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Effects of TYMV infection on leaf pigments in Brassica pekinensis Rupr
Physiological
Plant Pathology
(1974)
4, 379-387
Effects of TYMV infection on leaf pigments in Brassica pekinensis Ru p r. E. S. CROSBIE Department University (Acceptedfor
and R. E. F. MATTHEWS
of Cell BioloQ, Auckland,
of Auckland,
publiGation
December
New
Zealand
1973)
The effects are described of turnip yellow mosaic virus (TYMV) infection on the production of photosynthetic pigments in Chinese cabbage plants. Chlorophylls a and b and the four carotenoid pigments were analysed in plants following their infection with three strains of TYMV. The results support the view that infection reduces the amount of pigments in the plant mainly by inhibition of synthesis rather than by destruction of pre-existing pigments. In inoculated leaves, pale green and yellow green strains of the virus reduced the concentration of chlorophylls more than the carotenoids. A severe white strain of the virus reduced the concentration of all six pigments to a similar extent in all tissues tested.
INTRODUCTION In the yellows and mosaic diseases induced by many plant viruses it is obvious that infection leads to a reduction in photosynthetic pigments. However, there has been little detailed investigation of this effect ofvirus infection since the study by Peterson & McKinney [9] on the effects of four strains of tobacco mosaic virus (TMV) on pigments in tobacco. Turnip yellow mosaic virus (TYMV) is of particular interest with respect to the photosynthetic pigments since it is known to induce numerous cytological and biochemical changes in the chloroplasts, some of which appear to be closely connected with virus replication (reviewed in [7, 81). As part of a general study of the induction of disease by TYMV, we report here the effects of infection on the six chlorophyll and carotenoid pigments found in Brassica pekinensis. In the following paper [3] we relate the changes observed in the amount of pigments to the effects of infection on plant growth. Leaves of Chinese cabbage plants showing mosaic disease due to infection with TYMV may contain islands of dark green tissue which appear to be cytologically and biochemically normal and which contain little or no virus [I, 101. We have established that the pigment content of such dark green islands is similar to that of healthy leaf. As a leaf showing mosaic disease ages, the dark green areas tend to “break down” giving rise to numerous small areas in which virus replicates. These areas show pigment changes similar to those found in local lesions. Experiments with very small yellow leaves excised from the apex of diseased plants showed that a latent mosaic pattern exists before pigment development. 25
330
E. S. Crosbie and R. E. F. Matthews
MATERIALS
AND
METHODS
Plants and virus The host plant, Brassica pekinensis (Rupr.) var. Wong Bok (Chinese cabbage), was grown from stock commercial seed and transferred to 4-inch pots at the two-cotyledon stage. In some experiments the plants were later transplanted to T-inch square pots to ensure that growth was not checked by shortage of nutrients. Plants were grown in the glasshouse at 21 & 3 “C under natural light conditions. In winter the light was supplemented by four hours of artificial lighting, morning and evening, provided by Phillips 400 W HLRG lamps. Three strains of virus (severe white, pale green and yellow-green), were derived by serial selection of appropriate leaf material from plants infected with the stock culture of TYMV [Z]. Fresh extracts of infected leaf tissue were used as inocula. Plants were infected by mechanical inoculation of three older leaves at a stage when plants had developed a total of four to six well-expanded leaves. Analysis
of pigments in unfractionated
extracts
Small amounts (approximately 20 mg) of leaf lamina were ground in a KontesDual1 homogenizer in 2 ml of 95 y0 acetone. After clarification of the supernatant by standing for 0.5 h (or a brief, low speed centrifugation) the absorbance was read at 663, 645, 630 and 445 nm in a Cary model 15 spectrophotometer. Concentration of carotenoids was calculated directly from the absorbance at 445 nm. For the chlorophylls the formulae of Strain & Svec [13] were applied. Chromatograjhic
fractionation
of pigments
Extraction of pigments, preparation of the extract and subsequent chromatography were carried out as described in [12]. When analysing either chlorophylls or carotenoids a two-dimensional chromatogram was preferred because it provided better resolving power. This was particularly important when analysing pigments from infected tissue as a wide range of pigment concentration was encountered. For most samples, 0.4 to O-5 g of tissue was used and the volumes of extraction and “transfer” solvents were adjusted accordingly. The chromatographic solvents were : petroleum spirit (b.p. 80 to 100 “C) : n-propanol, 99 : 1, v/v, in the first dimension and petroleum spirit : chloroform, 3 : 1, v/v, in the second dimension. Ten ~1 of pigment concentrate was applied to Whatman 3 mm paper as a l-cm band. For carotenoid analysis chromatography was carried out for 40 min in the first dimension and O-5 h in the second dimension. At this time the pigments were sufficiently resolved and the spots showed minimal lateral spread. Spots were located by examination under U.V. light. The spots were cut out and the pigment eluted from the paper at four degrees by two successive extractions with 3 ml of acetone. The extracts were combined and evaporated to dryness at 32 degrees on a vacuum evaporator. The pigment was taken up in O-5 ml of chloroform and analysed spectrophotometrically. Estimation
of virus concentration
Virus concentration ultracentrifuge [3].
