Previous studies documented the differential effects of wounding and insect feeding on the primary and defense metabolism of higher plants, based on the monitoring of model genes and proteins, or, more recently, on the use of 'omics' approaches for a systemic analysis of mRNA transcript or protein complements. Transcriptomics has been instrumental over the years to decipher complex physiological processes in plant-insect systems, which often implicate dynamic cross-talks between defense pathways and the regulation of numerous genes in host plant tissues (e.g.
[21, 22, 42, 53–56]). Studies also confirmed the usefulness of proteomics in recent years, for the elucidation of plant-insect interactions at the proteome and metabolic levels (e.g.
[35–40]). Our data pointing to the onset of distinct protein regulation patterns in mechanically wounded and insect-treated plants despite similar transcriptional control patterns here illustrate the often described discrepency between transcriptomic and proteomic data generated from complex biological systems, including plant-insect systems
. From a biological standpoint, they may reflect in part the regulatory role of metabolic effectors in the oral secretions of attacking herbivores, and confirm the relevance of proteins as useful biomarkers for a realistic account of the situation in vivo.
About thirty proteins had their concentration increased or decreased by at least twofold in wounded or potato beetle-treated potato leaves under our experimental conditions, similar to Giri et al.
 reporting the modulation of 18 proteins, out of approximately 500 monitored, in leaves of N. attenuata challenged with M. sexta larvae. Most interestingly, none of the proteins modulated here was up- or downregulated by all three stress treatments, and most of them were up- or downregulated by only one treatment (see Figure
3). These observations, together with the unaltered normalized volumes observed on 2-D gels for more than 90% of the proteins detected, underline the remarkable stability of the host plant's leaf proteome under various stress conditions. They also underline, in accordance with the treatment-specific expression patterns observed for Pin-II and protein P4 mRNA transcripts (Figure
1), the onset of stress-specific responses in planta. The differential, even diverging expression patterns observed for a number of proteins following different stress treatments (Table
2) highlight, in particular, the diversity of possible responses, not only involving the identity and expression rate of several genes and proteins, but also their accumulation trend in plant tissues.
Experimental biases influencing data interpretation, such as the focus on abundant proteins during 2-DE, the adoption of a conservative twofold threshold for protein spot selection or the use of a wound treatment not exactly reproducing the injury pattern observed during insect feeding, cannot be excluded de facto. It is well known that leaf damage pattern, intensity and duration may significantly impact stress perception and wound hormone (e.g. jasmonic acid) accumulation in wounded plants, with a likely impact on stress metabolic pathways and defense gene inductions
[20, 57]. Nevertheless, a number of observations support the hypothesis of distinct effects for the three treatments assessed. For instance, the limited and specific effects of potato aphids could be expected a priori given the feeding habits of these insects and their limited impact on the structural integrity of host leaf tissues
. Proteomic data indicating the upregulation of a PR-4 protein (Spot 504) upon aphid feeding was also in line with previous reports on plant-aphid interactions and those models proposing different recognition schemes in planta in response to phloem sap feeding and chewing arthropods
[28, 45, 58, 59].
Interpretation issues may remain more problematic for the mechanical wound treatment, but a number of observations, such as the detection of an Asp protease inhibitor isoform that is upregulated exclusively in wounded leaves (Spot 426) and the diverging accumulation trends of photosystem I reaction center subunit II (Spot 444) in leaves subjected to wounding and potato beetle feeding, indeed suggest differential, treatment-specific effects. Most convincingly, RT PCR data showed comparable repressing effects for wounding and potato beetle treatments on the transcription of some photosynthesis-related genes, despite clearly divergent accumulation trends on 2-D gels for the corresponding proteins (Spots 104, 304, 444 and 451). These findings suggest overall the onset of stress-specific gene and protein control mechanisms in the host plant involving a combination of regulatory events common to different stress cues, and treatment-specific regulatory events leading to distinct responses at the proteome level. In the present case, the differential metabolic effects of wounding and potato beetles could have been the result of common transcriptional regulation events triggered by wounding and the 'wound hormone' jasmonic acid
, combined with specific effects of the potato beetle regurgitant
[42, 43] post-translationally altering the turnover of some regulated proteins in planta.
