**3.4 Main photosynthetic genes targeted by biotic stress**

Curiously, the gene coding for the Rubisco enzyme, an absolutely vital component of the carbon assimilation machinery in plants, was found to be one of the primary photosynthetic genes targeted by herbivore attack, in addition to genes coding for the components of the antenna complexes in both photosystems (Logemann et al., 1995; Ehness et al., 1997; Hermsmeier et al., 2001; Hahlbrock et al., 2003; Hui et al., 2003; Montesano et al., 2004; Zou et al., 2005). Proteomic investigations provided further evidence of the vulnerability of the CO2 fixing process to insect attack by showing that herbivory, or even the application of the insect´s saliva, also reduced the abundance of Rubisco activase (RCA) in *Nicotiana attenuata* and Arabidopsis (Giri et al., 2006; Thivierge et al., 2010). RCA is a key regulatory enzyme of photosynthetic carbon assimilation that modulates the activity of Rubisco by facilitating the removal of inhibiting sugar phosphates from its active site (Portis, 1995). Additional findings derived from the proteomic experimental approaches indicated that Rubisco large subunit and RCA, in addition to PS I P700 apoprotein A1 suffered several caterpillar-specific modifications, including the conversion of Cys192 in Rubisco to the the thiolate anion, which may lead to decreased enzyme activity and protein degradation, and adverse modifications, in RCA, of the protein domains involved in ATP binding (Thivierge et al., 2010).

Friend or Foe? Exploring the Factors that Determine the Difference

accumulation of autotoxic defensive allelochemicals (see below).

down-regulation of photosynthesis (Halitschke et al., 2011).

**4.2 Positive or neutral effects of insect herbivory on photosynthesis** 

Between Positive and Negative Effects on Photosynthesis in Response to Insect Herbivory 173

the time, this variability stems from characteristic factors of a given plant insect interaction, including damage intensity (e.g. total vs. partial defoliation; dispersed vs. concentrated damage; phloem feeding vs. defoliation) and location (e.g. proximity to veins), type of tissue that is preferentially damaged (as mentioned above) and the way tissue is damaged (e.g. chewing vs. scraping; crushing vs. piercing, etc.). Another important factor that influences the outcome of herbivory on photosynthesis has to do whether insect damage induces the

Resistance and tolerance represent two general strategies of plant defense against herbivores, although interactions between these two strategies are assumed to occur under certain conditions, i.e. when the resources available for defense are limited or when both defensive strategies are physiologically costly (Leimu & Koricheva, 2006). Resistance involves the reduction of the amount of herbivore damage whereas tolerance leads to a reduction of the impact of herbivory on plant fitness (Rausher et al., 1993; Stowe et al., 2000). Resistance traits include mechanical and chemical characters that reduce herbivore performance (antibiosis) or preference (antixenosis). Conversely, proposed mechanisms for tolerance/compensation are re-growth stimulation, elevated rates of photosynthesis in remaining leaves of partially defoliated plants, increased branching through the release of apical dominance, alteration of phenology or plant architecture, production of new leaf area, utilization of high pre-herbivory stored carbon resources or the ability to reallocate them to less vulnerable tissues, resorption of nutrients from senescent/damaged leaves, especially nitrogen (N) and phosphorus (P), alteration of the external light environment and higher reproductive efficiency through increased percentage of fruit set (Mabry & Wayne, 1997; Hjalten et al., 1993; Strauss & Agrawal, 1999; Hochwender et al., 2000; Tiffin, 2000; Anten et al., 2003; Silla & Escudero, 2003; Leimu & Koricheva, 2006; Schwachtje et al., 2006). Compensatory ability in plants varies widely across species, and the degree in which it is manifested depends on the amount of leaf lost, with complete rather than fragmented defoliation usually being more conductive to an increased rate of net photosynthesis in the remaining or newly formed leaves (Welter, 1989). The mode of herbivore damage and herbivore type may also determine whether the overall effect on photosynthesis in the plant. This was elegantly evidenced in a recent report showing that herbivory on *N. attenuata* by *Tupiocoris notatus*, a cell-content feeder, (or by application on wounded plants of its salivary secretions), induced an elevated photosynthetic activity, and consequent CO2 assimilation, that appeared to compensate for lost tissue and for the fitness costs associated with the deployment of direct and indirect defenses. This compensatory effect was shown to be specific for this insect, since feeding by chewing *Manduca sexta* larvae resulted in a strong

