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Plant Signaling & Behavior

Volume 6, 2011 - Issue 7
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Addendum

The multiple functions of plant serine protease inhibitors

Defense against herbivores and beyond

Markus Hartl Department of Molecular Ecology, Max Planck Institute for Chemical Ecology; Jena, GermanyCorrespondencemarkus_hartl@gmx.at
,
Ashok P. Giri Department of Molecular Ecology, Max Planck Institute for Chemical Ecology; Jena, Germany
,
Harleen Kaur Department of Molecular Ecology, Max Planck Institute for Chemical Ecology; Jena, Germany
&
Ian T. Baldwin Department of Molecular Ecology, Max Planck Institute for Chemical Ecology; Jena, Germany
Pages 1009-1011
Received 17 Mar 2011
Accepted 17 Mar 2011
Published online: 01 Jul 2011

Addendum

The multiple functions of plant serine protease inhibitors

Defense against herbivores and beyond

Abstract

Plant protease inhibitors (PIs) are a diverse group of proteins which have been intensely investigated due to their potential function in protecting plants against herbivorous insects by inhibiting digestive proteases. Although this mechanism has been well documented for a number of single PIs and their target enzymes, whether this mechanism protects plants in nature remains unclear. Moreover, many plants express a number of different PIs and it was unknown if these proteins work synergistically as defenses or if they also have other functions. We recently identified four serine PIs (SPI) of Solanum nigrum and demonstrated that they differ substantially in substrate specificity, accumulation patterns, and their effect against different natural herbivorous insects in field- and glasshouse experiments. These differences suggest that SPIs have at least partially diversified to provide protection against different attackers. Although we could not detect effects on plant development or growth when silencing SPIs, gene- and tissue-specific expression patterns suggest multiple functions in generative tissues, including a possible involvement in development.

This article refers to:

The Function of PIs, Their Diversity and Evolution

Directed and tightly controlled proteolysis is a key cellular requirement for all domains of life. In Arabidopsis, the more than 800 proteases from 60 families highlight the importance of proteolytic processes in plants.1 Such proteolytic machinery requires tight control to prevent unwanted degradation of proteins. Protease inhibitors (PIs) represent one possible way to achieve such control and there is evidence that plant PIs protect specific tissues, act as storage proteins and regulate the activity of proteases and direct their release.2,3 While these in planta physiological functions have been discussed, the majority of research on the function of PIs has been on their roles in a plant's interactions with other organisms, mainly in defense against insect herbivores by inhibiting digestive enzymes in the herbivore guts.3–5

Genes that play dual roles in plant defense and plant development and growth face an interesting evolutionary dilemma: their physiological functions will be conserved by stabilizing selection, while their defensive functions will be diversified by positive selection pressure resulting from the co-evolutionary arms race that plants have with their herbivorous and microbial attackers. Thus it is unlikely that a single PI gene, essential for growth and development, will simultaneously evolve to function as an important and evolutionary stable defense mechanism. However, if such a gene was duplicated, the different selection pressures could favor sub- and neo-functionalization that would allow the PI gene to escape this adaptive conflict.6,7 Interestingly, the PI-II family of serine protease inhibitors (SPI), classified as I20 in the MEROPS database,8 might provide an example of this evolutionary process. It is distributed throughout the Magnoliophyta largely as a single copy, e.g., in Arabidopsis, maize, rice and poplar, suggesting a common ancestral gene (Pfam domain: PF02428).9,10 The function of PI-II homologs in these species is unknown, although Arabidopsis expression profiles suggest a possible role in flower development and defense against pathogens (Genevestigator, gene ID: AT1G72060).11 In contrast, approximately 90% of the known PI-II members to date are of solanaceous origin, displaying a stunning diversity of 78 different protein sequences, comprising 288 domain forms in 30 species.10 Obviously the ancestral gene was duplicated early in the evolution of the Solanaceae,9,12 laying the foundation for gene diversification in this family.