was
estimated
by
analysis
of leaf
extracts
in the
analytical
Effects
of TYMV
infection
on leaf pigments
381
RESULTS Pigments present in healthy Chinese cabbage leaves Six major pigments were identified by their spectral characteristics following chromatography of extracts from healthy leaf lamina. Two major green spots were identified as chlorophyll a and b. A single spot contained the a and b carotenes while the xanthophylls consisted of neoxanthin, violaxanthin and lutein. An “insoluble” spot of variable intensity was found at the origin. Strain et al. [12] considered this to be one of four lutein spots. However, this component from our chromatograms had an absorption spectrum similar to that of violaxanthin, but a mixture of violaxanthin, neoxanthin and lutein could also have produced a very similar spectrum. Thus the composition of the insoluble spot was not established. Occasionally two minor spots were seen on the chromatogram at the positions where Strain et al. [12] indicated two other lutein spots in extracts from various plants. Table 1 summarizes the pigment composition of healthy leaf tissue. The low content of lutein may result, in part, from some of the lutein running in the minor spots noted above. On the other hand, a low content of lutein may be characteristic of the genus Brassica [Cl. TABLE
The photosynthetic
(1)
pg/g fresh wt + standard deviation
Pigment Neoxanthin Violaxanthin Lutein Carotenes Chlorophyll Chlorophyll
a b
24.9+ 30.6+ 35.5+ 57.8+ 982 +43 367 *I3
2.6 2:6 2.5 I.1
pigments
1
of Brassica
pekinensis
(2)
o/O of total B. pekinensis 1.7 2.0 2.4 3.8 65.6 24.5
pigment Spinach” 2.2 2.2 7.9 5.0 57.6 25.2
o/O of total B. pekiruxsis 17.8 21.8 24.1 36.3
(3) carotenoids B. campest&” 11.0 13.0 31 38
a Adapted from [5]. b From [S]. Ten samples of healthy mature leaf lamina from Chinese cabbage were analysed for their chlorophyll content by spectrophotometric analysis of the acetone extracts. A single 2-g leaf sample was ground and the filtered acetone extract divided equally into four. These were then independently analysed for their carotenoid content spectrophotometrically after chromatographic fractionation of the saponified pigments. (1) The pigment content of a healthy Chinese cabbage leaf. (2) A comparison of the pigment content of B. pekinensis with spinach. (3) A comparison of the carotenoid content of B. pekinensis with that of B. campestris.
Analyses carried out on successive leaves of healthy plants indicated that there is a very rapid synthesis of pigments as the leaves unfold and expand, followed by a period before leaf senescence when pigment concentration remains fairly constant. The relative amounts of the four carotenoids remained constant in mature and senescent healthy leaves. Effects of TYMV Table virus.
infection on pigments in inoculated
leaves
2 summarizes the data on pigment content in local lesions for three strains of Infection with the two “milder” strains reduced the content of chlorophylls
382
E. S. Crosbie
and
R. E. F. Matthews
more than that of the carotenoids. The severe white strain had no significant differential effect on the two groups of pigments. Other analyses, not detailed here, showed that for any particular strain of virus at a given time after inoculation the individual carotenoid pigments were each reduced by about the same amount compared with corresponding healthy tissue. TABLE The effect
of infection
2
with three strains of TTMV on the pigments of Chinese cabbage leaves Virus Pale green 15=
Pigment Chlorophyll Chlorophyll Total
in local lesions
strain
Yellow 8
green
Severe 8
white 10
a b
45b 48
72 74
84 83
85 77
carotenoids
54
83
79
76
0 Time in days after inoculation. b Plants were inoculated on half leaves with virus, the other half leaves being rubbed with water. Virus did not invade the control half leaf during the period of the experiment. A I-mm diam. punch was used to remove tissue from the central region of developing lesions and from corresponding healthy tissue. Pigment concentrations were similar in the healthy tissue for the three strains. Pigment concentration is expressed as a percentage of that in the corresponding healthy leaf.
Effects of TZTMV infection on pigments in systemicallyinfected leaves On a single Chinese cabbage plant, successively younger leaves may be affected differently by TYMV infection. Under our conditions about three leaves above the inoculated leaves remained free of virus for some time. Expanding leaves above this group of leaves became invaded by virus about 3 to 4 days after inoculation and showed vein clearing after approximately 6 days. Still younger leaves show a mosaic pattern as soon as they emerged from the apical region. For a detailed study of the effects of TYMV on pigment production we selected the second oldest leaf to show the initial vein-clearing symptoms. These leaves were rapidly and evenly invaded by the virus. They were usually 3 to 4 cm long and were expanding rapidly when invaded by virus. Two separate experiments were carried out to examine the effect of a severe white strain and a pale green strain of the virus on the leaf pigments. Figure 1 illustrates the growth of the leaf and virus multiplication for the severe white strain at various times after inoculation. Virus multiplication was rapid between days 8 to 10 and leaf stunting became evident during this period. Figure 2 shows that the concentration of photosynthetic pigments in systemically infected leaves began to be reduced compared with healthy control tissue after 8 to 10 days but the effect did not become marked until day 12. The fall in concentration of pigments shown in Fig. 2 could be due to destruction of existing pigments, or to dilution of pigments by leaf expansion, or a combination of these factors. In virus-infected leaves the amount of each pigment per leaf reached a plateau value about day 12. Pigment content in healthy leaves continued to rise rapidly (Fig 3). Thus the fall in concentration shown in Fig. 2 was due to a cessation of net
Effects
of TYMV
synthesis pigments.
infection
of pigments
on leaf
383
pigments
and dilution
by leaf expansion
rather
than
a destruction
of
120 3 G e 80c
P ;i
A/ / A’ /’
4oI
p-’/, p *-.,,- v-m.,., / ,,Q 8
/
,
,
,
14
,
Time aftejfinaeubtian (days)2o
FIG. 1. Increase in leaf area and virus content of Chinese after inoculation with a severe white strain of TYMV. (A-A) cm2/infected leaf, (V - . - . - . 7) virus, mg/g fresh wt of infected
8
i-24
‘-‘-.-.-.v
20 8 14 20 8 Time after inoculation(days)
6
cabbage leaves at various times cm2/healthy leaf, (A - - - - A) leaf.
14
20
FIG. 2. Effect of systemic infection with a severe white strain of TYMV on the concentration of photosynthetic pigments. Concentrations are expressed in pg of pigment/g fresh wt of lamina. Plants were the same as those of Fig. 1. (a) (v----v) Healthy leaf chlorophyll a, (m----m) healthy leaf chlorophyll b, (V - . - . - . V) infected leaf chlorophyll a, (O - - - - q ) infected leaf chlorophyll b. (b) (v----v) Healthy leaf neoxanthin, (V - - - - V) infected leaf neoxanthin, (O-O) healthy leaf lutein, (0 - . - . - . 0) infected leaf lutein. (c) (A-A) Healthy leaf violaxanthin, (A - - - - A) infected leaf violaxanthin, (+--•+) healthy leaf catroenes, (0 -. - . -. 0) infected leaf carotenes.
