13 Phloem Feeding Insect Stress and Photosynthetic Gene Expression

The ability to photosynthesise (i.e., to utilize solar energy for conversion into chemical energy) is a distinguishing characteristic unique to plants, algae and photoautotrophic bacteria. It is believed that photosynthesis was already well established at least 3.5 billion (Gyr) years ago in ancient organisms, with similar capabilities as that of modern cyanobacteria (Schidlowski, 1984, 1988; Blakenship, 1992). However, it is only much later (i.e., between 2.3 to 2.7 Gyr ago), with the advent of oxygen-evolving photosynthesis, that advanced life became possible (Buick, 1992; Björn & Govindjee, 2009). For sunlight to be converted into chemical energy, it must first be absorbed by organisms through the use of pigments. The primary light absorbing pigments, located in the thylakoid membrane of chloroplasts of eukaryotic cells, are Chlorophyll a (Chl a) and Chlorophyll b (Chl b). These pigments are located in the thylakoid membrane of the chloroplast, and absorb different light wavelengths so as to accumulate energy in the form of excited electrons. Secondary pigments, such as carotenoids (carotenes and xanthophyll), are located in the chloroplast membrane and outer membrane in order to absorb residual light wavelengths not efficiently absorbed by the primary pigments (Blankenship, 1992; Nelson & Yocum, 2006; Björn & Govindjee, 2009). This conversion of solar to chemical energy is a complex process and involves a large number of pigments and electron transfer proteins, collectively known as a photosynthetic unit (i.e., photosynthetic reaction centre) (Buttner et al., 1992). In a photosynthetic system the pigments serve as an antenna, collecting light and transferring the energy to the reaction centre, where the reactions leading to chemical energy conversion take place. The photosynthetic reaction centres, or the cores of light harvesting systems, consist of special protein-chlorophyll complexes which play a major role in the energy conversion process (Buttner et al., 1992). Oxygenic photosynthesis of chloroplasts involves two photosystems: the oxygen-evolving photosystem II (PSII) that originated from purple


Introduction
The ability to photosynthesise (i.e., to utilize solar energy for conversion into chemical energy) is a distinguishing characteristic unique to plants, algae and photoautotrophic bacteria.It is believed that photosynthesis was already well established at least 3.5 billion (Gyr) years ago in ancient organisms, with similar capabilities as that of modern cyanobacteria (Schidlowski, 1984(Schidlowski, , 1988;;Blakenship, 1992).However, it is only much later (i.e., between 2.3 to 2.7 Gyr ago), with the advent of oxygen-evolving photosynthesis, that advanced life became possible (Buick, 1992;Björn & Govindjee, 2009).For sunlight to be converted into chemical energy, it must first be absorbed by organisms through the use of pigments.The primary light absorbing pigments, located in the thylakoid membrane of chloroplasts of eukaryotic cells, are Chlorophyll a (Chl a) and Chlorophyll b (Chl b).These pigments are located in the thylakoid membrane of the chloroplast, and absorb different light wavelengths so as to accumulate energy in the form of excited electrons.Secondary pigments, such as carotenoids (carotenes and xanthophyll), are located in the chloroplast membrane and outer membrane in order to absorb residual light wavelengths not efficiently absorbed by the primary pigments (Blankenship, 1992;Nelson & Yocum, 2006;Björn & Govindjee, 2009).This conversion of solar to chemical energy is a complex process and involves a large number of pigments and electron transfer proteins, collectively known as a photosynthetic unit (i.e., photosynthetic reaction centre) (Buttner et al., 1992).In a photosynthetic system the pigments serve as an antenna, collecting light and transferring the energy to the reaction centre, where the reactions leading to chemical energy conversion take place.The photosynthetic reaction centres, or the cores of light harvesting systems, consist of special protein-chlorophyll complexes which play a major role in the energy conversion process (Buttner et al., 1992).Oxygenic photosynthesis of chloroplasts involves two photosystems: the oxygen-evolving photosystem II (PSII) that originated from purple bacteria and the ferredoxin reducing photosystem I (PSI) that originated from the green sulphur bacteria (Figure 1) (Xiong et al., 2000;Dent et al., 2001).

