Multiple interactions of NaHER1 protein with abscisic acid signaling in Nicotiana attenuata plants

Abstract

Previously, we identified a novel herbivore elicitor-regulated protein in Nicotiana attenuata (NaHER1) that is required to suppress abscisic acid (ABA) catabolism during herbivore attack and activate a full defense response against herbivores. ABA, in addition to its newly defined role in defense activation, mainly controls seed germination and stomatal function of land plants. Here we show that N. attenuata seeds silenced in the expression of NaHER1 by RNA interference (irHER1) accumulated less ABA during germination, and germinated faster on ABA-containing media compared to WT. Curiously, epidermal cells of irHER1 plants were wrinkled, possibly due to the previously demonstrated increase in transpiration of irHER1 plants that may affect turgor and cause wrinkling of the cells. We conclude that NaHER1 is a highly pleiotropic regulator of ABA responses in N. attenuata plants.

Keywords: :

• Nicotiana attenuata
• abscisic acid (ABA)
• ABA metabolism
• germination

Until recently, the plant hormone ABA was mainly thought to regulate a plant’s water regime and its responses to drought, as well as being one of the crucial factors controlling seed germination.^1,2 However, the recognition of ABA’s role in plant-pathogen interactions,³ and more recently in plant-insect interactions,⁴ reveals a larger and highly pleiotropic role played by ABA. This diversity of effects speaks to the importance of hormonal cross talk in plant development, and suggests a role of ABA in mediating tradeoffs between growth and defense.

While searching for genes regulated by specific herbivore-associated elicitors (HAEs) in the ecological model plant, Nicotiana attenuata, we identified NaHER1, a protein of previously unknown function. Upon herbivore attack or during simulated herbivory treatments, the levels of transcripts of this gene were strongly increased,⁴ suggesting a role in plant defense. From an RNAi-based loss of function analysis of transgenic plants, the NaHER1 protein was found to contribute to ABA homeostatsis by inhibiting the catabolism of the hormone, resulting in increases in leaf ABA levels. From this, we inferred that an increase in ABA was a prerequisite for activating a full defense response in N. attenuata against its herbivores. Second, plants silenced in the expression of NaHER1 were clearly impaired in their responses to ABA, first noticed as reduced growth under drought stress, and under closer examination, as higher transpiration rates during leaf desiccation after ABA treatments.⁴

Here we explored additional potential roles of NaHER1 in seed germination, as changes in ABA levels and sensitivity are known to be important factors involved in seed germination.² In addition, other hormones and signaling molecules such as gibberellic acid (GA), jasmonic acid (JA), reactive oxygen species (ROS), nitric oxide (NO), ethylene, cytokinins, and their cross talk, are known to affect significantly seed germination.^5-8 The content of endogenous ABA is known to rapidly decline during germination; however, decreases in endogenous ABA are not always associated with increased germination rates.⁹

In N. attenuata, seed germination is known to be very complex and tightly controlled. It is timed so that seedlings emerge in the immediate post-fire environment,¹⁰ providing the germinating seedlings a nutrient-rich environment in which to grow, but also one of intense intra- and inter-specific competition from other plants scrambling to occupy this niche as well as exposure to attack from local herbivore communities that rain in on these vigorously growing plants.¹¹ N. attenuata seeds can stay dormant in soil for up to 150 y between fires, only germinating after being exposed to smoke, which in turn increases endogenous GA and sensitivity to GA,¹² decreasing ABA levels, as well as having ABA and 4 additional terpenes that leach from the litter and inhibit germination removed by pyrolysis.¹³ While elevated endogenous ABA levels in N. attenuata seeds reduce germination,¹² as has been shown in other plants, the exact mechanism remains unknown.

Some proteins are known to regulate ABA levels in N. attenuata leaves during water stress, such as the homeodomain-leucine zipper type I protein NaHD20¹⁴ and the NaMAPK4 (mitogen-activated protein kinase 4) protein which plays a pivotal role in ABA signaling.¹⁵ The NaHER1 gene, which strongly and specifically responds to herbivore cues, such as N-linolenoyl-L-glutamate (C18:3-Glu) contained in oral secretions of Manduca sexta larvae,¹⁶ appears to be a novel ABA regulator. Because the function of NaHER1 is to inhibit the catabolism of ABA during herbivory, the ABA pools of irHER1 plants are typically depleted by unfettered catabolism. However, it remained unknown if this ABA deficiency extends to the seeds and germination of irHER1 plants. Following the main work on defense responses,⁴ additional experiments were conducted to clarify this point.

The biosynthesis of ABA is known to be tissue- and time-specific.¹⁷ At first, we found no differences in ABA levels in dry seeds between WT and irHER1 (Fig. 1A, 0h). In contrast, ABA levels in irHER1 seeds were significantly lower at 24 and 48 h after sterilizing and placing the seeds on germination media, following standard GA and smoke pre-treatments (Fig. 1A). Thus, at the minimum level, the role of NaHER1 indeed extends to seed function, but in an herbivory-independent manner. Since ABA controls many other aspects of plant physiology, e.g., post-germination embryo development, dormancy, stomata opening, water relations, photosynthesis, root geotropism, growth, and adaptation to biotic and abiotic stresses,¹⁸ NaHER1 could play additional roles in plant physiology and ecology.