In Fig. 4 the concentration of each percentage of that in the corresponding effect on all six pigments.
pigment healthy
in infected tissue is expressed as a tissue. Infection had a very similar
334
E. S. Crosbie
and
R. E. F. Matthews
25-
6-
8
14
8
20
14
8
20
Time after inoculqtion
14
20
(da);-9
FIG. 3. Effect of systemic infection with a severe white strain of TYMV on the amounts of pigment/leaf. Plants were the same as those used for the experiment of Fig. 1. The graphs show the pigments as pg/leaf. (a) (v---v) Chlorophyll a, healthy leaf, (V -. -. -. V) chlorophyll a, infected leaf, (m----m) chlorophyll b, healthy leaf, (O -. -. - . q ) chlorophyll b, infected leaf. (b) (v-v) Neoxanthin, healthy leaf, (V - . - . - . V) neoxanthin, infected leaf, (O-O) lutein, healthy leaf, (0 - . - . - . 0) lutein, infected leaf. (c) (+-+) Carotenes, healthy leaf, (0 - . - . - . 0) carotenes infected leaf, (&----A) violaxanthin, healthy leaf, (A - . - . -. A) violaxanthin, infected leaf.
\
i
i ii;
x.
‘. 30-
i\ :-A., I. I 8
I
I I 14
\ C. I
. I I 20 8
I
Time
‘.. T-‘-
I after
I I 14
\‘.
.
-.-._. I
k
0
I 20
inoculation
I 8
I
‘4‘he. \
_ . - .-. .
I I 14
+ I
I 20
(days)
FIG. 4. The concentration of photosynthetic pigments in leaf infected with a severe white strain of TYMV, expressed as a percentage of the concentration of the same pigment in the healthy leaf (experiment of Fig. 1). (a) (v----v) Chlorophyll a, (D - - - . - . W) chlorophyll b, (b) (v-7) neoxanthin, (0 -. - .-. 0) lutein, (c) (A- .- -- .A) violaxanthin, (+---+) carotenes.
In a similar experiment with a pale green strain of the virus, infection reduced the net rate of production of all six pigments to about 40 to 60 per cent of corresponding healthy leaves (Fig. 5), rather than causing a cessation of pigment production.
Pigments present in dark green islands from
leaves showing mosaic disease
The data in the first three columns of Table 3 suggest that dark green in leaves showing mosaic disease contains normal proportions
island tissue of the six
Effects
of TYMV
infection
385
on leaf pigments
photosynthetic pigments. The value of 26 pg/g fresh wt for violaxanthin in the healthy In addition it should be mentioned tissue was abnormally low in this experiment. that it was not possible to select healthy leaf tissue corresponding precisely to dark green island tissue so that the data in Table 3 do not conclusively demonstrate a higher than normal pigment content for dark green tissue.
32 16 8 7
II
17
7
II
Time after inoculation FIG. 5. Accumulation of photosynthetic a pale green strain of TYMV. Amounts Chlorophyll a, healthy leaf, (V - . -. - . v) healthy leaf, (u-.-.-. q ) chlorophyll 6, (v-.-.-. 8) neoxanthin, infected leaf, infected leaf. (c) (+--4) Carotenes, (A----& violaxanthin, healthy leaf, (A
Ii (days)
pigments in a healthy leaf and in a leaf infected with of pigment are shown as &leaf (a) (‘I----V) chlorophyll a, infected leaf, (H) chlorophyll b, infected leaf. (b) (v----v) Neoxanthin, healthy leaf, (o-e) lutein, healthy leaf, (o-a-.-. 0) lutein healthy leaf, (0 - . - . - . 0) carotenes, infected leaf, - - - - A) violaxanthin, infected leaf.
As the leaves showing mosaic patterns age, the dark green islands break down. Pale spots, similar in appearance to local lesions enlarge and coalesce. The last two columns of Table 3 suggest that a severe white strain affects all pigments to a similar extent in dark green tissue that is breaking down as it does in inoculated leaves (Table 2). TABLE
Concentration
Healthy tissue @g/g fresh wt)
Pigment Chlorophyll Chlorophyll Neoxanthin Violaxanthin Lutein Carotenes
a 6
3
of photosynthetic pigments in islands of dark green tissue of Chinese cabbage showing mosaic induced by a severe white strain of TYMV
1050 438 29 26 32 58
Newly formed green island (pg/g fresh wt) 1210 480 32 33 35 59
o/0 of healthy lamina 115 109 113 128 108 102
Older green island tissuea (w/g fresh 4
leaves
o/0 of healthy lamina
662 254 20 23 20 35
Pigments were analysed from healthy lamina, recently developed green island tissue older green island tissue that had begun to break down. Analysis of whole extracts performed to estimate the chlorophylls. Saponfied carotenoids were chromatographically fractionated prior to analysis. a This green island tissue was “breaking down” to give yellow spots containing virus text).