Chloroplast envelope membrane
Cell cytoplasm Photosystem I (PSI) reaction centre complex consists of 6 polypeptides containing two of subunit I, which associates with P700, subunit PSI-D, subunit PSI-E, quinones and fluorenones.TMP14 thylakoid membrane phosphoprotein (14 kDa), a novel subunit of the plant PSI (Khrouchtchova et al., 2005) is found second, after PSI-D, as phosphorylation subunits of PSI (Hansson & Vener, 2003).).It is probably involved in the interaction with LHCII and together with PSI-D ensures PSI's function by accepting electrons from PSII (Khrouchtchova et al., 2005).Photosystem I P700 is bound by PsaA and PsaB in PSI and function as the primary electron donor.PSI converts photonic excitation into a charge separation, which transfers an electron from the donor P700 chlorophyll pair to the spectroscopically characterized acceptors A0, A1, FX, FA and FB in turn.Photosystem I P700 induction ensures electron excitation and reduction might force the synthesis of reactive oxygen intermediates (ROIs) for the hypersensitive response (i.e., oxidative burst during plant defence) (Grotjohann & Fromme, 2005).Each PSI P700 antenna molecule consists of twenty chlorophyll a molecules and a cytochrome 522 heme (Bengis & Nelson, 1977).PSI utilises photons at 700 nm wavelength to excite electrons collected from its antenna molecule P700.The electrons produced by PSI are transferred to PSII, where it is excited, captured by ferredoxin and finally used to reduce NADP + to NADPH.ATP is produced from ADP and pyrophosphate via chemiosmosis.The energy for this process is produced by three hydrogen ions, which supply the energy by passing from the thylakoid to the stroma of the chloroplast.Both ATP and NADPH are subsequently used in the light-independent reactions of the PSII complex, to convert carbon dioxide into glucose using the hydrogen atom extracted from water by PSII, and releasing oxygen as a by-product (Fromme, 1996;Nelson & Yocum, 2006).

LHCII
Photosystem II (PSII) reaction centre complex on the other hand consists of D-1 and D-2 polypeptides, five chlorophyll a, two pheophytin a, one B-carotene, and one or two cytochrome b-559 heme-molecules (Nanba & Satoh, 1987).In PSII, the P680 reaction centre captures photons, and the light energy is used for oxidation (splitting) of water molecules.Upon electron release, the water molecule is broken into oxygen gas and released into the atmosphere.The resulting hydrogen ions are then used to power ATP synthesis.The electrons, excited at the antenna molecule P680, are passed down a chain of electrontransport proteins while receiving extra electrons from PSI.More hydrogen ions are pumped across the membrane as these electrons flow down the chain providing more protons for ATP synthesis.Chloroplast ATP synthase (cpATPase) is found to be essential for photosynthesis (Maiwald et al., 2003) by playing a direct role in the translocation of protons across the membrane as a key component of the proton channel.Nine different polypeptides make up the cpATPase, which consists of intrinsic CF 0 and extrinsic CF 1 segments.The Ftype ATPase CF 1 segment functions as the catalytic core and the CF 0 segment functions as the membrane proton channel (Cramer et al., 1991;Groth & Strotmann, 1999).The electrons are then transported as NADPH molecules to enzymes that build sugar from water and carbon dioxide (Nanba & Satoh, 1987).

