Susceptibility of Tobacco Leaves to Photoinhibition following Infection with Two Strains of Tobacco Mosaic Virus under Different Light and Nitrogen Nutrition Regimes

Sensitivity to photoinhibition under high light stress (2000 [mu]mol photons m-2 s-1 for 2 h in air) and recovery from this stress were examined in leaves of control, uninfected tobacco (Nicotiana tabacum cv Xanthi) leaves and in leaves in tobacco plants infected with tobacco mosaic virus (TMV) when grown under low light (150-200 [mu]mol photons m-2 s-1) or high light (1200 [mu]mol photons m-2 s-1) with high (8.0 mM) or low (0.5 mM) nitrate supply. Photoinhibition was monitored using the dark-adapted fluorescence parameters variable fluorescence/maximum fluorescence, an indicator of photosynthetic efficiency that correlated well with the quantum yield of photosynthetic oxygen evolution, and initial fluorescence, potentially an indicator of photoinhibitory damage. Susceptibility to photoinhibition was greater in low light- and low nitrogen-grown control plants than in high light- or high nitrogen-treated plants. Compared with uninfected controls, infection with the masked strain PV42 increased susceptibility to photoinhibition only in plants grown under low light/low nitrogen conditions. In expanding leaves, infection with severe strain TMV PV230 markedly accelerated photoinhibition under these conditions and under high light/low nitrogen conditions, even before visible symptoms were evident. High nitrogen levels during growth protected against this accelerated photoinhibitory response to virus infection during light stress and generally promoted recovery, at least prior to symptom development. As symptoms developed, the yellow regions provided evidence for chronic photoinhibitory damage, prior to and during the stress treatment, irrespective of growth conditions. Green regions of leaves showing visible symptoms were generally indistinguishable from control, uninfected plants during photoinhibitory stress and recovery. In developed leaves that remained free of visible symptoms during the experiments, in spite of the accumulation of about the same amounts of virus protein (S. Balachandran, C.B. Osmond, A. Makino [1994] Plant Physiol 104: 1043-1050) infection led to an acceleration of photoinhibition during stress treatments, especially in low light/low nitrogen treatments, in which chronic photoinhibitory damage was evident. These studies suggest a role for photoinhibitory damage in the acceleration of visible symptom development following TMV PV230 infection of expanding leaves, as well as in acceleration of senescence in developed leaves without visible symptoms.     

Diagnosis of the Earliest Strain-Specific Interactions between Tobacco Mosaic Virus and Chloroplasts of Tobacco Leaves in Vivo by Means of Chlorophyll Fluorescence Imaging

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 (Nicotiana tabacum cv Xanthi). The 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 II (PSII) components in vitro, leading to photoinhibition and photooxidation of chlorophyll in infected cells and the development of visible chlorotic-mosaic symptoms. Although mechanisms leading to the low, unquenched fluorescence condition are not known, the intermediate high, unquenched fluorescence condition is consistent with impaired PSII electron transport as measured in vitro. Fluorescence lesions appear more rapidly and develop more extensively in high light, consistent with the faster and larger extent of symptom formation in high-light-grown leaves than in low-light-grown leaves.     

Association of TMV coat protein with chloroplast membranes in virus-infected leaves

Summary The polypeptide composition of extracts of chloroplasts from tobacco leaves systemically infected with different strains of Tobacco Mosaic Virus (TMV) was analyzed by one- and two-dimensional gel electrophoresis. There were no changes in the protein profiles of chloroplasts from infected leaves when compared to control leaves except for the presence of coat protein (CP) of TMV, identified by immunoblotting. When protease-treated intact chloroplasts isolated on Percoll gradients were osmotically disrupted the CP could be detected in both stroma and membrane fractions. The majority of the CP associated with the thylakoid membranes (about 1–5% of the total thylakoid proteins) was in the form of free molecules while stroma contained aggregated or assembled CP (about 0.1% of the soluble proteins). Thylakoid-associated CP was insensitive to protease digestion unless the membranes were first treated with a detergent, indicating that the CP was embedded inside or otherwise complexed with the thylakoid membranes.Chloroplasts isolated from leaves infected with TMV-PV42, a symptomless strain, contained approximately 10–50 times less CP than did chloroplasts isolated from leaves bearing mosaic symptoms induced by other strains of TMV (U1, PV230 or PV39). A possible role of CP in symptom development is discussed.     