63 58 68 90 62 60 and was
(see
E. S. Crosbie
386
and R. E. F. Matthews
Inhibition of chlorophyll a synthesisin veryyoung leaves Leaves near the apex of Chinese cabbage plants that have been growing in large pots for several weeks are shielded from light by the outer leaves. Such leaves of healthy plants and of plants infected with a severe white strain of TYMV are similar in colour, both being pale yellow or cream. When such leaves O-5 to 2.5 cm. long were excised and placed in moist conditions in a glasshouse at 22 “C or under artificial (lighting 2000 lumens/sq. ft) for 1 to 2 days the leaves ceased to expand but chlorophyll synthesis occurred very rapidly. Healthy leaves became uniformly green. Leaves from infected plants rapidly developed a prominent mosaic pattern. The experiment summarized in Table 4 shows that leaves in the size range 1 to 2.5 cm, when first dissected from the diseased plant, contained about 100 p-g of chlorophyll a/g fresh wt. This is about one-tenth of the concentration in a mature leaf. After 24 h illumination the green islands had reached about half maximal size, while the chlorotic background (diseased) tissue had not significantly increased in pigment content. TABLE Chlorophyll
development
in young
Whole Tissue 1.5 to 2.5 cm leaves I.0 to 1.5 cm leaves
(to) 123 96
4
infected
leaves after exposure
to light
Chlorophyll a content &g/g fresh wt) leaf Chlorotic tissue Green island ‘kZ&
(2;;
129
473
tissue
Chinese cabbage plants inoculated with a severe white strain of TYMV were grown in 7-inch square pots of soil for 6 weeks. Leaves from the apex were then removed and analysed immediately for chlorophyll a by whole-extract analysis (to). A comparable group of excised leaves were placed on damp filter paper in a Petri dish kept in a glasshouse for 24 h (ts4). After this time the leaves had developed green islands surrounded by a chlorotic background. The green islands were dissected from the chlorotic tissue and both tissues were analysed for their chlorophyll content. DISCUSSION
In both inoculated and systemically infected leaves, the severe white strain of TYMV caused quantitatively similar reductions in the concentrations of all six photosynthetic pigments. These effects became marked only after virus synthesis was substantially completed suggesting that effects on pigments are a secondary consequence of infection by TYMV. Two separate classes of compounds-the chlorophylls and the carotenoids-and their two separate biosynthetic pathways are involved. Extra components on chromatograms that might be interpreted as virus-induced degradation products of the leaf pigments were not observed. The data support the view that infection mainly causes a block in the synthesis of pigments rather than an enhanced rate of destruction as shown in Table 4. This experiment also demonstrates that the mosaic pattern is established at a very early stage of leaf ontogeny as we have suggested previously from more indirect evidence [I, 101. The disappearance of pigments from local lesion areas in inoculated leaves could be due to blocked synthesis together with residual leaf expansion and a normal rate of chlorophyll destruction. Godnev &
Effects
of TYMV
infection
387
on leaf pigments
Shlik [4] calculated that half the chlorophyll in mature tobacco leaves was renewed every 13 days. The phenomenon of “turnover” in young leaves is well established but may not always be significant in mature leaves [II]. It is well known that the local lesions produced by TYMV in Chinese cabbage (even by a virus culture consisting mainly of one strain) are highly variable in macroscopic appearance. This variability could well be due to the effects of environmental conditions on the rate of breakdown and synthesis of the pigments in the healthy tissue surrounding the lesions. Differences between strains of virus, however, must also play a part in bringing about the variability in the appearance of local lesions. Milder strains of the virus (pale green and yellow green) caused more reduction in the chlorophylls than the carotenoids (Table 2), whereas a severe white strain affected all pigments to about the same extent. In the systemically infected leaf, the pale green strain of TYMV caused an approximately equal reduction in the amount of all six pigments (Fig. 5). As would be expected, the effect was less marked than with a severe white strain. Although the pigment changes appear to be secondary as far as virus synthesis is concerned they are an important part of the disease process, considering the plant as a whole. This aspect is discussed in the following paper in relation to data on plant growth [3]. We wish to thank
Mrs Judy
Douglas
and Mr
C. Whittingham
for skilled
assistance.
REFERENCES 1. CHALCROPT, J. P. & MATTHEWS, R. E. F. (1966). Cytological changes induced by turnip yellow mosaic virus in Chinese cabbage leaves. Virology 28, 555-562. 2. CHALCROFT, J. P. & MATTHEWS, R. E. F. (1967). Role of virus strains and leaf ontogeny in the production of mosaic patterns by turnip yellow mosaic virus. Virology 33, 659-673. 3. CROSBIE, E. S. & MATTHEWS, R. E. F. (1974). Effects of TYMV infection on the growth of Bras&a pekinensis Rupr. Physiological Plant Pathology 4, 389-400. 4. GODNEV, T. N. & SHLIK, A. A. (1955), r4C in the study of the biosynthesis of chlorophyll. First International Conference on the Peaceful Uses of Atomic Energy, 12, 358-363. 5. GREGORY, R. P. F. (1971). The Biochemistry of Photosynthesis. Wiley-Interscience, London, New York and Toronto. 6. KONISHI, K., OGAWA, T., ITOH, M. & SHIBATA, K. (1968). Minor carotenoid components in the chloroplasts of higher plants. Plant and Cell Physiology 9, 519-527. 7. MATTHEWS, R. E. F. (1970). Plant Virolo~. Academic Press, New York. 8. MATTHEWS, R. E. F. (1973). Induction of disease by plant viruses with special reference to turnip yellow mosaic virus. A&al Reviews of Phyto@athoio& 11, 147-170. 9. PETERSON, P. D. & MCKINNEY. H. H. ( 1938). The influence of four mosaic diseases on the Aolastid pigments and chlorophylls in tobacco Iekes. Phytopathology 28, 329-342. 10. REID, M. S. & MATTHEWS, R. E. F. (1966). On the origin of the mosaic induced by turnip yellow mosaic virus. Virolop 28, 563-570. 11. ROBERTS, D. W. A. & PERKINS, H. J. (1962). Chlorophyll biosynthesis and turnover in wheat leaves. Biochimica et bio#hysz’ca Acta 58, 499-506. 12. STRAIN, H. H., SHERMA, J., BENTON, F. L. & KATZ, J. J. (1965). Two way paper chromatography of the chloroplast pigments of leaves. Biochimica et biophysia Acta 109, 16-22. 13. STRAIN, H. H. & SVEC, W. A. (1966). Extraction, separation and isolation. In The Chlorophylls, ed. by L. P. Vernon and G. R. Seely, pp. 2 l-66. Academic Press, New York.