Photosynthetic genes respond to biotic stressors
Plants are constantly locked in an evolutionary arms race with their biological attackers, whether they be viral, bacterial or fungal pathogens, parasitic plants or herbivorous insects, therefore imposing the need to evolve defensive strategies to overcome this onslaught.
Although there are a few examples of compensatory stimulation of photosynthesis (Trumble et al., 1993), most reports suggest that a decline in photosynthetic capacity is inevitable and this may represent the "hidden fitness costs" to defence (Fouché et al., 1984;Zangerl et al., 2002Zangerl et al., , 2003;;Heng-Moss et al., 2003;Bilgin et al., 2008Bilgin et al., , 2010;;Nabity et al., 2009).A recent study by Bilgin et al. (2010), reported that photosynthesis associated genes were downregulated in seven different dicotyledonous and one coniferous plant species upon exposure to twenty different forms of biotic damage, regardless of the type of biotic attack.The hosts seem able to down-regulate these genes as an adaptive response to biotic attack, since a reduction in gene expression does not necessarily translate into loss of function.
Once invasion by an attacker has been recognized, through the detection of various effectors (either pathogen-associated molecular patterns (PAMPs), microbe-associated molecular patterns (MAMPs), viral coat proteins or insect salivary elicitors), the host must balance competing demands for metabolic resources between either supporting defence versus sustaining cellular maintenance, growth and reproduction (Berger et al., 2007a,b).Plant defence can be costly in terms of plant growth and fitness (Tian et al., 2003;Zavala & Baldwin, 2004), as in addition to the mobilization of an array of defensive strategies (i.e., upregulation of a suite of defence response genes and production of chemical defence responses), the plant usually also has to cope with a reduction in effective biomass, and a decline in photosynthetic capacity in the remaining leaf tissue (Zangerl et al., 2002;Bilgin et al., 2008Bilgin et al., , 2010;;Nabity et al., 2009).

Regulated gene Host species Aphid species
Platform1 Ref2

291
PFIs achieve these benefits, at some cost to their host by inducing genes, involved in carbon assimilation and mobilization, so as to increase their own sugar uptake, whilst at the same time depleting sugars and creating localized metabolic sinks (Moran & Thompson, 2001;Zhu-Salzman et al., 2004).PFIs also modify nitrogen allocation in their hosts by upregulating genes involved in nitrogen assimilation.In particular, genes encoding enzymes required for the synthesis of tryptophan and other essential amino acids are up-regulated to fulfil to the dietary requirements of the PFI (Heidel & Baldwin, 2004;Zhu-Salzman et al., 2004;Botha et al., 2010).

Linking photosynthesis and plant defence
The linkage between photosynthesis and host defence was recently demonstrated by silencing two central photosynthetic proteins, i.e., ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase in Nicotiana attenuata using virusinduced gene silencing (VIGS) (Mitra & Baldwin, 2008).Silencing of these genes improved the performance of a native generalist (Spodoptera littoralis) and specialist (Manduca sexta) herbivorous larvae on transformed host plants (Mitra & Baldwin, 2008).Similarly, it was shown that independent silencing of the TMP 14 kDa thylakoid membrane phosphoprotein, PSI P700 apoprotein, and Fructose-1,6-bisphosphatase in near-isogenic (NILs) Triticum aestivum lines using VIGS, also affected host resistance to Diuraphis noxia in varying degrees (Jackson, 2010).In the latter study, no significant decrease in aphid fecundity was observed in the susceptible Tugela plants after silencing with any of the genes when compared with the uninfested Tugela plants (Table 2).Silencing with BSMV:TMP14 caused a significant increase in aphid fecundity when aphids were fed on the resistant Tugela-Dn1 plants, while silencing with BSMV:FBPase caused an increase in aphid fecundity in resistant Tugela-Dn2 plants.A significant increased aphid fecundity was also observed upon silencing of Tugela-Dn2 plants with BSMV:P700, but in Tugela-Dn1 plants no significant increase in aphid fecundity was observed (Table 2).(Wang et al., 2004) b SA2199/6* 'Tugela' (Dn2 + ) -confers tolerance to Diuraphis noxia (Wang et al., 2004) Table 2. Summary of Diuraphis noxia fecundities when feeding on three near isogenic wheat lines before and after gene silencing.Aphid fecundity is indicated as number of aphids per plant 10 d.p.i.(n=3)(From Jackson, 2010).