Reduced Photosystem II Activity and Accumulation of Viral Coat Protein in Chloroplasts of Leaves Infected with Tobacco Mosaic Virus

We previously reported (A Reinero, RN Beachy 1986 Plant Mol Biol 6:291-301) that coat protein (CP) of tobacco mosaic virus (TMV) accumulates in chloroplasts of systemically infected leaves. To determine the significance of such interaction we examined electron transport rates in chloroplasts containing different levels of TMV-CP. Tobacco (Nicotiana tabacum L.) plants were infected with either a TMV strain inducing chlorosis or with a strain inducing mild symptoms, and both the accumulation pattern of TMV-CP inside chloroplasts as well as the rates of photosynthetic electron transport were followed. The CP of the TMV strain inducing chlorosis was detected inside chloroplasts 3 days after infection, and thereafter accumulated at a rapid rate, first in the stroma and then in the thylakoid membranes. On the other hand, the CP of the TMV strain that caused only mild symptoms accumulated in chloroplasts to lower levels and little CP was associated with the thylakoids. In vivo and in vitro measurements of electron transport revealed that photosystem II activity was inhibited in plants infected with the aggressive TMV strain while no reduction was observed in plants infected with the mild strain. The capacity of chloroplasts to synthesize proteins was equivalent in organelles isolated from healthy and virus-infected leaves. The possibility that a large accumulation of TMV-CP inside chloroplasts may affect photosynthesis in virus-infected plants by inhibiting photosystem II activity is discussed.     

Selective inhibition of photosystem II in spinach by tobacco mosaic virus: an effect of the viral coat protein

Leaves of Spinacia oleracea inoculated with tobacco mosaic virus (TMV) strain PV230 develop mild chlorotic and mosaic symptoms of infection. Thylakoid membranes isolated from these infected leaves showed a reduced Fv/Fm ratio for chlorophyll fluorescence kinetics, at 25 degrees C. The photosystem II (PS II)-mediated electron-transport rate was inhibited 50%, whereas PS I activity was unaffected by virus infection. Protein analysis indicated that TMV coat protein was associated with thylakoids, in particular with the PS II fraction. The results demonstrate that TMV-infected S. oleracea shows inhibition of photosynthetic electron transport through PS II. We propose that the inhibition of photosynthetic activity results from the association of viral coat protein with the PS II complex.     

Depletion of the photosystem II core complex in mature tobacco leaves infected by the flavum strain of tobacco mosaic virus

The flavum strain of Tobacco mosaic virus (TMV) differs from the wild-type (wt) virus by causing strong yellow and green mosaic in the systemically infected developing leaves, yellowing in the fully expanded leaves, and distinct malformations of chloroplasts in both types of infected tissues. Analysis of the thylakoid proteins of flavum strain-infected tobacco leaves indicated that the chlorosis in mature leaves was accompanied by depletion of the entire photosystem II (PSII) core complexes and the 33-kDa protein of the oxygen evolving complex. The only change observed in the thylakoid proteins of the corresponding wt TMV-infected leaves was a slight reduction of the alpha and beta subunits of the ATP synthase complex. The coat proteins of different yellowing strains of TMV are known to effectively accumulate inside chloroplasts, but in this work, the viral movement protein also was detected in association with the thylakoid membranes of flavum strain-infected leaves. The mRNAs of different enzymes involved in the chlorophyll biosynthesis pathway were not reduced in the mature chlorotic leaves. These results suggest that the chlorosis was not caused by reduction of pigment biosynthesis, but rather, by reduction of specific proteins of the PSII core complexes and by consequent break-down of the pigments.     