26
Plant Pathology
(1974)
4, 379-387
Effects of TYMV infection on leaf pigments in Brassica pekinensis Ru p r. E. S. CROSBIE Department University (Acceptedfor
and R. E. F. MATTHEWS
of Cell BioloQ, Auckland,
of Auckland,
publiGation
December
New
Zealand
1973)
The effects are described of turnip yellow mosaic virus (TYMV) infection on the production of photosynthetic pigments in Chinese cabbage plants. Chlorophylls a and b and the four carotenoid pigments were analysed in plants following their infection with three strains of TYMV. The results support the view that infection reduces the amount of pigments in the plant mainly by inhibition of synthesis rather than by destruction of pre-existing pigments. In inoculated leaves, pale green and yellow green strains of the virus reduced the concentration of chlorophylls more than the carotenoids. A severe white strain of the virus reduced the concentration of all six pigments to a similar extent in all tissues tested.
INTRODUCTION In the yellows and mosaic diseases induced by many plant viruses it is obvious that infection leads to a reduction in photosynthetic pigments. However, there has been little detailed investigation of this effect ofvirus infection since the study by Peterson & McKinney [9] on the effects of four strains of tobacco mosaic virus (TMV) on pigments in tobacco. Turnip yellow mosaic virus (TYMV) is of particular interest with respect to the photosynthetic pigments since it is known to induce numerous cytological and biochemical changes in the chloroplasts, some of which appear to be closely connected with virus replication (reviewed in [7, 81). As part of a general study of the induction of disease by TYMV, we report here the effects of infection on the six chlorophyll and carotenoid pigments found in Brassica pekinensis. In the following paper [3] we relate the changes observed in the amount of pigments to the effects of infection on plant growth. Leaves of Chinese cabbage plants showing mosaic disease due to infection with TYMV may contain islands of dark green tissue which appear to be cytologically and biochemically normal and which contain little or no virus [I, 101. We have established that the pigment content of such dark green islands is similar to that of healthy leaf. As a leaf showing mosaic disease ages, the dark green areas tend to “break down” giving rise to numerous small areas in which virus replicates. These areas show pigment changes similar to those found in local lesions. Experiments with very small yellow leaves excised from the apex of diseased plants showed that a latent mosaic pattern exists before pigment development. 25
330
E. S. Crosbie and R. E. F. Matthews
MATERIALS
AND
METHODS
Plants and virus The host plant, Brassica pekinensis (Rupr.) var. Wong Bok (Chinese cabbage), was grown from stock commercial seed and transferred to 4-inch pots at the two-cotyledon stage. In some experiments the plants were later transplanted to T-inch square pots to ensure that growth was not checked by shortage of nutrients. Plants were grown in the glasshouse at 21 & 3 “C under natural light conditions. In winter the light was supplemented by four hours of artificial lighting, morning and evening, provided by Phillips 400 W HLRG lamps. Three strains of virus (severe white, pale green and yellow-green), were derived by serial selection of appropriate leaf material from plants infected with the stock culture of TYMV [Z]. Fresh extracts of infected leaf tissue were used as inocula. Plants were infected by mechanical inoculation of three older leaves at a stage when plants had developed a total of four to six well-expanded leaves. Analysis
of pigments in unfractionated
extracts
Small amounts (approximately 20 mg) of leaf lamina were ground in a KontesDual1 homogenizer in 2 ml of 95 y0 acetone. After clarification of the supernatant by standing for 0.5 h (or a brief, low speed centrifugation) the absorbance was read at 663, 645, 630 and 445 nm in a Cary model 15 spectrophotometer. Concentration of carotenoids was calculated directly from the absorbance at 445 nm. For the chlorophylls the formulae of Strain & Svec [13] were applied. Chromatograjhic
fractionation
of pigments
Extraction of pigments, preparation of the extract and subsequent chromatography were carried out as described in [12]. When analysing either chlorophylls or carotenoids a two-dimensional chromatogram was preferred because it provided better resolving power. This was particularly important when analysing pigments from infected tissue as a wide range of pigment concentration was encountered. For most samples, 0.4 to O-5 g of tissue was used and the volumes of extraction and “transfer” solvents were adjusted accordingly. The chromatographic solvents were : petroleum spirit (b.p. 80 to 100 “C) : n-propanol, 99 : 1, v/v, in the first dimension and petroleum spirit : chloroform, 3 : 1, v/v, in the second dimension. Ten ~1 of pigment concentrate was applied to Whatman 3 mm paper as a l-cm band. For carotenoid analysis chromatography was carried out for 40 min in the first dimension and O-5 h in the second dimension. At this time the pigments were sufficiently resolved and the spots showed minimal lateral spread. Spots were located by examination under U.V. light. The spots were cut out and the pigment eluted from the paper at four degrees by two successive extractions with 3 ml of acetone. The extracts were combined and evaporated to dryness at 32 degrees on a vacuum evaporator. The pigment was taken up in O-5 ml of chloroform and analysed spectrophotometrically. Estimation
of virus concentration
Virus concentration ultracentrifuge [3].