Environmental conditions and the timing of the herbivory event are also influential factors. Thus, compensation to damage in terms of timing of herbivory is usually more effective when required early in the growing season or before the reproductive phase has started. For example, a study performed in Lebanese cucumber (*Cucumis sativus*) to compare the ability to compensate for foliar herbivory at both the pre-flowering and flowering stages found that damage produced before flowering allowed plants to compensate more efficiently, in terms of vegetative biomass and fruit production, for leaf losses that sometimes reached 80% of the total leaf area in the plant groups examined. Higher compensation was correlated with a

A valid explanation for the slower growth and down-regulation of photosynthetic-related genes elicited by herbivore damage is that it may be required to liberate resources, e.g. the high proportion of leaf N that is invested in photosynthetic proteins, primarily Rubisco, for defense-related processes (Baldwin, 2001). Another possibility is that these changes could represent a variety of the *scorched earth* strategies used by plants to buffer the impact of herbivory, based on the premise that a reduction in growth and nutrient availability, resulting from the combined effects of decreased photosynthesis, inhibited nitrate assimilation and diminished levels of amino acids and of the main dietary protein (i.e Rubisco) in leaves, will reduce the nutritional quality of the plant to the feeding insect, and consequently, the degree of attractiveness for subsequent damage (Hermsmeier et al., 2001; Hahlbrock et al., 2003; Schwachtje et al., 2006).

Silencing of either *RCA* or *Rubisco* in *N. attenuata* suppressed photosynthetic capacity, as expected, but uncovered further refinements regarding the defensive role played by a reduced expression of these genes in plants subjected to insect damage (Giri et al., 2006; Mitra et al., 2008). Surprisingly, insect performance of both specialist and generalist insect pests was increased in RCA-silenced plants, a result attributed mostly to an impairment of the JA-Ile/leucine signaling pathway required for the expression of defense-related genes coding for trypsin protease inhibitors or for enzymes needed for biosynthesis of defensive metabolites such as diterpene glycosides (Mitra et al., 2008). The negative effect exerted by RCA silencing on JA-Ile/leucine signaling and related herbivore resistance traits was hypothesized to occur in response to reduced ATP levels produced in carbon and energy depleted plants having decreased photosynthetic rates, since ATP is needed for the adenylation of JA, the first step in the amino acid conjugation process needed to regulate this hormone's activity (Staswick et al., 2002). On the other hand, no negative effects on JA signaling were detected in Rubisco-silenced plants, which nevertheless suffered greater damage when confronted by larvae of a specialist insect. These were proposed to have a higher tolerance than generalist insects to the protein deficiency resulting from Rubisco silencing due to their improved ability to detoxify plant defenses (Green et al., 2001). One of the principal conclusions reached from the results of the above series of experiments was that the nature of the photosynthetic genes affected as a consequence of insect herbivory will have important repercussions in their relation to plant defense.

### **4. Plant responses to herbivory from the phostosynthetic perspective**

#### **4.1 The profound impact of insect herbivory on plants**

Herbivory can negatively affect ecosystems by decreasing photosynthesis and net primary production. Estimates of global crop production losses caused by foliage-feeding insects typically range from 5% to 30%, with losses estimated to exceed 50% were it not for the widespread application of pesticides (Mattson & Addy, 1975; Oerke & Dehne, 1997). Additional losses range from 2 to 15% in forests and from 4 to 24% in old-fields and grasslands, while insect outbreaks have been known to reduce net primary productivity by 70% to 100% in some terrestrial ecosystems (Cyr & Pace, 1993). Insect herbivory reduces leaf area or depletes leaf fluids by mining and cell content feeding. It can also be selective, targeting other tissues such the phloem, xylem (Haile et al., 1999; Macedo et al., 2003a, b; Heng-Moss et al., 2006), or the stems (Macedo et al., 2005, 2007). Insect feeding typically reduces photosynthesis, although evidence showing positive or neutral effects (i.e. tolerance or compensatory responses; see below) on photosynthesis has also been reported. Most of