In line with an earlier study reporting the induction of several defense-related genes in potato leaves challenged with potato beetle larvae
, defense-related proteins such as PR-proteins (e.g. proteins P2 and P4) and wound-inducible protease inhibitors were upregulated in leaves by wounding, potato aphid and/or potato beetle treatments. Of interest was, by contrast, the downregulation of a Kunitz Asp protease inhibitor in potato beetle-treated plants (Spot 469) (Figures
5). Asp protease inhibitors are well-characterized wound-/jasmonate-inducible proteins in potato leaves
, known to inhibit digestive Asp proteases in the Colorado potato beetle larval midgut
[63, 64]. From an ecological viewpoint, the downregulation of an Asp protease inhibitor and the slight repression of a second isoform despite a twofold upregulation in wounded leaves (Spot 426) (Figure
5) could represent an advantage for the insect in vivo. The repression of Ser protease inhibitor-encoding genes in wounded tomato leaves treated with potato beetle regurgitant has been reported earlier
, as well as the repression of two trypsin inhibitor-encoding genes in Arabidopsis leaves by oral secretions of the lepidopteran herbivore Spodoptera littoralis
. The biological significance of protease inhibitor downregulation in potato leaves remains equivocal in the present case given the number and diversity of protease inhibitor isoform- (including Kunitz inhibitor isoform-) encoding genes in the potato genome
, but it is tempting to speculate about a possible evasive strategy of the insect to elude detrimental digestive protease inhibition. In a similar way, the downregulation of an ATP synthase β subunit (Spot 104) (Figure
5) in potato leaves could contribute to attenuate the impact of host plant defenses and help maintain the insect's fitness, given the role attributed to host plant ATP synthase fragments as "non-self" triggers of defense responses upon herbivory
The downregulation of photosynthesis-related proteins in potato beetle-treated plants is more difficult to interpret. Proteomic and transcriptomic studies have already documented the post-translational modification
 or the downregulation of rubisco, rubisco activase or other photosynthesis-related proteins in insect-challenged leaves or in wounded leaves treated with insect oral secretions
[35, 39, 67]. Several hypotheses have been proposed to explain these observations in terms of metabolic strategies to sustain plant's or herbivore's fitness. A number of authors have suggested the need for a reallocation of carbon resources towards defense responses in the host plant
[67–69]. The degradation of rubisco, in this perspective, would provide the plant with an important source of amino acids for newly synthesized defense proteins
[52, 70], while also lowering the nutritive value of leaf tissues by limiting dietary protein availability to the aggressor
Two alternative, although non-exclusive hypotheses could explain the observed effects: (1) the secretion of biochemical effectors in the insect regurgitant which might limit energy resources available to the host plant or promote the accumulation of compounds, such as reactive oxygen species, toxic to plant cells
[72, 73]; and (2) a general disturbance of the whole plant system under stress conditions which might negatively affect photosynthesis and induce compensatory responses. The strong and specific downregulation of chloroplastic l-ascorbate peroxidase (Spot 349) and photosystem I proteins (Spots 444, 451 and 461) observed here following potato beetle feeding (Figure
5) is compatible with the hypothesis of reduced energy production and increased accumulation of toxic oxidizing molecules. Specific upregulation of the 60-kDa chaperonin α-subunit rubisco chaperone (Spot 72) and protein disulfide isomerase (Spot 76) upon potato beetle treatment, along with the maintenance of rubisco activase (Spot 172) content, support the idea of compensatory responses to sustain protein biosynthesis, folding and assembly. Overexpression of the 60-kDa rubisco chaperone, also observed in tobacco leaves attacked by M. sexta larvae
, could represent a general strategy for the plant to preserve rubisco assembly under stress conditions. Work is underway to assess the net impacts of mechanical wounding and potato beetle herbivory on photosynthesis in potato leaves, keeping in mind the striking plasticity of major physiological processes in plants. Work is also underway to further dissect the inducing effects of the potato-potato beetle system components, including the plant itself. Studies have described in recent years the elicitor activity of plant-derived compounds in plant-herbivore systems, including plant protein fragments coming back to the host plant via insect regurgitant
[14, 15, 75–77].