was
estimated
by
analysis
of leaf
extracts
in the
analytical
Effects
of TYMV
infection
on leaf pigments
381
RESULTS Pigments present in healthy Chinese cabbage leaves Six major pigments were identified by their spectral characteristics following chromatography of extracts from healthy leaf lamina. Two major green spots were identified as chlorophyll a and b. A single spot contained the a and b carotenes while the xanthophylls consisted of neoxanthin, violaxanthin and lutein. An “insoluble” spot of variable intensity was found at the origin. Strain et al. [12] considered this to be one of four lutein spots. However, this component from our chromatograms had an absorption spectrum similar to that of violaxanthin, but a mixture of violaxanthin, neoxanthin and lutein could also have produced a very similar spectrum. Thus the composition of the insoluble spot was not established. Occasionally two minor spots were seen on the chromatogram at the positions where Strain et al. [12] indicated two other lutein spots in extracts from various plants. Table 1 summarizes the pigment composition of healthy leaf tissue. The low content of lutein may result, in part, from some of the lutein running in the minor spots noted above. On the other hand, a low content of lutein may be characteristic of the genus Brassica [Cl. TABLE
The photosynthetic
(1)
pg/g fresh wt + standard deviation
Pigment Neoxanthin Violaxanthin Lutein Carotenes Chlorophyll Chlorophyll
a b
24.9+ 30.6+ 35.5+ 57.8+ 982 +43 367 *I3
2.6 2:6 2.5 I.1
pigments
1
of Brassica
pekinensis
(2)
o/O of total B. pekinensis 1.7 2.0 2.4 3.8 65.6 24.5
pigment Spinach” 2.2 2.2 7.9 5.0 57.6 25.2
o/O of total B. pekiruxsis 17.8 21.8 24.1 36.3
(3) carotenoids B. campest&” 11.0 13.0 31 38
a Adapted from [5]. b From [S]. Ten samples of healthy mature leaf lamina from Chinese cabbage were analysed for their chlorophyll content by spectrophotometric analysis of the acetone extracts. A single 2-g leaf sample was ground and the filtered acetone extract divided equally into four. These were then independently analysed for their carotenoid content spectrophotometrically after chromatographic fractionation of the saponified pigments. (1) The pigment content of a healthy Chinese cabbage leaf. (2) A comparison of the pigment content of B. pekinensis with spinach. (3) A comparison of the carotenoid content of B. pekinensis with that of B. campestris.
Analyses carried out on successive leaves of healthy plants indicated that there is a very rapid synthesis of pigments as the leaves unfold and expand, followed by a period before leaf senescence when pigment concentration remains fairly constant. The relative amounts of the four carotenoids remained constant in mature and senescent healthy leaves. Effects of TYMV Table virus.
infection on pigments in inoculated
leaves
2 summarizes the data on pigment content in local lesions for three strains of Infection with the two “milder” strains reduced the content of chlorophylls
382
E. S. Crosbie
and
R. E. F. Matthews
more than that of the carotenoids. The severe white strain had no significant differential effect on the two groups of pigments. Other analyses, not detailed here, showed that for any particular strain of virus at a given time after inoculation the individual carotenoid pigments were each reduced by about the same amount compared with corresponding healthy tissue. TABLE The effect
of infection
2
with three strains of TTMV on the pigments of Chinese cabbage leaves Virus Pale green 15=
Pigment Chlorophyll Chlorophyll Total
in local lesions
strain
Yellow 8
green
Severe 8
white 10
a b
45b 48
72 74
84 83
85 77
carotenoids
54
83
79
76
0 Time in days after inoculation. b Plants were inoculated on half leaves with virus, the other half leaves being rubbed with water. Virus did not invade the control half leaf during the period of the experiment. A I-mm diam. punch was used to remove tissue from the central region of developing lesions and from corresponding healthy tissue. Pigment concentrations were similar in the healthy tissue for the three strains. Pigment concentration is expressed as a percentage of that in the corresponding healthy leaf.
Effects of TZTMV infection on pigments in systemicallyinfected leaves On a single Chinese cabbage plant, successively younger leaves may be affected differently by TYMV infection. Under our conditions about three leaves above the inoculated leaves remained free of virus for some time. Expanding leaves above this group of leaves became invaded by virus about 3 to 4 days after inoculation and showed vein clearing after approximately 6 days. Still younger leaves show a mosaic pattern as soon as they emerged from the apical region. For a detailed study of the effects of TYMV on pigment production we selected the second oldest leaf to show the initial vein-clearing symptoms. These leaves were rapidly and evenly invaded by the virus. They were usually 3 to 4 cm long and were expanding rapidly when invaded by virus. Two separate experiments were carried out to examine the effect of a severe white strain and a pale green strain of the virus on the leaf pigments. Figure 1 illustrates the growth of the leaf and virus multiplication for the severe white strain at various times after inoculation. Virus multiplication was rapid between days 8 to 10 and leaf stunting became evident during this period. Figure 2 shows that the concentration of photosynthetic pigments in systemically infected leaves began to be reduced compared with healthy control tissue after 8 to 10 days but the effect did not become marked until day 12. The fall in concentration of pigments shown in Fig. 2 could be due to destruction of existing pigments, or to dilution of pigments by leaf expansion, or a combination of these factors. In virus-infected leaves the amount of each pigment per leaf reached a plateau value about day 12. Pigment content in healthy leaves continued to rise rapidly (Fig 3). Thus the fall in concentration shown in Fig. 2 was due to a cessation of net
Effects
of TYMV
synthesis pigments.
infection
of pigments
on leaf
383
pigments
and dilution
by leaf expansion
rather
than
a destruction
of
120 3 G e 80c
P ;i
A/ / A’ /’
4oI
p-’/, p *-.,,- v-m.,., / ,,Q 8
/
,
,
,
14
,
Time aftejfinaeubtian (days)2o
FIG. 1. Increase in leaf area and virus content of Chinese after inoculation with a severe white strain of TYMV. (A-A) cm2/infected leaf, (V - . - . - . 7) virus, mg/g fresh wt of infected
8
i-24
‘-‘-.-.-.v
20 8 14 20 8 Time after inoculation(days)
6
cabbage leaves at various times cm2/healthy leaf, (A - - - - A) leaf.
14
20
FIG. 2. Effect of systemic infection with a severe white strain of TYMV on the concentration of photosynthetic pigments. Concentrations are expressed in pg of pigment/g fresh wt of lamina. Plants were the same as those of Fig. 1. (a) (v----v) Healthy leaf chlorophyll a, (m----m) healthy leaf chlorophyll b, (V - . - . - . V) infected leaf chlorophyll a, (O - - - - q ) infected leaf chlorophyll b. (b) (v----v) Healthy leaf neoxanthin, (V - - - - V) infected leaf neoxanthin, (O-O) healthy leaf lutein, (0 - . - . - . 0) infected leaf lutein. (c) (A-A) Healthy leaf violaxanthin, (A - - - - A) infected leaf violaxanthin, (+--•+) healthy leaf catroenes, (0 -. - . -. 0) infected leaf carotenes.