Does the photosynthetic compensation of the wheat host form part of Diuraphis noxia's defence strategy?
Diuraphis noxia feeding on susceptible wheat causes chlorosis (i.e., longitudinal chlorotic streaking) and leaf rolling in the leaves of susceptible wheat (Figure 2).Leaf chlorophyll content is reduced by D. noxia infestation (Heng-Moss et al., 2003;Botha et al., 2006).This results in decreased photosynthetic potential and the eventual collapse of the plant (Burd & Burton, 1992).Aphid damage has historically been ascribed to a phytotoxin injected during feeding, which is responsible for chloroplast disintegration (Fouché et al., 1984).Although, such a phytotoxin has never been described or isolated, ultrastructural studies revealed limited chloroplast breakdown in the leaves of resistant cultivars after aphid feeding ( Van der Westhuizen et al., 1998).Since cell fluorescence data has shown that D. noxia feeding causes reduced photosynthetic capacity even in intact chloroplasts (Haile et al., 1999), this chloroplast rupture mechanism seems unlikely.D. noxia feeding probably induces malfunctioning of the photosynthetic apparatus in the stacked region of the thylakoid membrane, but the exact site of interference has not been determined (Burd & Elliott, 1996;Heng-Moss et al., 2003).Chlorosis induced by D. noxia differs significantly from normal chlorophyll degradation during leaf senescence (Ni et al., 2001).D. noxia feeding stimulates an increase in the activity of Mg-dechelatase, a catabolic enzyme that converts chlorophyllide a to pheophorbide a as the final step in the chlorophyllase pathway (Ni et al., 2001;Wang et al., 2004) Total chlorophyll concentration assays indicate that D. noxia feeding causes a marked decrease in chlorophyll levels in Tugela, but that the reduction in the antibiotic near-isogenic line (NIL) Tugela-Dn1 is much less severe (Botha et al., 2006).Since the phenotypes afforded by different Dn genes vary  Dn1 confers antibiosis, Dn2 tolerance and Dn5 a combination of antibiosis and antixenosis (Wang et al., 2004)  it appears that the presence of these genes activate transcription of defence-related genes differently (Botha et al., 2008).Antibiotic Betta-Dn1 plants are also unable to compensate for chlorophyll loss, which has been attributed to an increase in defence compound production.Tolerant Betta-Dn2 plants have very stable chlorophyll content during D. noxia feeding, suggesting that they can compensate for chlorophyll loss in some way (Heng-Moss et al., 2003).
Susceptible wheat shows decreased levels of chlorophyll a upon infestation by D. noxia (Burd & Elliott, 1996;Ni et al., 2001;Wang et al., 2004) which indicates damage to PSI (Botha et al., 2006).If this is indeed the case, it has serious implications for susceptible wheat under aphid attack.PSI catalyzes the electron transport from plastocyanin to ferredoxin (Haldrup et al., 2003).This reduced ferredoxin pool is mostly employed in generating NADPH for CO 2 assimilation, but is also used in regulating the activity of, among others, CF 1 -ATPase and several enzymes in the Calvin cycle (Ruelland & Miginiac-Maslow, 1999).Underreduced ferredoxin directly diminishes the plant's ability to synthesize ATP and carbohydrates.Studies by Van Eck (2007) using qRT-PCR analysis of the CF 1 -ATPase response to aphid feeding indicated an increased demand for ATPase transcripts as infestation progressed (Figure 3).Since damaged PSI can no longer act as electron acceptor from PSII via the cytochrome b 6-f complex, inefficient reoxidation of the reduced plastoquinone occurs, halting state transitions and resulting in an over-reduction of PSII (Burd & Elliott, 1996).This leads to photoinactivation of PSII, and thus the irreversible decline in functional PSII complexes, because the absorbed light energy exceeds the amount that can be employed in electron transport (Kornyeyev et al., 2006).An acute induction of a TMP 14 kDa thylakoid membrane phosphoprotein, a putative component of PSI, was observed in Tugela-Dn2 after infestation with RWA (Table 1, Figure 3) which indicates transcriptionally regulated photosynthetic compensation (Van Eck, 2007).An induction of TMP14 could be a strategy to overcome pest attack in order to keep PSI stable and energy production going, while a reduction of TMP14 might force energy to flow in a different direction.Thus, up-regulation of PSI complexes would ensure the integrity of electron transport from PSII during state 2 as well as increased levels of NADPH and possibly increased CO 2 assimilation.In growth tolerance experiments, the tolerant PI 262660 line containing the Dn2 gene maintained vigorous growth during aphid infestation when compared to the susceptible Arapahoe and antibiotic PI 137739 line (Haile et al., 1999).It is suggested that increased photosynthetic capacity via up-regulation of photosystem components may provide a mechanism for passive resistance against D. noxia feeding (Botha et al., 2006).

