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Short Communication |
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK
Correspondence
Michael G. Handford
mhandfor{at}uchile.cl
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ABSTRACT |
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Present address: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Ñuñoa, Casilla 653, Santiago, Chile. 
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Handford & Carr, 2006; Loebenstein & Akad, 2006). In directly inoculated leaves of susceptible plants, the alterations in carbohydrate metabolism frequently result in the occurrence of starch lesions or ringspots at viral infection sites and abnormal accumulation or depletion of this important storage compound in systemically infected tissues, which can be revealed by iodine staining (Holmes, 1931; Bawden, 1950; Cohen & Loebenstein, 1975; Roberts & Wood, 1982; Técsi et al., 1994).
Starch lesions or ringspots on virus-inoculated leaves develop before the appearance of visible disease symptoms. They consist of rings of cells with chloroplasts containing enlarged starch grains surrounding a zone of starch-depleted cells (Cohen & Loebenstein, 1975). In marrow cotyledons inoculated with the cucumovirus Cucumber mosaic virus (CMV), starch accumulation occurs in cells that are behind the replication front of the expanding infection zone. These starch-accumulating cells display increased photosynthetic capacity relative to uninfected cells or cells in which virus replication is actively occurring (Doke & Hirai, 1970; Técsi et al., 1996). The starch-depleted cells within the ringspot show enhanced respiratory activity and starch hydrolase activity, with decreased photosynthetic efficiency (Técsi et al., 1996).
It is unclear if or how the complex biochemical changes reflected in the distribution of starch in the developing viral infection site influence the progress of viral infection. In this study we investigated the role of starch accumulation itself using pgm1 mutant plants of Arabidopsis thaliana (L.) Heynh (ecotype Col-0). These plants cannot synthesize starch due to a defect in the gene encoding the plastidic phosphoglucomutase, an enzyme required for starch biosynthesis (EC 5.4.2.2
[EC]
; Caspar et al., 1985). Three viruses that infect A. thaliana and induce recognizable disease symptoms were used for the study. These were the Tobamovirus Turnip vein-clearing virus (TVCV; Melcher, 2003) and the Fny strain of CMV (Roossinck & Palukaitis, 1990), which are both positive single-stranded RNA viruses, and the Cabb B-JI strain of the caulimovirus Cauliflower mosaic virus (CaMV), a double-stranded DNA pararetrovirus (Cecchini et al., 1998).
To confirm that virus infection induces starch lesions in A. thaliana, wild-type plants at the six-leaf stage (17 days post-seeding) were mock-inoculated or inoculated on the two oldest true leaves with CMV at 10 µg ml–1 and grown at 22 °C in a 16 h light/8 h dark (diurnal) regime for various periods. Inoculated leaves were harvested 2 h into the light period, decolourized in boiling 80 % (v/v) ethanol and stained with iodine solution (5.7 mM I2, 43 mM KI). By 2 days post-inoculation (p.i.) discrete regions of starch were detectable on leaves inoculated with CMV, but not on mock-inoculated leaves (Fig. 1a). The virus-induced starch lesions expanded into well defined rings of starch accumulation by 3 days p.i., but by 6 days p.i. distinct regions of starch accumulation were not apparent in stained, virus-infected leaves (Fig. 1a). TVCV is a very close relative of Tobacco mosaic virus (TMV), and the spatial and temporal development of starch lesions in wild-type A. thaliana inoculated with CMV are similar to these observed on leaves inoculated with TMV (Handford & Carr, 2006) and to those seen in CMV-infected marrow cotyledons (Técsi et al., 1994).
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; Caspar & Pickard, 1989). Iodine staining was used to confirm that under these growth conditions, leaves of wild-type A. thaliana plants accumulate high levels of starch, while leaves of the mutant plants do not react with the stain (Fig. 1b
). Following viral infection, neither wild-type nor mutant plants maintained under continuous light developed starch lesions; at all time points, the decolourized leaves of these plants stained with iodine solution appeared like the 6 days p.i. samples shown in Fig. 1(a). Thus, starch lesion development can be blocked by altering the environment or by creating a defect in the biochemical pathway responsible for starch biosynthesis. We examined the effect of preventing the build-up or breakdown of starch on systemic infection and symptom induction by three highly distinct viruses.
Wild-type and pgm1 mutant plants cultivated under continuous illumination at 22 °C grew and developed similarly (Fig. 2a, Table 1). Although wild-type plants developed faster under continuous illumination (reaching the six-leaf stage at 12 days post-seeding, rather than 17 days), they accumulated less biomass over that time, probably due to the disruption of the plant circadian system (Dodd et al., 2005). Plants were inoculated at the six-leaf stage, grown under continuous light or diurnal conditions and analysed at 12 or 17 days p.i., respectively. The virus-induced symptoms were analysed by observing the physical appearance of the leaves (wrinkling, distortion, chlorosis, etc.) and then in terms of the effects of virus infection on plant growth.