Comparative ecophysiology of leaf and canopy photosynthesis

Leaves and herbaceous leaf canopies photosynthesize efficiently although the distribution of light, the ultimate resource of photosynthesis, is very biased in these systems. As has been suggested in theoretical studies, if a photosynthetic system is organized such that every photosynthetic apparatus photosynthesizes in concert, the system as a whole has the sharpest light response curve and is most adaptive. This condition can be approached by (i) homogenization of the light environment and (ii) acclimation of the photosynthetic properties of leaves or chloroplasts to their local light environments. This review examines these two factors in the herbaceous leaf canopy and in the leaf. Changes in the inclination of leaves in the canopy and differentiation of mesophyll into palisade and spongy tissue contribute to the moderation of the light gradient. Leaf and chloroplast movements in the upper parts of these systems under high irradiances also moderate light gradients. Moreover, acclimation of leaves and chloroplasts to the local light environment is substantial. These factors increase the efficiency of photosynthesis considerably. However, the systems appear to be less efficient than the theoretical optimum. When the systems are optically dense, the light gradients may be too great for leaves or chloroplasts to acclimate. The loss of photosynthetic production attributed to the imperfect adjustment of photosynthetic apparatus to the local light environment is most apparent when the photosynthesis of the system is in the transition between the light-limited and light-saturated phases. Although acclimation of the photosynthetic apparatus and moderation of light gradients are imperfect, these markedly raise the efficiency of photosynthesis. Thus more mechanistic studies on these adaptive attributes are needed. The causes and consequences of imperfect adjustment should also be investigated.     

SOME EFFECTS OF VIRUS INFECTION ON LEAF WATER CONTENTS OF NICOTIANA SPECIES

Infection with tobacco mosaic virus decreases the water content which detached tobacco leaves attain when kept for 20 hr. in conditions of minimum water stress, and does so more when the plants are kept in light before inoculation than when they are kept in darkness. No such effects of infection during the first day after inoculation were obtained with tobacco leaves infected with either tobacco etch virus or potato virus X , or with Nicotiana glutinosa leaves infected with tobacco mosaic virus. These results, like those showing early effects of TMV on respiration and photosynthesis of tobacco leaves, suggest that inoculation with TMV affects deeper leaf tissues than the epidermis earlier in tobacco leaves than in other leaves, and earlier than other viruses in tobacco leaves.     

两种热带雨林树苗对环境光强变化的生理响应和适应机制

干季末雨季初以西双版纳热带雨林中木奶果和玉蕊2种树苗为材料,研究了将生长于12.5%自然光（相当于小林窗的光强）和36%自然光（相当于大林窗的光强）下的这2种树苗分别移至36%自然光和12.5%自然光下之后各自叶片最大净光合速率（Pmax）、叶绿素荧光参数、光合色素含量、比叶重（LMA）以及叶片悬挂角（MA）的变化过程,探讨了2种植物幼苗在生长环境光强改变后其形态和生理生态特性做出的相应调整以适应新的光环境的过程与机制。结果表明,这2种树苗均不适宜生长在大林窗的强光环境下,但木奶果对光环境的增强表现出一定的耐受力,光合适应潜力强于玉蕊,2种树苗对低光环境都能较好地适应;新叶的生成在整株植物对生长光环境变化的适应过程中也起到至关重要的作用。     

Photodamage of the photosynthetic apparatus and its dependence on the leaf developmental stage in the npq1 Arabidopsis mutant deficient in the xanthophyll cycle enzyme violaxanthin de-epoxidase

The npq1 Arabidopsis mutant is deficient in the violaxanthin de-epoxidase enzyme that converts violaxanthin to zeaxanthin in excess light (xanthophyll cycle). We have compared the behavior of mature leaves (ML) and developing leaves of the mutant and the wild type in various light environments. Thermoluminescence measurements indicated that high photon flux densities (>500 micromol m(-2) s(-1)) promoted oxidative stress in the chloroplasts of npq1 ML, which was associated with a loss of chlorophyll and an inhibition of the photochemical activity. Illuminating leaf discs in the presence of eosin, a generator of singlet oxygen, brought about pronounced lipid peroxidation in npq1 ML but not in wild-type leaves. No such effects were seen in young leaves (YL) of npq1, which were quite tolerant to strong light and eosin-induced singlet oxygen. Non-photochemical energy quenching was strongly inhibited in npq1 YL and ML and was not improved with high-light acclimation. Our results confirm that the xanthophyll cycle protects chloroplasts from photooxidation by a mechanism distinct from non-photochemical energy quenching and they reveal that the absence of xanthophyll cycle can be compensated by other protective mechanisms. npq1 YL were observed to accumulate considerable amounts of vitamin E during photoacclimation, suggesting that this lipophilic antioxidant could be involved in the high phototolerance of those leaves.     