In Fig. 4 the concentration of each percentage of that in the corresponding effect on all six pigments.
pigment healthy
in infected tissue is expressed as a tissue. Infection had a very similar
334
E. S. Crosbie
and
R. E. F. Matthews
25-
6-
8
14
8
20
14
8
20
Time after inoculqtion
14
20
(da);-9
FIG. 3. Effect of systemic infection with a severe white strain of TYMV on the amounts of pigment/leaf. Plants were the same as those used for the experiment of Fig. 1. The graphs show the pigments as pg/leaf. (a) (v---v) Chlorophyll a, healthy leaf, (V -. -. -. V) chlorophyll a, infected leaf, (m----m) chlorophyll b, healthy leaf, (O -. -. - . q ) chlorophyll b, infected leaf. (b) (v-v) Neoxanthin, healthy leaf, (V - . - . - . V) neoxanthin, infected leaf, (O-O) lutein, healthy leaf, (0 - . - . - . 0) lutein, infected leaf. (c) (+-+) Carotenes, healthy leaf, (0 - . - . - . 0) carotenes infected leaf, (&----A) violaxanthin, healthy leaf, (A - . - . -. A) violaxanthin, infected leaf.
\
i
i ii;
x.
‘. 30-
i\ :-A., I. I 8
I
I I 14
\ C. I
. I I 20 8
I
Time
‘.. T-‘-
I after
I I 14
\‘.
.
-.-._. I
k
0
I 20
inoculation
I 8
I
‘4‘he. \
_ . - .-. .
I I 14
+ I
I 20
(days)
FIG. 4. The concentration of photosynthetic pigments in leaf infected with a severe white strain of TYMV, expressed as a percentage of the concentration of the same pigment in the healthy leaf (experiment of Fig. 1). (a) (v----v) Chlorophyll a, (D - - - . - . W) chlorophyll b, (b) (v-7) neoxanthin, (0 -. - .-. 0) lutein, (c) (A- .- -- .A) violaxanthin, (+---+) carotenes.
In a similar experiment with a pale green strain of the virus, infection reduced the net rate of production of all six pigments to about 40 to 60 per cent of corresponding healthy leaves (Fig. 5), rather than causing a cessation of pigment production.
Pigments present in dark green islands from
leaves showing mosaic disease
The data in the first three columns of Table 3 suggest that dark green in leaves showing mosaic disease contains normal proportions
island tissue of the six
Effects
of TYMV
infection
385
on leaf pigments
photosynthetic pigments. The value of 26 pg/g fresh wt for violaxanthin in the healthy In addition it should be mentioned tissue was abnormally low in this experiment. that it was not possible to select healthy leaf tissue corresponding precisely to dark green island tissue so that the data in Table 3 do not conclusively demonstrate a higher than normal pigment content for dark green tissue.
32 16 8 7
II
17
7
II
Time after inoculation FIG. 5. Accumulation of photosynthetic a pale green strain of TYMV. Amounts Chlorophyll a, healthy leaf, (V - . -. - . v) healthy leaf, (u-.-.-. q ) chlorophyll 6, (v-.-.-. 8) neoxanthin, infected leaf, infected leaf. (c) (+--4) Carotenes, (A----& violaxanthin, healthy leaf, (A
Ii (days)
pigments in a healthy leaf and in a leaf infected with of pigment are shown as &leaf (a) (‘I----V) chlorophyll a, infected leaf, (H) chlorophyll b, infected leaf. (b) (v----v) Neoxanthin, healthy leaf, (o-e) lutein, healthy leaf, (o-a-.-. 0) lutein healthy leaf, (0 - . - . - . 0) carotenes, infected leaf, - - - - A) violaxanthin, infected leaf.
As the leaves showing mosaic patterns age, the dark green islands break down. Pale spots, similar in appearance to local lesions enlarge and coalesce. The last two columns of Table 3 suggest that a severe white strain affects all pigments to a similar extent in dark green tissue that is breaking down as it does in inoculated leaves (Table 2). TABLE
Concentration
Healthy tissue @g/g fresh wt)
Pigment Chlorophyll Chlorophyll Neoxanthin Violaxanthin Lutein Carotenes
a 6
3
of photosynthetic pigments in islands of dark green tissue of Chinese cabbage showing mosaic induced by a severe white strain of TYMV
1050 438 29 26 32 58
Newly formed green island (pg/g fresh wt) 1210 480 32 33 35 59
o/0 of healthy lamina 115 109 113 128 108 102
Older green island tissuea (w/g fresh 4
leaves
o/0 of healthy lamina
662 254 20 23 20 35
Pigments were analysed from healthy lamina, recently developed green island tissue older green island tissue that had begun to break down. Analysis of whole extracts performed to estimate the chlorophylls. Saponfied carotenoids were chromatographically fractionated prior to analysis. a This green island tissue was “breaking down” to give yellow spots containing virus text).