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). TVCV caused mild stunting and perturbations in leaf morphology in wild-type plants grown in a day/night regime (Fig. 2a; Lartey et al., 1997). Under continuous light, TVCV infection induced distinctly different symptoms in wild-type compared with pgm1 mutant plants: no chlorosis was observed in the mutant plants and leaf shape was rounded, rather than elongated, compared with the mock-inoculated plants (Fig. 2a). In wild-type A. thaliana, the Fny strain of CMV induced stunting, leaf distortion, chlorosis and, in some plants, symptoms included premature inflorescence production (bolting) (Fig. 2a; Mayers et al., 2005). The impact of CMV on leaf appearance was more pronounced in wild-type plants grown in continuous light (Fig. 2a). By contrast, in pgm1 mutant plants chlorosis was less apparent and, as with the plants infected with TVCV, CMV-infected mutant plants produced rounded rather than elongated leaves (Fig. 2a). Consistent with previous reports (Cecchini et al., 1998; Love et al., 2005), we observed that infection of wild-type plants with the Cabb-B JI strain of CaMV caused distorted leaf shape and chlorosis in older leaves and vein-clearing and stunting of newly emerged leaves (Fig. 2a). Chlorosis, vein-clearing and distortion were more apparent on CaMV-infected wild-type plants grown under continuous light, but less severe on pgm1 mutant plants, which also appeared less stunted (Fig. 2a).
The wild-type, virus-infected plants grown under continuous light did not show a significant degree of growth inhibition compared with mock-inoculated plants, which was in contrast to the inhibition of growth seen in plants infected and grown under diurnal conditions (Table 1). Most notably, whereas CaMV infection of wild-type plants grown in a day/night regime decreased shoot biomass significantly, CaMV-inoculated wild-type and pgm1 plants under continuous light grew well. This was mirrored by increases of 
60 and 40 % in shoot biomass for CaMV-inoculated pgm1 and wild-type plants, respectively, compared with mock-inoculated controls grown under continuous illumination. These differences were statistically significant (P=0.001, pgm1) or approaching statistical significance (P=0.060, wild-type). Consistent with recent findings by Dodd et al. (2005), it appears that in this case, the disruption of the circadian system of the plant combined with infection by CaMV significantly perturbs the accumulation of shoot biomass.
To determine if growth under continuous light or inhibition of starch accumulation affected the yield of virus, total soluble protein was extracted from virus- or mock-inoculated plants at 12 or 17 days p.i., for plants grown under continuous or diurnal light regimes, respectively. Protein was extracted, quantified and subjected to Western blotting analysis, as described previously (Chivasa et al., 1997). Rabbit polyclonal antisera against the coat proteins (CP) of TVCV (using anti-TMV CP serum; Wong et al., 2002), CMV or CaMV were used as appropriate and antibody binding was detected using an anti-rabbit IgG horseradish peroxidase conjugate and an enhanced luminol-based chemiluminescence reagent mix (Perkin-Elmer). We found that accumulation of TVCV, CMV (Fig. 2b) and CaMV (data not shown) was similar in wild-type and pgm1 plants and was not affected by the light regime. These results demonstrate that for all three viruses the effects of infection on symptoms or growth shown in Fig. 2(a) or Table 1 cannot be attributed to differences in viral load.
It is apparent from the data that a combination of environmental and metabolic factors is differentially influencing the susceptibility of A. thaliana to viral attack. In plants grown under a normal day/night cycle, such as the virus-infected wild-type A. thaliana plants in this study (Fig. 1), CMV-infected marrow cotyledons (Técsi et al., 1994, 1996) or CaMV-infected turnip and A. thaliana (Love et al., 2005), changes in starch deposition may reflect perturbations in photosynthetic, biosynthetic and respiratory pathways. Conceivably, this might affect the supply of fixed carbon for synthesis of virus-specific gene products within the virus-infected cells and the import of fixed carbon into virus-infected tissues from uninfected areas of the plant. In this study, a starch-depleted mutant line of plants cannot exhibit such changes, yet the accumulation of three different viruses was unaffected. This demonstrates unequivocally that catabolism of starch is not required to meet the demand for carbon for biosynthesis of virus-specific products. In CMV-infected marrow cells, starch accumulation required direct illumination of those cells, and not the import of photosynthates from neighbouring uninfected cells, yet virus accumulation was unaffected in the dark (Técsi et al., 1994). The susceptibility to several viruses of pgm1 supports these findings, in that it is not the build-up of starch per se which is a prerequisite for a successful infection, nor is it increased availability of free sugars in tissues where starch synthesis is not possible (as in pgm1; this study) or starch is broken down (as in dark-grown plants; Técsi et al., 1994).
Free sugars can be potent signals in plant metabolism, gene expression and development (Rolland et al., 2006), and in pgm1 mutant plants the conversion of these potential signals to starch is inhibited. Recent work by Love et al. (2005), using A. thaliana sugar response mutants, indicated that sugar signalling does not play a simple role in the modulation of symptoms in response to CaMV, at least in plants grown under diurnal (16 h day/8 h night) conditions. Nevertheless, we have shown that an environmental influence on symptom severity, in this case continuous light, is being modulated, directly or indirectly, by blocking the incorporation of sugars into starch and that this occurred in plants infected by three diverse viruses. The findings raise the possibility that cross-talk between proposed disease-signalling pathways (Cecchini et al., 2002) and sugar signalling occurs, but only in plants grown under conditions of stress, in which additional stress-related signalling pathways are operating.
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ACKNOWLEDGEMENTS |
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Received 11 July 2006;
accepted 14 September 2006.
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