Regulating plant homeostasis
The production of ROIs is a by-product of normal cellular processes, such as photosynthesis and respiration, but can also be produced in response to a variety of environmental conditions, i.e., light, cold, drought, as well as pathogen and pest attack.The latter event is known as the hypersensitive response and has proven to be effective against sedentary insects, such as PFIs that target a specific tissue.However, for an effective hypersensitive response-based programmed cell death to occur, a cascade of events have to occur, including production of ROIs and associated downstream defensive responses.Indeed, increases in the activity of oxidative enzymes such as peroxidases, polyphenol oxidases and lipoxygenases were observed after Diuraphis noxia feeding ( Van der Westhuizen et al., 1998;Ni et al., 2000Ni et al., , 2001;;Ni & Quisenberry, 2003).This increase occurs not only at the site of feeding, but it also spreads systemically ( Van der Westhuizen et al. 1998).Thus, if this spreading is not kept at bay, it can be lethal to the host.ROIs are partially reduced forms of atmospheric oxygen (O 2 ), and typically result from the excitation of O 2 to form singlet oxygen O 2 1 or from the transfer of one, two or three electrons to O 2 to form, respectively, a superoxide radical (O 2 -), hydrogen peroxide (H 2 O 2 ) or a hydroxyl radical (HO -).Unlike atmospheric oxygen, ROIs are capable of unrestricted oxidation of various cellular components and can lead to the oxidative destruction of the cell (Asada, 1999).A variety of mechanisms exist for the dissipation of excess excitation that may give rise to the generation of reductants and the production of ROIs, act as signalling agents, or to serve as alternative  electron acceptors to avoid over-reduction and potentially the generation of toxic intermediates (Avenson et al., 2005;Mullineaux & Karpinski, 2002;Foyer & Noctor, 2005).It is thus suggested that cellular homeostasis will be maintained as long as the mechanisms for redox poising are in place, otherwise uncontrolled cellular damage will follow leading to death of the host (Schelbe et al., 2005).

SunlightFig. 1 .
Fig. 1.Indicated are Photosystems I and II's location and their respective functions.The thylakoid membrane with PSI and PSII are indicated with the energy flow through the Calvin cycle (Modified from Dent et al., 2001).

Fig. 3 .
Fig.3.qRT-PCR expression profiles of ATPase, where the expression level is calculated relative to the expression level of the uninfested, susceptible Tugela (at 0 h.p.i.) sample and is normalized to the expression of the unregulated chloroplast 16S rRNA transcript.

Table 1 .
Genes involved in photosynthesis under regulation after aphid feeding.Underlined genes are down-regulated, non-underlined genes are mostly up-regulated.
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