63 58 68 90 62 60 and was
(see
E. S. Crosbie
386
and R. E. F. Matthews
Inhibition of chlorophyll a synthesisin veryyoung leaves Leaves near the apex of Chinese cabbage plants that have been growing in large pots for several weeks are shielded from light by the outer leaves. Such leaves of healthy plants and of plants infected with a severe white strain of TYMV are similar in colour, both being pale yellow or cream. When such leaves O-5 to 2.5 cm. long were excised and placed in moist conditions in a glasshouse at 22 “C or under artificial (lighting 2000 lumens/sq. ft) for 1 to 2 days the leaves ceased to expand but chlorophyll synthesis occurred very rapidly. Healthy leaves became uniformly green. Leaves from infected plants rapidly developed a prominent mosaic pattern. The experiment summarized in Table 4 shows that leaves in the size range 1 to 2.5 cm, when first dissected from the diseased plant, contained about 100 p-g of chlorophyll a/g fresh wt. This is about one-tenth of the concentration in a mature leaf. After 24 h illumination the green islands had reached about half maximal size, while the chlorotic background (diseased) tissue had not significantly increased in pigment content. TABLE Chlorophyll
development
in young
Whole Tissue 1.5 to 2.5 cm leaves I.0 to 1.5 cm leaves
(to) 123 96
4
infected
leaves after exposure
to light
Chlorophyll a content &g/g fresh wt) leaf Chlorotic tissue Green island ‘kZ&
(2;;
129
473
tissue
Chinese cabbage plants inoculated with a severe white strain of TYMV were grown in 7-inch square pots of soil for 6 weeks. Leaves from the apex were then removed and analysed immediately for chlorophyll a by whole-extract analysis (to). A comparable group of excised leaves were placed on damp filter paper in a Petri dish kept in a glasshouse for 24 h (ts4). After this time the leaves had developed green islands surrounded by a chlorotic background. The green islands were dissected from the chlorotic tissue and both tissues were analysed for their chlorophyll content. DISCUSSION
In both inoculated and systemically infected leaves, the severe white strain of TYMV caused quantitatively similar reductions in the concentrations of all six photosynthetic pigments. These effects became marked only after virus synthesis was substantially completed suggesting that effects on pigments are a secondary consequence of infection by TYMV. Two separate classes of compounds-the chlorophylls and the carotenoids-and their two separate biosynthetic pathways are involved. Extra components on chromatograms that might be interpreted as virus-induced degradation products of the leaf pigments were not observed. The data support the view that infection mainly causes a block in the synthesis of pigments rather than an enhanced rate of destruction as shown in Table 4. This experiment also demonstrates that the mosaic pattern is established at a very early stage of leaf ontogeny as we have suggested previously from more indirect evidence [I, 101. The disappearance of pigments from local lesion areas in inoculated leaves could be due to blocked synthesis together with residual leaf expansion and a normal rate of chlorophyll destruction. Godnev &
Effects
of TYMV
infection
387
on leaf pigments
Shlik [4] calculated that half the chlorophyll in mature tobacco leaves was renewed every 13 days. The phenomenon of “turnover” in young leaves is well established but may not always be significant in mature leaves [II]. It is well known that the local lesions produced by TYMV in Chinese cabbage (even by a virus culture consisting mainly of one strain) are highly variable in macroscopic appearance. This variability could well be due to the effects of environmental conditions on the rate of breakdown and synthesis of the pigments in the healthy tissue surrounding the lesions. Differences between strains of virus, however, must also play a part in bringing about the variability in the appearance of local lesions. Milder strains of the virus (pale green and yellow green) caused more reduction in the chlorophylls than the carotenoids (Table 2), whereas a severe white strain affected all pigments to about the same extent. In the systemically infected leaf, the pale green strain of TYMV caused an approximately equal reduction in the amount of all six pigments (Fig. 5). As would be expected, the effect was less marked than with a severe white strain. Although the pigment changes appear to be secondary as far as virus synthesis is concerned they are an important part of the disease process, considering the plant as a whole. This aspect is discussed in the following paper in relation to data on plant growth [3]. We wish to thank
Mrs Judy
Douglas
and Mr
C. Whittingham
for skilled
assistance.
REFERENCES 1. CHALCROPT, J. P. & MATTHEWS, R. E. F. (1966). Cytological changes induced by turnip yellow mosaic virus in Chinese cabbage leaves. Virology 28, 555-562. 2. CHALCROFT, J. P. & MATTHEWS, R. E. F. (1967). Role of virus strains and leaf ontogeny in the production of mosaic patterns by turnip yellow mosaic virus. Virology 33, 659-673. 3. CROSBIE, E. S. & MATTHEWS, R. E. F. (1974). Effects of TYMV infection on the growth of Bras&a pekinensis Rupr. Physiological Plant Pathology 4, 389-400. 4. GODNEV, T. N. & SHLIK, A. A. (1955), r4C in the study of the biosynthesis of chlorophyll. First International Conference on the Peaceful Uses of Atomic Energy, 12, 358-363. 5. GREGORY, R. P. F. (1971). The Biochemistry of Photosynthesis. Wiley-Interscience, London, New York and Toronto. 6. KONISHI, K., OGAWA, T., ITOH, M. & SHIBATA, K. (1968). Minor carotenoid components in the chloroplasts of higher plants. Plant and Cell Physiology 9, 519-527. 7. MATTHEWS, R. E. F. (1970). Plant Virolo~. Academic Press, New York. 8. MATTHEWS, R. E. F. (1973). Induction of disease by plant viruses with special reference to turnip yellow mosaic virus. A&al Reviews of Phyto@athoio& 11, 147-170. 9. PETERSON, P. D. & MCKINNEY. H. H. ( 1938). The influence of four mosaic diseases on the Aolastid pigments and chlorophylls in tobacco Iekes. Phytopathology 28, 329-342. 10. REID, M. S. & MATTHEWS, R. E. F. (1966). On the origin of the mosaic induced by turnip yellow mosaic virus. Virolop 28, 563-570. 11. ROBERTS, D. W. A. & PERKINS, H. J. (1962). Chlorophyll biosynthesis and turnover in wheat leaves. Biochimica et bio#hysz’ca Acta 58, 499-506. 12. STRAIN, H. H., SHERMA, J., BENTON, F. L. & KATZ, J. J. (1965). Two way paper chromatography of the chloroplast pigments of leaves. Biochimica et biophysia Acta 109, 16-22. 13. STRAIN, H. H. & SVEC, W. A. (1966). Extraction, separation and isolation. In The Chlorophylls, ed. by L. P. Vernon and G. R. Seely, pp. 2 l-66. Academic Press, New York.
26