Figure 1. Silencing NaHER1 affected ABA levels in seeds during germination in Nicotiana attenuata. Seeds were sterilized, placed on GB5 media, collected after the designated times, and analyzed by LC-MS/MS for ABA (A). Sterilized seeds were placed on GB5 media supplied with 1µM ABA and germination rate was collected (B). Comparisons were performed by ANOVAs, Fisher’s PLSDs test: ** p ≤ 0.01.

Because ABA levels were reduced in irHER1 seeds (Fig. 1A), most likely because NaHER1 suppresses local ABA catabolism in WT seeds, we compared germination rates of irHER1 and WT seeds. Initially, we did not observe any significant changes in germination behavior of irHER1 seedlings under laboratory conditions, which reflected the relatively small changes in ABA content found in the germinating seeds (Fig. 1A). Moreover, decreases in endogenous ABA are not always associated with increased germination rates.⁹ However, when ABA levels are enhanced by exogenously applying 1µM ABA in the standard Gamborg’s B5 media, irHER1 seedlings showed accelerated germination. At 10 d post germination, germination rates of irHER1 seeds were about 2-fold of those found in WT (Fig. 1B). These results suggest that NaHER1 gene might affect the ABA levels, and the long-term dormancy of seeds in natural seed banks that is sustained by the terpenes leaching from the litter of dominate vegetation before fires.¹³ Since N. attenuata is a fire-chasing annual that occupies the post-fire nitrogen-rich soils,¹⁰ the tight regulation of germination to time growth in the post fire niche could be influenced and/or regulated by NaHER1.¹⁹

We also carefully inspected cells on leaf surfaces. Surprisingly, epidermal leaf cells in irHER1 plants were wrinkled, as if the cells had lost their turgor and deflated (Fig. 2, upper panels). Although we do not have a direct explanation for this phenotype, it was even more obvious when the leaves were painted with a nail polish and the obtained surface imprints were studied under a light microscope on a flat surface (Fig. 2, lower panels). This phenotype is consistent with the altered transpiration rates and function of NaHER1 in normal plant development under non-stress conditions.⁴

Figure 2. Leaf surface of wild type and irHER1 plants. Bright field microscopy image (upper row) of leaf surface of WT and irHER1–6/4 plants cultivated in the glasshouse shows wrinkled leaf surface of irHER1 leaf. Surface relief of nail polish strips (lower row) painted and peeled from the leaves after drying and photographed on a flat surface of a microscope slide emphasizes the uneven surface of irHER1 leaf (red arrows).

The regulation of ABA is undoubtedly one of the critical survival strategies in plants²⁰ that requires homeostatic mechanisms which are environmentally sensitive. Endogenous ABA levels are mainly controlled at the level of biosynthesis and catabolism.²¹ Oxidation and conjugation are the 2 main pathways to inactivate ABA in which the ABA 8'-hydroxylase and the glucosyltransferases are the 2 key genes.^22-24 Although we already demonstrated at least 2 functions of NaHER1 during herbivory⁴ and seed germination, how NaHER1 actually regulates ABA catabolism remains unresolved. Ongoing work, including microarray experiments, will assist the future identification of the NaHER1-regulated gene networks and provide additional functional hints for a full understanding of NaHER1 in plant defense and development.

Materials and Methods

Plant material, growth conditions and plant treatments

Seeds of Nicotiana attenuata Torr. ex Watson wild type (WT) and previously established N. attenuata transgenic seeds (irHER1) silenced in the expression of NaHER1⁴ were germinated on Gamborg’s B5 medium (Duchefa) as described previously.²⁵

ABA analysis

ABA was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a Varian 1200 Triple-Quadrupole-LC-MS system (Varian, Palo Alto, CA, USA) as described previously.⁴

Statistical analyses

Analysis of variance (ANOVA) followed by post hoc Fisher's protected least significant difference (PLSD) were used. Comparisons of means were calculated at a minimal 0.05 level of significance.

Microscopy and surface imaging

N. attenuata leaves were freshly removed from the plants and directly observed on a Leica laser microdissection microscope system (Leica, Bensheim, Germany), or the leaf surface was observed indirectly by nail polish method as follows. A thin layer of commercial transparent nail polish was painted on the leaf surface and allowed to dry completely. A small piece of transparent sellotape was placed over each dry nail polish patch, gently peeled together with the attached polish layer, and re-taped on a clean microscope slide. The imprints were observed as above.

Acknowledgments

This work was funded by the Max Planck Society and ERC advanced grant, ClockworkGreen (No. 293926) to ITB. Son Truong Dinh was supported by the Vietnam Ministry of Agricultural and Rural Development and Deutscher Akademischer Austauschdienst (DAAD) and the Max Planck Society. We thank Dr John T Christeller for critically reading the manuscript and for his helpful suggestions.

10.4161/psb.26365

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.