Stress inducible proteinase inhibitor diversity in Capsicum annuum

Manasi Mishra 0 Neha Mahajan 0 Vaijayanti A Tamhane 0 1 Mahesh J Kulkarni 0 Ian T Baldwin 2 Vidya S Gupta 0 Ashok P Giri 0 0 Plant Molecular Biology Unit, Division of Biochemical Sciences, CSIR-National Chemical Laboratory , Dr. Homi Bhabha Road, Pune, MS 411 008 , India 1 Present address: Institute of Bioinformatics and Biotechnology, University of Pune , Pune, MS 411 007 , India 2 Department of Molecular Ecology, Max Planck Institute for Chemical Ecology , Jena 07745 , Germany Background: Wound-inducible Pin-II Proteinase inhibitors (PIs) are one of the important plant serine PIs which have been studied extensively for their structural and functional diversity and relevance in plant defense against insect pests. To explore the functional specialization of an array of Capsicum annuum (L.) proteinase inhibitor (CanPIs) genes, we studied their expression, processing and tissue-specific distribution under steady-state and induced conditions. Inductions were performed by subjecting C. annuum leaves to various treatments, namely aphid infestation or mechanical wounding followed by treatment with either oral secretion (OS) of Helicoverpa armigera or water. Results: The elicitation treatments regulated the accumulation of CanPIs corresponding to 4-, 3-, and 2-inhibitory repeat domains (IRDs). Fourty seven different CanPI genes composed of 28 unique IRDs were identified in total along with those reported earlier. The CanPI gene pool either from uninduced or induced leaves was dominated by 3-IRD PIs and trypsin inhibitory domains. Also a major contribution by 4-IRD CanPI genes possessing trypsin and chymotrypsin inhibitor domains was specifically revealed in wounded leaves treated with OS. Wounding displayed the highest number of unique CanPIs while wounding with OS treatment resulted in the high accumulation of specifically CanPI-4, -7 and 10. Characterization of the PI protein activity through two dimensional gel electrophoresis revealed tissue and induction specific patterns. Consistent with transcript abundance, wound plus OS or water treated C. annuum leaves exhibited significantly higher PI activity and isoform diversity contributed by 3- and 4-IRD CanPIs. CanPI accumulation and activity was weakly elicited by aphid infestation yet resulted in the higher expression of CanPI-26, -41 and 43. Conclusions: Plants can differentially perceive various kinds of insect attacks and respond appropriately through activating plant defenses including regulation of PIs at transcriptional and post-translational levels. Based on the differentially elicited CanPI accumulation patterns, it is intriguing to speculate that generating sequence diversity in the form of multi-IRD PIs is a part of elaborative plant defense strategy to obtain a diverse pool of functional units to confine insect attack. - Background Plants have evolved elaborate defense strategies composed of constitutive and inducible responses in order to cope with herbivore challenges. The induced defenses commence only when herbivore-derived signals are perceived by the plants. A wide array of studies has reported the induction of direct and indirect plant defenses in response to herbivory and other biotic stresses [1-4]. Insect damage, mechanical wounding and/or elicitors in insect oral secretions (OS), such as fatty acid amino acid conjugates, volicitin, inceptins, caeliferins, and glucose oxidase, stimulate the local and systemic release of signaling intermediates like systemin and/or jasmonic acid; these then amplify the defense cascade throughout the plant [5-7]. Though the major consequence of herbivory is wounding, plants' responses to insect feeding are more complex due to the elicitors present in insect OS [8]. Defense responses entail the regulated activation of plant defense genes and the suppression of growth-related genes [8,9]. As a result, defensive metabolites and/or proteins accumulate in plants within the local tissues damaged by herbivores as well as systemically in undamaged tissues. The accumulation of trypsin and chymotrypsin-like proteinase inhibitors (PIs) throughout the aerial tissues of tomato and potato plants was demonstrated to be a direct consequence of insect-mediated damage or mechanical wounding [10]. Thus, serine PIs represent one of the best examples of locally and systemically induced responses in Solanaceous plants [11-16]. The constitutive expression of PIs, which has been reported to occur in storage organs and the reproductive tissues of plants, may fulfill anti-insecticidal as well as other endogenous functions in planta [4,13,16-18]. Most Solanaceae members contain the multi-gene family encoding Pin-II type PIs [4,16,19], which possesses considerable sequence diversity resulting from variations in tandem sequence repeats, domain duplications and circularly permuted domain organizations [20]. A distinct feature of these PIs is the presence of tandem repeats of a 50-amino-acid polypeptide called inhibitory repeat domain (IRD), which can vary from 1 to 8 with interconnecting linker peptides. Each IRD contains 8 conserved cysteines (Cys) along with a reactive site for targeting a serine protease. Gene duplication events have resulted in the evolution of the multi-domain Pin-II family of PIs with structurally and functionally divergent IRDs [21]. Horn et al. [22] isolated a set of IRDs resulting from the differential proteolysis at the linker peptide separating the subunits of a 7-domain precursor from methyl-jasmonate-elicited N. attenuata leaves. The sequence variability in the multi-gene family of Pin-II PI proteins, their regulated expression and their posttranslational processing are together responsible for generating a PI cocktail effective in defense and/or endogenous function [4,16,23]. Several different PI proteins and genes with 1- to 4-IRDs have been identified and characterized from C. annuum (CanPIs) tissues [16,24-29]. There was substantial variability in the induced expression of CanPIs upon aphid infestation, virus infection, chewing by insects and mechanical wounding. The abundance of transcripts did not always result in higher CanPI proteins, though they were well correlated in lepidopteran-infested C. annuum leaves. Furthermore, these studies indicated that many CanPIs are expressed simultaneously, but the significance of such PI expression diversity in C. annuum remains unclear. In order to examine the potential functional specificities of the various isoforms of CanPI in C. annuum, we addressed the following questions: (i) Does elicitation increase PI isoform diversity? (ii) How specialized is the induction response to a particular treatment? Following experimental inductions of C. annuum leaves, we investigated the diversity in CanPI transcript and protein profiles. Sequencing revealed 24 novel CanPI transcripts, increasing the total known to 47. Selective analysis of PI activity in proteomes using 1D and 2D electrophoresis followed by mass spectrometry revea (...truncated)