In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of f luorescently labeled dextrans along with sizefractionated Cucurbita maxima phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual f luorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20-100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from Ricinus communis, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules.

The consequences of biotic stress have been poorly understood, partly because its application is difficult to control and partly because its physiological consequences are highly variable. Many plant viruses are recognised on the basis of leaf symptoms that depend on localised changes to chloroplast structure and function. This paper reviews recent progress in understanding early interactions between plant viruses and the photosynthetic apparatus, using chlorophyll fluorescence analysis of novel, defined algal‐virus systems and using high resolution imaging of chlorophyll fluorescence and other photosynthetic processes in higher plant systems. We then consider the consequences of viral effects on photosynthetic functioning for whole plants and populations with an emphasis on the potential interactions with other environmental factors. Early responses indicated by increase in both non‐photochemical quenching of fluorescence and increased reduction state of the primary electron transport acceptor QA suggest that, not surprisingly, both photoprotective and photoinhibitory processes contribute to the accelerated local demise of the photosynthetic apparatus and symptom development. In other cases, localised accumulations of carbohydrate and source‐sink imbalance following infection may inhibit gene expression, leading to altered levels of chloroplast protein complexes and enzymes of photosynthetic metabolism coincident with symptom development. Recent experiments suggest that much of the variability in plant responses to biotic stress may result from interactions with other environmental factors, such as light intensity and nutrition. Experiments suggest that virus infections may have greater effects on fitness and competitive ability in low N, high light environments than in shaded, high nutrient conditions. Some ecological implications of these observations are discussed.

Fluorescence imaging was used to diagnose early stages of the strain-specific interactions between tobacco mosaic virus (strain PV230) and chloroplasts following infection of tobacco leaves (Nicofiana tabacum cv Xanthi). l h e earliest indication of interaction in tissues that ultimately become chlorotic was a reduction in chlorophyll fluorescence, and there was little fluorescence quenching compared with adjacent healthy tissues. Subsequently, fluorescence increased but remained unquenched. In the late stages fluorescence declined again in chlorotic regions as the chloroticmosaic symptoms developed. These in vivo data showing altered fluorescence yields confirm strain-specific interaction of virus coat protein with photosystem I1 (PSII) koids inhibits PSII electron transport (Hodgson et al., 1989) and leads to chronic photoinhibition and destruction of chloroplasts.It is impossible to predict where isolated chlorotic regions of infection will arise in a leaf. Changes in Chl fluorescence in the earliest stages of infection have the potential to locate areas in which visible symptoms develop and to indicate mechanisms involved in development of chlorotic-mosaic symptoms (Osmond et al., 1990). Subsequent development of equipment, and especially of data analysis software, has made it possible for us to make quantitative analyses of Chl fluorescence quenching with unit pixel resolution. In the present study, we describe the in vivo analysis of visible symptom formation using fluorescence emission characteristics and video imaging. Using these fluorescence imaging techniques with leaves, we show that the earliest in vivo sign of infection is a reduction of fluorescence yield, prior to enhancement of unquenched fluorescence, and light-dependent development of chlorotic-mosaic symptoms following photoinhibitory damage. A preliminary study based on these techniques has been reported (Balachandran et al., 1992). Symptom development in tobacco (Nicotiana tabacum) following infection with severe strains of TMV has long remained enigmatic (Matthews, 1971). Previous experiments (Balachandran et al., 1994) showed that in expanding leaves visible symptom development largely determined the extent of impaired photoacclimation in tobacco leaves. Furthermore, visible symptom development was due to chronic photoinhibition and subsequent photooxidation of Chl (Balachandran and Osmond, 1994). We believe that these events may begin with an association of virus coat protein with chloroplasts that are dividing and developing at the time of infection Beachy, 1986, 1989; Van Loon et al., 1990). Although mechanisms remain obscure, it is evident that this association of TMV coat protein with PSII centers in thylaSupported by Duke University, Australian National University, and U.S. Department of Agriculture grant 90-37280-5612 to C.B.O. * Present address: Section of Plant Biology, Division of Biological MATERIALS A N D M E T H O D SWild-type tobacco (Nicotiana tabacum cv Xanthi) plants were grown and inoculated with TMV strain PV230 as d...

The influence of the 30 kDa movement protein of tobacco mosaic virus (TMV‐MP) on carbon partitioning in trans‐genie tobacco plants (Nicotiana tabacum cv. Xanthi) expressing the TMV‐MP was investigated. Using reciprocal grafting of transgenic tobacco plants expressing this movement protein and vector control plants, as well as transgenic tobacco plants expressing the TMV‐MP in phloem cells only, we showed that the interactive site involved in carbon allocation to roots is localized to the mesophyll tissue. Biomass partitioning experiments conducted on transgenic plants, in which various deletion mutant forms of the TMV‐MP (two of which included deletions in the domain responsible for increasing the size exclusion limit) were expressed, revealed that the TMV‐MP exerts its influence on carbon allocation via a mechanism that is completely independent of the TMV‐MP‐induced increase in the plasmodesmal size exclusion limit. Furthermore, small N‐ and C‐terminal deletions in the MP revealed the complexity of the interactions likely to be involved between the MP and an endogenous regulatory mechanism. We propose that the TMV‐MP interferes with an endogenous signal transduction pathway that involves macromolecular trafficking through plasmodesmata to regulate biomass partitioning between the source and various sink tissues.