Induction of Cold Acclimation in Cornus stolonifera Michx

A warm (20 to 15 Celsius day or night) preconditioning treatment enhanced cold acclimation of Cornus stolonifera bark under short-day conditions when plants were preconditioned for at least 4 weeks. Warm preconditioning inhibited the acclimation of plants subjected to long photoperiods. Removing leaves from plants exposed to low temperatures and short days inhibited acclimation. Removal of buds did not affect acclimation. Plants did not acclimate unless they were exposed to at least 4 weeks of short photoperiods prior to defoliation. Plants began to acclimate to cold at the time of growth cessation but not before. When half of the leaves were removed from plants, the defoliated and foliated branches both acclimated as well as branches on completely foliated plants. Girdling the phloem between foliated and defoliated branches prevented acclimation of the latter regardless of the position of the girdle in relation to the root system and the defoliated branch. When all of the leaves of plants were covered with aluminum foil to exclude light after 0 or 4 weeks of exposure to short days, the results resembled a defoliation study, i.e., plants with leaves covered at the start of the experiment failed to acclimate, and those covered after 4 weeks acclimated to some extent but less than uncovered control plants. Under longday conditions plants with all leaves covered failed to acclimate, and plants with none or half of their leaves covered acclimated equally and to a limited extent. Under short-day conditions, however, the covered branches of partially covered plants acclimated more than their uncovered counterparts or branches of totally uncovered plants.     

烟草花叶病毒对烟草叶片光合特征和POD表达的影响

以烤烟(Nicotiana tabacum L.)品种'中烟5号'为实验材料,对烟草健康株与感染烟草花叶病毒(TMV)株的叶绿素、光合速率、光合速率对光强的响应曲线、光暗反应荧光特征、POD活性及其表达等进行研究,以探讨TMV感染对烟草植株生理生态特征的影响.结果显示:病株的叶绿素a(Chl a)和叶绿素b(Chl b)含量显著低于健康株,但Chl a/Chl b值基本相同;病株暗中初始荧光(F0)、暗中最大荧光(Fm)、暗中可变荧光(Fv)、光下初始荧光(F0′)、光下最大荧光(Fm′)、光下可变荧光(Fv′)、非光化学猝灭系数(NPQ)、PSⅡ捕光效率(Fv′/Fm′)、PSⅡ实际光化学效率(ФPSⅡ)及光饱和点显著低于健康株;净光合速率在光强较大(＞1 500 μmol·m-2·s-1)时病株比健康株低,光强适中(1 500 μmol·m-2·s-1左右)时两者相差不大,光强较弱(＜1 500 μmol·m-2·s-1左右)时病株比健康株高;病株叶片的过氧化物酶(POD)活性显著升高,POD同工酶中一些大分子量蛋白分子表达量加大.研究表明,感染TMV使烟草植株对光抑制更为敏感,叶片的荧光激发能力和热耗散能力下降,PSⅡ反应中心捕光效率和光化学反应效率降低,光合电子传递能力和碳同化能力受到抑制;POD活性提高和表达量增加可能是诱导烟草抗病性的一个关键生理过程.     

