NaJAZh regulates a subset of defense responses against herbivores and spontaneous leaf necrosis in Nicotiana attenuata plants

Abstract

The JASMONATE ZIM DOMAIN (JAZ) proteins function as negative regulators of jasmonic acid signaling in plants. We cloned 12 JAZ genes from native tobacco (Nicotiana attenuata), including nine novel JAZs in tobacco, and examined their expression in plants that had leaves elicited by wounding or simulated herbivory. Most JAZ genes showed strong expression in the elicited leaves, but NaJAZg was mainly expressed in roots. Another novel herbivory-elicited gene, NaJAZh, was analyzed in detail. RNA interference suppression of this gene in inverted-repeat (ir)JAZh plants deregulated a specific branch of jasmonic acid-dependent direct and indirect defenses: irJAZh plants showed greater trypsin protease inhibitor activity, 17-hydroxygeranyllinalool diterpene glycosides accumulation, and emission of volatile organic compounds from leaves. Silencing of NaJAZh also revealed a novel cross talk in JAZ-regulated secondary metabolism, as irJAZh plants had significantly reduced nicotine levels. In addition, irJAZh spontaneously developed leaf necrosis during the transition to flowering. Because the lesions closely correlated with the elevated expression of programmed cell death genes and the accumulations of salicylic acid and hydrogen peroxide in the leaves, we propose a novel role of the NaJAZh protein as a repressor of necrosis and/or programmed cell death during plant development.

Figure 1.

Phylogenetic tree of tobacco, rice, tomato, and Arabidopsis JAZ proteins. The phylogenetic tree was constructed using previously reported tobacco, rice, tomato, and Arabidopsis JAZ proteins and 12 JAZ protein sequences from N. attenuata identified in this paper. Protein sequences were aligned with ClustalW, Gonnet matrix gap penalty of 10, and extension penalty of 0.2. The phylogenetic tree was constructed from the alignment using MEGA 5.05 software using the maximum likelihood bootstrap method (1,000 replicates). Black circles indicate the positions of N. attenuata proteins in the phylogram.

Figure 2.

Basal and induced expression of individual JAZ genes in N. attenuata. The accumulation of NaJAZ transcripts in local treated leaves, systemic leaves, and roots was determined by microarrays (n = 3) after elicitation of the leaves with wounding (W+W) or simulated herbivory (W+OS); control plants remained untreated. Control and W+OS samples were harvested at 0, 1, 5, 9, 12, 17, and 21 h post elicitation; because samples from the W+W treatment were collected only at 0, 1, 5, and 17 h post elicitation, the time points of the W+W treatments are not connected with lines in the graphs.

Figure 3.

Silencing of the NaJAZh gene affects the expression of several other NaJAZ genes in N. attenuata. A, Transcript abundances of the NaJAZh gene were determined by real-time qPCR in three independent silenced irJAZh lines before and after elicitation with W+OS. B, Transcript abundances of other NaJAZ genes determined by qPCR in untreated (Control) and 1-h W+OS-elicited (1h WOS) leaves of irJAZh plants. C, NaJAZh and NaJAZg expression in systemic roots of irJAZh plants. Barsin indicate EF1a-normalized relative transcript abundances ± se (n = 3). Different letters (a–d) indicate significant differences among the combination of genotypes (wild type [WT] versus independent irJAZh lines irJAZh-264, -267, and -368) and treatments determined by ANOVA (P ≤ 0.05).

Figure 4.

The performance of larvae of the specialist herbivore M. sexta is strongly suppressed on irJAZh plants. The performance of M. sexta larvae was observed on the wild type (WT) and NaJAZh-silenced lines at two stages of development. A, M. sexta neonates were placed on rosette-stage leaves of wild-type and irJAZh-368 plants. The mean fresh mass (FM) ± se of irJAZh-368 caterpillars (n = 20) was significantly smaller at all time points as determined by Student’s t test (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). B, M. sexta performance (n = 10) on early-flowering-stage wild-type plants and three independent JAZh-silenced lines (irJAZh-264, -267, and -368) that developed necrotic lesions during the experiment. Significant differences between genotypes were determined at each time point by ANOVA (* P ≤ 0.05).

Figure 5.

NaJAZh silencing does not alter JA-Ile levels induced by simulated herbivory. Rosette-stage plants (the wild type [WT] and NaJAZh-silenced lines irJAZh-264, -267, and -368) were treated with simulated herbivory (W+OS) and harvested before and 1, 2, and 3 h after elicitation. Mean ± se levels of JA, JA-Ile, ABA, and SA (n = 3) were determined by LC-ESI-MS/MS using internal deuterium-labeled hormone standards. JA levels were significantly higher at 1 h after elicitation in irJAZh lines, but other hormones showed no significant differences compared with wild-type plants. Asterisks indicate significant differences among the wild type and independent irJAZh lines determined by ANOVA (* P ≤ 0.05). FM, Fresh mass.

Figure 6.

NaJAZh silencing enhanced constitutive and inducible levels of TPIs and DTGs but suppressed nicotine accumulation. Rosette-stage plants (the wild type [WT] and NaJAZh-silenced lines irJAZh-264, -267, and -368) were treated with simulated herbivory (W+OS), and treated leaves were harvested before and 24, 48, and 72 h after elicitation. Mean ± se levels of TPIs determined by radial diffusion assay (A) and DTGs determined by HPLC (B) were significantly higher at each time point in irJAZh compared with wild-type plants. Mean ± se levels of nicotine (C) determined by HPLC were significantly lower in irJAZh compared with wild-type plants at every time point (ANOVA; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001; n = 3). There were no significant differences found in metabolite content when three independent irJAZh lines were compared. FM, Fresh mass.

