A maize landrace that emits defense volatiles in response to herbivore eggs possesses a strongly inducible terpene synthase gene

Abstract

Maize (Zea mays) emits volatile terpenes in response to insect feeding and egg deposition to defend itself against harmful pests. However, maize cultivars differ strongly in their ability to produce the defense signal. To further understand the agroecological role and underlying genetic mechanisms for variation in terpene emission among maize cultivars, we studied the production of an important signaling component (E)-caryophyllene in a South American maize landrace Braz1006 possessing stemborer Chilo partellus egg inducible defense trait, in comparison with the European maize line Delprim and North American inbred line B73. The (E)-caryophyllene production level and transcript abundance of TPS23, terpene synthase responsible for (E)-caryophyllene formation, were compared between Braz1006, Delprim, and B73 after mimicked herbivory. Braz1006-TPS23 was heterologously expressed in E. coli, and amino acid sequences were determined. Furthermore, electrophysiological and behavioral responses of a key parasitic wasp Cotesia sesamiae to C. partellus egg-induced Braz1006 volatiles were determined using coupled gas chromatography electroantennography and olfactometer bioassay studies. After elicitor treatment, Braz1006 released eightfold higher (E)-caryophyllene than Delprim, whereas no (E)-caryophyllene was detected in B73. The superior (E)-caryophyllene production by Braz1006 was positively correlated with high transcript levels of TPS23 in the landrace compared to Delprim. TPS23 alleles from Braz1006 showed dissimilarities at different sequence positions with Delprim and B73 and encodes an active enzyme. Cotesia sesamiae was attracted to egg-induced volatiles from Braz1006 and synthetic (E)-caryophyllene. The variation in (E)-caryophyllene emission between Braz1006 and Delprim is positively correlated with induced levels of TPS23 transcripts. The enhanced TPS23 activity and corresponding (E)-caryophyllene production by the maize landrace could be attributed to the differences in amino acid sequence with the other maize lines. This study suggested that the same analogous genes could have contrasting expression patterns in different maize genetic backgrounds. The current findings provide valuable insight not only into genetic mechanisms underlying variation in defense signal production but also the prospect of introgressing the novel defense traits into elite maize varieties for effective and ecologically sound protection of crops against damaging insect pests.

Keywords: (E)‐caryophyllene synthase; induced defense; maize landraces; natural enemy; plant–insect interactions; terpene biosynthesis.

Figure 1

Behavioral responses of the parasitic wasp Cotesia sesamiae in a four‐arm olfactometer bioassay to (a) headspace volatiles from maize (Zea mays) landrace Braz1006 (Brazil) with and without spotted stemborer (Chilo partellus) eggs, (b) authentic standard of (E)‐caryophyllene. Each parasitoid was observed for 12 min (N = 12). Mean (± SE) for time spent (min) in each part of the olfactometer is shown. Parasitoid responses were compared by ANOVA after conversion of the data into proportions and log‐ratio transformation. Different letters above the bars indicate statistically significant differences based on the Student–Newman–Keuls (SNK) test (p < .05)

Figure 2

A coupled gas chromatography–electroantennogram (GC ‐ EAG) recording of a female parasitic wasp, Cotesia sesamiae, showing responses to different compounds in the headspace volatiles from maize (Zea mays) landrace cv. Braz1006 (Brazil) exposed to egg deposition by the spotted stemborer (Chilo partellus). The upper trace represents EAG responses of the C. sesamiae antenna, whereas the lower traces represent the gas chromatography–flame ionization detector (GC–FID) responses of the headspace volatile sample from maize landrace with and without C. partellus eggs. Identified compounds elicited consistent responses from three or more antennae: (1) (R)‐linalool, (2) (E)‐4,8‐dimethyl‐1,3,7‐nonatriene (DMNT), (3) decanal, (4) (E)‐caryophyllene, (5) (E,E)‐4,8,12‐trimethyl‐1,3,7,11‐tridecatetraene (TMTT).

Figure 3

Coupled gas chromatography mass spectrometry (GC‐MS) analysis of volatiles from induced and control maize (Zea mays) plants (a) maize landrace Braz1006 (Brazil), (b) maize line Delprim, (c) maize line B73. (E)‐caryophyllene (peak 1) was released in large amount (395 ng/g of tissue powder) from Braz1006 compared to Delprim (47 ng/g of tissue powder) (N = 6; p < .05) after elicitor induction, while B73 did not produce. Other represented EAG‐active compounds were α‐bergamotene (peak 2), (E)‐β‐farnesene (peak 3), and (E,E)‐4,8,12‐trimethyl‐1,3,7,11‐tridecatetraene (TMTT) (peak 4). Control plants produced only trace amounts of these compounds. Six replicates of control and induced plant volatile samples were analyzed for each maize line

Figure 4

Transcript abundance of tps23 on control and induced leaves from maize (Zea mays) landrace Braz1006 and maize lines Delprim and B73. Transcript levels were determined from 14‐day‐old plant leaves treated with the elicitor (indanoyl isoleucine conjugates) solution for 24 hr. The tps23 gene expression was compared relative to the reference gene (APT1). Significance was calculated by one‐way ANOVA from three technical replicates of three biological samples using general linear model (GLM). Different letters above the bars indicate statistically significant differences based on Student–Newman–Keuls (SNK) test (p < .05)

Figure 5

Alignment of the amino acid sequences of TPS23 from the maize (Zea mays) landrace Braz1006 and maize lines Delprim and B73. The differences in the amino acid sequences resulting from different TPS23‐allels are highlighted in gray

Figure 6

Products of the terpene synthase TPS23. The enzyme was expressed in Escherichia coli, extracted, purified, and incubated with the substrate (E,E)‐ FPP. The resulting terpene products were collected with a solid‐phase microextraction (SPME) fiber and analyzed by gas chromatography–mass spectrometry. The major product (E)‐caryophyllene was identified by comparison of retention times and mass spectra with those of authentic standards