The protective functions of carotenoid and flavonoid pigments against excess visible radiation at chilling temperature investigated in Arabidopsis npq and tt mutants

The npq1 mutant of Arabidopsis thaliana (L.) Heynh. has no xanthophyll cycle due to a lack of functional violaxanthin de-epoxidase. Short-term exposure (<2 days) of detached leaves or whole plants to the combination of high photon flux density (1,000 micromol m(-2) s(-1)) and low temperature (10 degrees C) resulted in PSII photoinhibition which was more acute in npq1 than in the wild type. This increased photosensitivity of npql at chilling temperature was attributable to the inhibition of nonphotochemical energy quenching (NPQ) and not to the absence of zeaxanthin itself. In contrast to PSII, PSI was found to be phototolerant to chilling stress in the light in both genotypes. In the long term (10-12 days), PSII activity recovered in both npql and wild type, indicating that A. thaliana is able to acclimate to chilling stress in the light independently of the xanthophyll cycle. In npql, photoacclimation involved a substantial reduction of the light-harvesting pigment antenna of PSII and an improvement of photosynthetic electron transport. Chilling stress also induced synthesis of early light-inducedproteins (ELIPs) which, in the long term, disappeared in npql and remained stable in the wild type. In both genotypes, photoacclimation at low temperature induced the accumulation of various antioxidants including carotenoids (except beta-carotene), vitamin E (alpha- and -gamma-tocopherol) and non-photosynthetic pigments (anthocyanins and other flavonoids). Analysis of flavonoid-deficient tt mutants revealed that UV/blue-light-absorbing flavonols have a strong protective function against excess visible radiations. In contrast to the defect in npq1, the absence of flavonoids could not be overcome in the long term by compensatory mechanisms, leading to extensive photooxidative and photoinhibitory damage to the chloroplasts. Depth profiling of the leaf pigments by phase-resolved photoacoustic spectroscopy showed that the flavonoid-related photoprotection was due to light trapping, which decreased chlorophyll excitation by blue light. In contrast to flavonoids, the xanthophyll cycle and the associated NPQ seem to be mainly relevant to the protection of photosynthesis against sudden increases in light intensity.     

Changes in Glucose-6-Phosphate Dehydrogenase, Ribonucleases, Esterases and Contents of Viruses in Potato Virus Y Infected Tobacco Superinfected with Tobacco Mosaic Virus

Effects of the superinfection with tobacco mosaic virus (TMV) on susceptible tobacco plants infected with potato virus Y (PVY) were determined. Dynamic changes in the TMV and/or PVY contents, the ribonucleases (RNases), the phosphomonoesterase (PME), the phosphodiesterase (PDE) and the glucose-6-phosphate dehydrogenase (G6P DH) activities were studied. The PVY infection caused a substantial reduction in the multiplication of TMV. The content of TMV in the PVY inoculated leaves amounts to 6 and 9 % in the PVY systemically infected leaves when compared with single TMV. Surprisingly, the challenging virus (TMV) enhanced the content of inducing virus (PVY) in the locally inoculated leaves up to 130 – 141 %. In contrast, the reduction of PVY content down to 35 – 40 % by TMV was seen in the PVY systemically infected leaves. The activities of the RNase, the PME, the PDE and the G6P DH were increased (when compared with the healthy plants) during the acute phase of single virus multiplication (PVY or TMV). The increase in the activities of the enzymes in the leaves with mixed infection was at least as high as the sum of the increases of single infections. Moreover, a higher increase than the sum was seen for G6P DH and PDE (by about 20 – 35 %). This revised version was published online in July 2006 with corrections to the Cover Date.     

The study of a determinate growth orchid highlights the role of new leaf production in photosynthetic light acclimation

Plants usually respond to the changes of growth irradiance by a combination of the physiological modifications in their preexisting leaves and the production of new leaves. However, those with a determinate growth habit produce certain number of leaves in a growing season and cannot produce new leaves when light condition changes. We used an epiphytic orchid with only one leaf produced every growing season to examine whether and how determinate growth species adapt to changing environments after their preexisting leaves mature. Leaf photosynthesis and anatomy of Pleione aurita were investigated at full expansion and at 40 days after the fully expanded leaves were transferred from high to low light or from low to high light. Leaves show large physiological and morphological plasticity to light gradients at full expansion and the transferred leaves exhibited multiple physiological modifications, including reallocation of nitrogen between light harvesting and carbon fixation, and enhancement of thermal dissipation in their new environments, to optimize carbon assimilation and avoid photoinhibition. Irrespective of the various changes either to shade or sun, the sole preexisting leaf could not fully acclimate to new light environments due to the mesophyll thickness constraint. This leads to the consequence that only plants exposed to high light throughout the experiment had a positive annual biomass gain. Our results highlighted the importance of new leaf production in the carbon accumulation during photosynthetic light acclimation, and contribute new insights of epiphytes physiological responses to their highly dynamic arboreal habitat.     