Figure 7.

NaJAZh silencing strongly affects the accumulation and structural modification of DTGs in transgenic plants. Rosette-stage plants in the glasshouse were treated with simulated herbivory (W+OS) and harvested before and 24, 48, and 72 h after elicitation. Mean ± se relative amounts of individual DTGs were determined by LC-ESI-MS/MS. The malonylated DTGs (rows 3 and 4) strongly accumulated after treatment in all three independent irJAZh lines (irJAZh-264, -267, and -368) compared with wild-type (WT) plants, whereas core (row 2) and precursor (row 1) DTGs were highest in the irJAZh lines before treatment (constitutive levels at 0 h). Asterisks indicate significant differences among the wild type and three independent irJAZh lines determined by ANOVA (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001; n = 3). There were no significant differences found in metabolite content among three independent irJAZh lines. FM, Fresh mass.

Figure 8.

NaJAZh-silenced plants emit higher amounts of VOCs and GLVs. Volatiles were collected from the head space of wild-type (WT) and irJAZh (irJAZh-267 and -368) leaves for 24 h after connecting the volatile trap units to locally treated leaves 3 h after W+OS elicitation; control plants were collected in parallel but remained untreated. Samples were analyzed by gas chromatography-mass spectrometry with tetraline as an internal standard. Bars indicate normalized relative emissions of volatiles per cm² of leaf area ± se (n = 5). Different letters (a–c) indicate statistically significant differences in emissions among genotypes (the wild type versus two independent irJAZh lines) and treatments determined by ANOVA (P ≤ 0.05). n.d, Not detected.

Figure 9.

Elongated and mature irJAZh plants develop spontaneous necrosis on leaves. Necrotic spots on leaves of irJAZh plants (indicated by red arrows) appeared in a strict developmental sequence as irJAZh plants started to elongate and transitioned into the flowering stage of growth. Symptoms were first detected on cotyledons of irJAZh plants and gradually spread to the next developed but still fully green leaves. Necrotic lesions were not detected on flowers or capsules of the irJAZh plants. In contrast to irJAZh plants, wild-type (WT) plants developed natural senescence that was characterized by yellow color of the old leaves and necrosis of the yellow senescent leaves in the final stages of development.

Figure 10.

irJAZh plants accumulate ROS and express PCD markers during leaf necrosis. A, For DAB staining, leaves were detached from plants and either punched with a cork borer to wound (bottom panel) or leaves remained unwounded (top panel). The entire leaf was floated in DAB staining solution overnight in the dark and destained to visualize the brown DAB precipitate. B, For the Amplex Red assay, leaf extracts prepared from wounded leaves of irJAZh-368 and wild-type (WT) plants were incubated with Amplex Red reagent. Amplex signal intensity was determined by measuring sample fluorescence at excitation/emission = 530/590 nm. Mean ± se levels of H₂O₂ (n = 3) were calculated using H₂O₂ external calibration curves, and significant differences between wild-type and irJAZh plants were determined by Student’s t test at every measured time point (* P ≤ 0.05). C, Mean relative transcript abundances ± se of NaJAZh and PCD marker genes (Hin1, Hsr203, and VPE361) in developing N. attenuata plants (34–50 d post germination; n = 4). D, SA levels determined by LC-ESI-MS/MS in developing N. attenuata plants (34–50 d post germination; n = 4). Arrows in C and D indicate the date of the first appearance of necrotic symptoms of a leaf that during early rosette-stage growth (34 d post germination) occupied a node one position younger than the source-sink transition leaf (−1). Asterisks in C and D indicate significant differences between wild-type and irJAZh plants determined by Student’s t test (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). FM, Fresh mass.

Figure 11.

Rejuvenation of irJAZh plants delays the development of necrosis on the leaves. A, The extent of necrotic lesions was documented in wild-type (WT) and irJAZh (irJAZh-267 and -368) plants at 45 and 53 d post germination. Plants were cultivated either under the normal glasshouse fertilization regime (Peters Allrounder and Borax; left panel) or under high nitrogen supply rates (supplied with an additional 50 mL of 20 mm NH₄NO₃ every 2 d starting at 30 d post germination [DAG]; right panel). Arrows indicate the leaf that during early rosette-stage growth (34 d post germination) occupied a node one position younger than the source-sink transition leaf (−1) and was used to measure chlorophyll contents. B, Mean ± se chlorophyll content (n = 5) in wild-type and irJAZh (irJAZh-267 and -368) leaves determined between 35 and 43 d post germination. The arrow indicates the date of the first visible necrotic symptoms on the labeled leaf. Chlorophyll content started to decline when the first necrotic symptoms appeared on irJAZh plants grown under the normal glasshouse fertilization regime; compared with 39 d, the chlorophyll content at 41 d post germination significantly decreased in all plants grown under the normal fertilization regime (determined by Student’s t test; P ≤ 0.01). [See online article for color version of this figure.]

Figure 12.

Summary of JAZh function in N. attenuata plants. NaJAZh is a major repressor of JA-dependent defense responses, including direct (DTGs and TPI activity) and indirect (GLVs and VOCs) defenses. It is proposed that NaJAZh regulates nicotine accumulation via interaction with other JAZ genes in N. attenuata. Furthermore, NaJAZh is required for direct or indirect repression of ROS, SA, and PCD during plant development in the glasshouse; necrosis (PCD) is prevented by an unknown environmental factor in the field. The black lines indicate interactions established in this paper; gray dashed lines show predicted components of the JAZ signaling pathways.