after treatment with MeJA for 48h in leaves and root of maize (μg/g FW)

Location	Treatment	Protocatechuric acid	p -Hydroxybenzoic acid	Vanillic acid	Unknown compound
2nd leaf	CK	31.16±2.78ab	16.57±2.45a	37.88±6.30 b	ND
	MeJA 2μL/L	32.68±2.47a	22.88±7.08a	53.68±7.16 a	ND
	MeJA 4μL/L	21.98±5.23b	29.83±10.84a	62.07±11.10 a	ND
3rd leaf	CK	12.25±2.04a	13.25±3.28a	84.69±12.08b	ND
	MeJA 2μL/L	8.02±1.82a	11.57±3.79a	120.06±26.39a	ND
	MeJA 4μL/L	9.90±1.22a	21.15±4.60a	122.18±22.61a	ND
root	CK	ND	ND	21.75±5.48b	67.11±13.34c
	MeJA 2μL/L MeJA 4μL/L	22.87±7.35a 20.66±5.80a	ND ND	55.70±7.96a 43.51±9.45a	208.28±36.85a 130.46±29.87b

ND. Not detectable.
Data of the same column at same location sharing the same letter are not significant different at P=0.05

Defense related genes expression induced by MeJA

The expression patterns of several defense-related genes were analyzed in MeJA treated maize leaves and roots using RT-PCR (Figure 2). The expression of allene oxide cyclase (AOC) gene, one important enzyme in the biosynthesis of jasmonates, was induced in maize leaves and roots as consequence of 4μL/L MeJA treatment. Phenylalanine ammonia-lyase (PAL), an important enzyme in the biosynthesis of phenolic acid, showed the strong expression only in maize leaves at two MeJA treatment level. However, the BX1 gene, which is the first gene of DIMBOA biosynthesis, was not detected in shoot and root after MeJA induced for 48h. In contrast, BX9, the gene regulates the produce of DIMBOA-glucoside, showed a perfect expression in leaves at 2μL/L MeJA treatment level and in roots at 4μL/L MeJA treatment level. Further studies of temporal expression dynamics of BX1 genes showed that the highest transcript level of BX1 observed in leaves 3 hours after MeJA induced (Figure3). The transcript level dropped rapidly as time goes to 36h (Figure3). Combined with chemical analyses data, it seems that exogenous MeJA elicited maize jasmonate-signaling pathway, which induced the maize synthesizing hydroxamic acid and phenolic acid and allocated them to shoot and roots. Those secondary metabolites produced by maize under MeJA induced may serve as allelopathic agents.

Figure 2. Expression patterns of defense-related genes in maize seedlings treated with MeJA for 48h.
AOC, Allene oxide cyclase; PAL, Phenylalanine ammonia-lyase; GAPC, Internal standard of RT-PCR. 1-2, CK; 3-4, 2μL/LMeJA treatment; 5-6, 4μL/LMeJA treatment.

Figure 3. Temporal expression dynamics of BX1 genes in maize seedlings treated with MeJA (2μL/L).

Allelopathic effects of aqueous extract of MeJA-induced maize leave

Both of the two concentrations aqueous extracts (5% and 10%) of MeJA-treated maize showed a significant inhibitory effect on the root length of barnyard grass (reduced 4.7% and 6.8%) as compared with control (Figure 4,). The suppressive effects of the extracts increased as the concentration increased. However, the extracts showed almost the same degrees of a slightly inhibitory effect on the shoot growth of barnyard grass at two concentration levels (Figure 4).

Figure 4. Aqueous extract bioassays of 2nd leaves of maize treated by MeJA for 48h (A, 5%; B, 10%)

Conclusion

Chemical analysis, related gene expression and bioassay confirmed that JA-induced maize secondary compounds serve as allelopathic agents.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (Projects No. 30270270 and No. 30470335), and the Guangdong Provincial Natural Science Foundation (Projects No. 021043 and No. 000569).

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