The analysis of Kong and Ranganathan12 revealed that the active sites and the linker regions of PI-II inhibitors harbor the signatures of having been under positive selection pressure. Thus, if neo- or sub-functionalization had occurred in these genes, we would expect differences in substrate specificity, tissue localization, response to elicitors, or effects on herbivores.13 Moreover, if some of these PIs play a physiological role in planta, plants silenced for single inhibitors or their combinations should display different growth-related or developmental phenotypes. Given that gene diversification represents an ongoing evolutionary process, it is also possible that these genes are in the process of diversification and exhibit functional overlap or multiple roles.

Genetic and Functional Diversity of SPIs in Solanum nigrum

In our recent publication14 we profiled the SPIs of S. nigrum, tested their importance for plant defense and examined indications for neo- or sub-functionalization. Our research confirmed the importance of some of the identified SPIs for defense against herbivores, in some cases with overlapping or synergistic functions. However, we also demonstrated that the four different SPIs in S. nigrum differ substantially in their expression patterns, their substrate specificities, and their defensive properties. Although we could not identify any effects on growth and development when silencing individual PI forms using RNAi, the tissue-specific expression and the lack of anti-herbivore properties suggested functions other than defense in the case of some PIs (e.g., SPI1 and SPI2a). Interestingly, we also could not observe a defect in seed development when knocking down SPI2a and SPI2b expression, as has been shown for S. americanum,15 a close relative of S. nigrum. However, without knowing the exact mechanism by which PIs affect seed development, it will be difficult to understand this variability between species and the particular function of SPIs. A comparison of SPI profiles across different Solanum spp. revealed that they differ even between closely related species which might also explain different phenotypes in the silenced plants.

Sin et al.15 found that SaPIN2a is particularly expressed in ovules and young seeds. The specificity of SnSPI2a and SnSPI2b for subtilisin, which was also observed for SaPIN2a by Wang et al. suggests that these SPIs interact with a plant subtilase in the ovary.16 However, the constitutively high expression of SPIs in flowers could certainly serve a dual function. In addition to a putative involvement in seed development, SPIs might accumulate in flowers as a constitutive defense against herbivores to protect these highly fitness-relevant tissues. As a consequence we predicted different SPI accumulation patterns for the various flower tissues, in accordance with possible roles in defense or development. When we dissected flowers of S. nigrum to measure PI activity in different parts, we found the expression of the different SPIs to be tissue-specific (Fig. 1). Generative tissues exhibit much higher levels of PIs when compared to the corolla, which is consistent with the hypothesis of SPIs accumulating to form a constitutive defense barrier against herbivores in these tissues. However, by comparing the ratios of subtilisin and trypsin PI activity we found that anthers express high amounts of SnSPI2a and SnSPI2b (both subtilisin-specific) but very little SnSPI2c (trypsin-specific). Gynoecia seem to express high amounts of SPI2c and rather little SPI2a or SPI2b—in contrast to the findings by Sin et al. (2006) in S. americanum,15 who found SPI2a and SPI2b expression mostly localized in the gynoecia and the style. These different accumulation patterns suggest specialized functions of the different PIs in either their interaction with other organisms or in development in different tissues. The high PI concentration in anthers, for example, could limit the amount of pollen collected by pollinators or pollen robbers. On the other hand the high PI activity in ovaries and anthers suggests a common reason for their expression in sporogenic tissues: perhaps SPIs are not only involved in the formation of embryos and seeds but also in microsporogenesis and pollen formation. In this context, it is interesting to note that a serine-protease was found to be involved in microsporogenesis in anthers of Lilium longiflorum.17 Although we could not find any obvious effects on pollen viability (data not shown) it is possible that similarly subtle effects as in seed development occur.

In summary, these findings illustrate how much is still unknown about the multiple and specialized functions of PIs, which might extend far beyond a simple concept of general defense against herbivores. The evolutionary dynamics, functional overlaps and synergistic actions may complicate the analysis but offer a fascinating opportunity to examine the dynamic evolution of a gene family under different selection pressures. Integrating plant development, cell biology and ecology will be imperative to fully uncover the functions of SPIs.

Figures and Tables

Figure 1 Mean ± SE (n = 3) total protein concentration (A), trypsin PI activity (B) and subtilisin PI activity (C) in flower tissues of S. nigrum wild-type plants. FM, fresh mass.

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