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A suppression-subtractive-hybridization (SSH) strategy led to the identification of several genes whose expression was differentially modified in response to different larval phases present during the infestation process of tomato plants (Solanum lycopersicum) by virus-free whitefly Bemisia tabaci (Bt). The findings regarding photosynthetic gene expression were in accordance to previous studies reporting altered patterns of expression as a result of insect herbivory. However, the examination, in this study, of four stages of larval Bt development permitted the detection of phase-dependent changes in gene expression which appeared to target specific photosynthetic complexes. Thus, an upregulation of photosystem II genes in the latter two phases of Bt development contrasted with a general repression of genes belonging to the three other photosynthetic complexes, in addition to a number of genes coding for proteins associated with the oxygen evolving complex and the Calvin cycle. We propose that the contrasting pattern of expression led to an over-excitation of PSII and consequent oxidative damage, as suggested by the concomitant upregulation of oxidative stress genes, and could have contributed to the wide-spread necrosis observed in Bt-infested tomato plants at late stages of the plant-insect interaction.
Suppressed transcription of genes for Rubisco, the primary carboxylating en-zyme in C3 plants and other photosynthetic proteins, which is usually accompanied by a loss of photosynthetic efficiency, is a negative but frequently observed consequence of insect herbivory.1–4 The phenomenon involves the repression of several photosynthetic genes and is mostly characterized by a sharp reduction in photosystem II (PS II) operating efficiency and concomitant fall in the rate of CO2 exchange. Such a disruption of the photosynthetic process can have deleterious effects on plant performance, leading to important reductions in crop or net primary productivity, which have been reported to range between 5% and 70%, respectively.5–7 The process usually affects components of all four multi-subunit membrane protein-pigment complexes [PS II, photosystem I (PS I), cytochrome b6f and F-ATPase] required for oxygenic photosynthesis,8 and is modulated by the degree of tissue damage.
Recent studies on phloem-feeding insects (PFIs) have shown that they induce a very particular transcriptional reprogramming in their host plants, which differs from the responses produced when plants interact with other insect-feeding guilds, mainly because their feeding habit, designed to siphon nutrients directly from the phloem sap, tends to minimize the wound responses in their hosts.9–12 This is also true for the whitefly Bemisia spp., which rarely damage epidermal or mesophyll cells while probing their way to the phloem.13,14 Thus, in addition to their capacity to induce cell wall modifications, manipulate source-sink relations, and alter secondary metabolism in their hosts PFIs, even when present at low populations, can reduce photosynthetic activity concomitantly with a downregulated expression of photosynthesis-related genes.3,4,15–18 This is believed to occur, at least in part, as a plant strategy designed to suppress apparently less important functions in order to reallocate energy preferentially to defense responses.19,20
We recently used a suppression-subtractive-hybridization strategy to gain a deeper insight on the transcriptional changes occurring in tomato plants infected with virus-free whitefly (Bemisia tabaci, Bt) by concentrating on emblematic stages of the entire larval developmental period. The study included very early (adult feeding and oviposition) and late (fourth instar prior to adult emergence) stages that have been usually ignored in favor of the more active second- and third-instar stages in which feeding, and related changes in the plant host, appear to be more intense.21 In accordance to the above, Bt infestation also led to a disrupted expression of photosynthetic genes. Closer examination of photosynthesis-related genes indicated contrasting expression patterns between genes comprising the four multi-subunit membrane protein-pigment complexes (Table 1). Thus, the general upregulation of genes associated with the structure and function of PS II, observed predominantly in the latter stages of the infestation process (phases III and IV), contrasted with the wide-spread downregulation of those required for the function and structure of the remaining three complexes, in addition to genes coding for key Calvin cycle proteins. Such imbalance could have led to a potentially harmful disruption of electron transport, leading to an over excitation of the PS II complex and subsequent oxidative damage. Although reactive oxygen species (ROS) are mainly produced by PS I, PS II is known to contribute to their formation under certain conditions, such as a limitation of the electron transport reaction between both photosystems. This is known to occur at elevated PS II/PS I gene expression ratios, similar to those herewith reported. Under these conditions, PS II-derived ROS can be generated by the photo-reduction of molecular oxygen to superoxide anion radicals which can be further dismutated to hydrogen peroxide and lead to the subsequent formation of highly damaging hydroxyl radicals. Additional ROS accumulation by PS II, as hydrogen peroxide, can result from the incomplete oxidation of water.22 PS II-associated photo-oxidation is usually accompanied by the upregulation of oxidative stress genes that protect photosynthetic organisms against photo-oxidative stress.23 This was in accordance with the concomitant induction of several oxidative stress-related genes in phases III and IV of Bt's larval development (Table 1). Moreover, the blocked transport of an excess of electrons to their final acceptors, combined with a disrupted CO2 fixation caused by lower expression of genes coding for Rubisco small subunits and related regulatory enzymes, together with a probable reduction in ATP synthase activity caused by a downregulated expression of the delta subunit, could have contributed to the necrotic cell death often observed in Bemisia tabaci-infested plants. Experimental support for this scenario comes from studies performed mostly in marine organisms (phytoplankton, chlorophyte alga and a marine cyanobacterium), which undergo programmed cell death processes in response to a blockage in the electron flow during photosynthesis and/or as a consequence of a decline in photosynthetic efficiency leading to an accumulation of ROS. Also, chloroplast ROS-related cell death have been observed in higher plants such as pea and soybean,24 whereas a recent study demonstrated that the bacterial toxin coronatine was an instrumental factor contributing to, (1) the reduced expression of photosynthesis related genes, (2) the 1.5- to 2-fold reduction in maximum quantum efficiency of PS II, and (3) the generation ROS and accompanying necrotic cell death occurring during bacterial speck disease of tomato.25
Most experimental evidence gathered to date indicates that Bt and other PFIs can make use of several strategies, firstly, to avoid detection in order to gain access to a suitable plant host and, secondly, to manipulate and/or suppress defensive responses to favor infestation.12 In addition, the results herewith discussed suggest that Bt might also utilize the disruption of the sequential order required for the efficient functioning of the photosynthetic process as an additional invasive weapon. Such hypothesis will require supportive physiological and biochemical evidence to be validated.
Previously published online: www.landesbioscience.com/journals/psb/article/9663