ANATOMICAL AND PHYSIOLOGICAL ACCLIMATION OF FATSIA JAPONICA LEAVES TO IRRADIANCE

Anatomical and physiological leaf characteristics and biomass production of Fatsia japonica plants were studied. Plants were grown in a growth chamber at 300 μmol m^-2 s^-1 (high light) and 50 μmol m^-2 s^-1 (low light) photosynthetic photon flux density. Plants grown under high light showed a net maximum photosynthetic rate 44% higher than plants grown under low light; the light compensation point and the light saturation point were also higher in high-light plants. Photosynthetic oxygen evolution in isolated chloroplasts was about 40% higher in high-light plants. However, chlorophyll content on a dry weight basis, on a leaf area basis, and per chloroplast was greater in plants grown under low light. Leaf thickness in high-light plants was 13% higher than in low-light plants. The number of chloroplasts was 30% higher in high-light leaves, while chloroplast size was only slightly higher. Chloroplast ultrastructure was also affected by light. Leaf dry weight, leaf area, and biomass production per plant were drastically reduced under low light. Thus, F. japonica is a plant that is able to acclimate to different photosynthetic photon flux density by altering its anatomical and physiological characteristics. However, low-light acclimation of this plant has a considerable limiting effect on biomass production.     

Demonstration of the Specific Involvement of Coat Protein in Tobacco Mosaic Virus (TMV) Cross Protection using a TMV Coat Protein Mutant

The DT-1G mutant of tobacco mosaic virus (TMV) which has no coat protein was used to study the specific involvement of coat protein in TMV cross protection in N. sylvestris. Leaves of N. sylvestris previously inoculated with the mutantor the common strain of TMV were challenged with either turnip mosaic virus (TuMV) or a strain of TMV (TMV-N). Both TuMV and TMV-N produce necrotic lesions on N. sylvestris. About one-half as many lesions were produced by TuMV and TMV-N on leaves, inoculated with the DT-1G mutant compared with lesions produced by the same inoculum on control leaves. When leaves of N. sylvestris previously inoculated with the common strain of TMV were challenged with either TuMV or TMV-N, TuMV produced about one-half as many lesions as on control leaves whereas TMV-N produced about one-tenth as many lesions as on control leaves. A high level of non-specific resistance was induced by the mutant without coat protein, but it did not specifically protect against TMV.     

Two Apoplastic alpha-Amylases Are Induced in Tobacco by Virus Infection

α-Amylase activity (EC 3.2. 1.1) is greatly increased in leaves of tobacco (Nicotiana tabacum L. cv Samsun NN) infected with tobacco mosaic virus (TMV). The kinetics of enzyme induction during the hypersensitive reaction resemble those of other hydrolases known to be pathogenesis-related proteins of tobacco. Two α-amylases were purified from TMV-infected leaves and shown to have features in common with well-characterized pathogenesis-related proteins: they are acidic monomers that can be separated upon electrophoresis on basic native gels, and they are found in the apoplastic compartment of the cell. This extra-cellular localization was demonstrated by comparing the α-amylase partition between the intercellular wash fluid and the cell extract with that of proteins of known cellular compartmentalization. These data indicate an active secretion of both α-amylases produced in tobacco upon TMV infection.     

TOBACCO MOSAIC VIRUS IN CHLOROPLAST AND CYTOPLASM OF INFECTED TOBACCO LEAF

Chloroplasts containing Tobacco Mosaic Virus (TMV) were isolatedfrom TMV inoculated tobacco leaves, and TMV was extracted fromthem. The preparations from isolated chloroplasts of infectedleaves showed 30% increase in the optical density at 260 mµover those from healthy leaves. Most of infectivity was observedin chloroplast fraction 40 hr after inoculation. Thereafter,infectivity in the chloroplast fraction decreased and that ofcytoplasm increased with time. (Received April 6, 1964; )