A valid explanation for the slower growth and down-regulation of photosynthetic-related genes elicited by herbivore damage is that it may be required to liberate resources, e.g. the high proportion of leaf N that is invested in photosynthetic proteins, primarily Rubisco, for defense-related processes (Baldwin, 2001). Another possibility is that these changes could represent a variety of the *scorched earth* strategies used by plants to buffer the impact of herbivory, based on the premise that a reduction in growth and nutrient availability, resulting from the combined effects of decreased photosynthesis, inhibited nitrate assimilation and diminished levels of amino acids and of the main dietary protein (i.e Rubisco) in leaves, will reduce the nutritional quality of the plant to the feeding insect, and consequently, the degree of attractiveness for subsequent damage (Hermsmeier et al., 2001;

Silencing of either *RCA* or *Rubisco* in *N. attenuata* suppressed photosynthetic capacity, as expected, but uncovered further refinements regarding the defensive role played by a reduced expression of these genes in plants subjected to insect damage (Giri et al., 2006; Mitra et al., 2008). Surprisingly, insect performance of both specialist and generalist insect pests was increased in RCA-silenced plants, a result attributed mostly to an impairment of the JA-Ile/leucine signaling pathway required for the expression of defense-related genes coding for trypsin protease inhibitors or for enzymes needed for biosynthesis of defensive metabolites such as diterpene glycosides (Mitra et al., 2008). The negative effect exerted by RCA silencing on JA-Ile/leucine signaling and related herbivore resistance traits was hypothesized to occur in response to reduced ATP levels produced in carbon and energy depleted plants having decreased photosynthetic rates, since ATP is needed for the adenylation of JA, the first step in the amino acid conjugation process needed to regulate this hormone's activity (Staswick et al., 2002). On the other hand, no negative effects on JA signaling were detected in Rubisco-silenced plants, which nevertheless suffered greater damage when confronted by larvae of a specialist insect. These were proposed to have a higher tolerance than generalist insects to the protein deficiency resulting from Rubisco silencing due to their improved ability to detoxify plant defenses (Green et al., 2001). One of the principal conclusions reached from the results of the above series of experiments was that the nature of the photosynthetic genes affected as a consequence of insect herbivory will

Hahlbrock et al., 2003; Schwachtje et al., 2006).

have important repercussions in their relation to plant defense.

**4.1 The profound impact of insect herbivory on plants** 

**4. Plant responses to herbivory from the phostosynthetic perspective** 

Herbivory can negatively affect ecosystems by decreasing photosynthesis and net primary production. Estimates of global crop production losses caused by foliage-feeding insects typically range from 5% to 30%, with losses estimated to exceed 50% were it not for the widespread application of pesticides (Mattson & Addy, 1975; Oerke & Dehne, 1997). Additional losses range from 2 to 15% in forests and from 4 to 24% in old-fields and grasslands, while insect outbreaks have been known to reduce net primary productivity by 70% to 100% in some terrestrial ecosystems (Cyr & Pace, 1993). Insect herbivory reduces leaf area or depletes leaf fluids by mining and cell content feeding. It can also be selective, targeting other tissues such the phloem, xylem (Haile et al., 1999; Macedo et al., 2003a, b; Heng-Moss et al., 2006), or the stems (Macedo et al., 2005, 2007). Insect feeding typically reduces photosynthesis, although evidence showing positive or neutral effects (i.e. tolerance or compensatory responses; see below) on photosynthesis has also been reported. Most of the time, this variability stems from characteristic factors of a given plant insect interaction, including damage intensity (e.g. total vs. partial defoliation; dispersed vs. concentrated damage; phloem feeding vs. defoliation) and location (e.g. proximity to veins), type of tissue that is preferentially damaged (as mentioned above) and the way tissue is damaged (e.g. chewing vs. scraping; crushing vs. piercing, etc.). Another important factor that influences the outcome of herbivory on photosynthesis has to do whether insect damage induces the accumulation of autotoxic defensive allelochemicals (see below).
