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Effects of TYMV infection on growth of Brassica pekinensis Rupr
Physiological
Plant Pathology
(1974)
4, 389-400
Effects of TYMV infection on growth of Brassica pekinensis Rupr. E. S. CROSBIE and R. E. F. MATTHEWS Department of Cell Biology, University of Auckland, Auckland, (Accepted for publication,
December
New
Zealand
1973)
The effect of turnip yellow mosaic virus (TYMV) infection on the growth of various parts of Chinese cabbage plants was studied in relation to virus synthesis, the production of leaf pigments and various macromolecules. Mild and severe strains of the virus affected overall growth to about the same extent, but growth form and distribution of chlorophyll were affected differently. Among the host components studied, the first to be affected by infection were chlorophyll and Fraction I protein. Late in infection the reduction of ribosomes and proteins on a weight/plant basis was about 20 times greater than the weight of virus produced. Dark green islands of tissue of the mosaic caused by a severe white strain of the virus made a significant contribution to the total chlorophyll of the diseased plant. The proportion of lamina that became dark green tissue in new leaves could be increased by removal of old expanded leaves that had escaped infection.
INTRODUCTION The symptoms of many plant virus diseases have recently been summarized [6]. Descriptions of these diseases have usually been confined to the visual symptoms, and to general statements concerning overall effects on growth. In a few studies changes in the external morphology of the plant have been observed in detail during disease development (e.g. with tobacco mosaic virus in tobacco [7]). However there is very little quantitative information concerning the development of disease in different parts of the plant in relation to virus replication, and to the effects of infection on important classes of molecules and organelles in the cell. The work described below forms part of a general study of the relationship between the replication of turnip yellow mosaic virus (TYMV) and disease production in Chinese cabbage. The stock culture of TYMV induces a typical mosaic disease in systemically infected Chinese cabbage plants. Islands of tissue in the mosaic contain different strains of the virus that affect the chloroplasts more or less severely. As the disease develops, dark green islands of tissue appear in the mosaic. These islands contain little or no virus and appear to be cytologically and metabolically normal [Z, 51. After a period of weeks these dark green islands may “break down”, a process involving the appearance of many yellow local lesion-like areas containing virus. In the work described here we have measured the effects of infection with a mild and a severe strain of TYMV on the growth of various parts of the Chinese cabbage plant during the,development of disease. We have attempted to relate these changes to the production of virus and to changes in important classes of compounds and
390
E. S. Crosbie
and
R. E. F. Matthews
organelles in the leaf. We have also assessed the significance of dark green islands in the mosaic (and of older leaves which escape infection for a time) for the survival and growth of diseased plants.
MATERIALS
AND
METHQDS
Growth of Chinese cabbage plants (Bras&a pekinensis Rupr. var. Wong of TYMV and pigment analyses were as described previously [4]. Analysis
of Fraction
I protein
and Fraction
Ilproteins,
TTMV,
Bok)
cultures
83 S and 68 S ribosomes
Fresh leaf lamina (0.5 to 0.8 g) was mixed with twice its weight of extraction medium (modified from [3] : 0.01 M-Tris-HCl buffer, pH 7.2, 0.02 M-MgC1, and 0.003 M-mercaptoethanol. The mixture was ground in a small pestle and mortar until there were no visible pieces of leaf remaining. The extract was then subjected to a low speed centrifugation and the resulting supernatant fluid was examined in a Model E analytical ultracentrifuge using Schlieren optics. Photographs showing the ribosomes and virus boundaries were recorded after centrifugation at 40 000 rev/min for 8 min. Fraction I and Fraction II components were recorded after a further 16 min at 52 000 rev/min. From a photographic record of the schlieren pattern the concentrations of the components were estimated by planimetry, with correction for radial dilution. The area units were converted to absolute concentrations by comparison with values for a known concentration. Measurement
of areas
Total leaf lamina area was measured by weighing paper facsimile cutouts of leaves using paper of known weight per unit area. Green islands were outlined on the paper and estimated in the same way. Only islands larger than about 2 mm diam were measured. Fresh weights Leaves, petioles plus midrib and stems were excised, grouped as described below and weighed immediately. Root fresh weight was obtained after flushing the soil from the roots with water. Excess water was removed by placing the roots on an absorbent surface for half an hour before they were weighed. Definition
of leaf groups used
In growth experiments it was not practicable to consider every leaf separately. Leaves were pooled in groups as follows. Inoculated leaves. Two well-expanded leaves of each plant were inoculated mechanically. Two or three much smaller and older leaves were removed from each plant at the time of inoculation and discarded. Sub-systemic leaves. Under our conditions of growth the group of about three leaves immediately above the inoculated leaves did not become infected when the virus moved into the younger leaves. Some leaves of this group remained uninfected for a period of weeks, or sometimes until they senesced. Others became infected by a slow
Effects
of TYMV
infection
391
invasion of the virus from the base of the leaf, beginning after systemic infection of younger leaves was well advanced. Systemic leaves. These are the leaves in which the characteristic mosaic pattern of TYMV infection develops. They were numbered from the oldest (i.e. S, is the oldest systemically infected leaf on the plant). For sampling the S leaves were usually taken in two groups:-S,-, that do not develop green islands and S,, that do.
RESULTS
After preliminary trials a major growth experiment was carried out using four sets of Chinese cabbage plants. The objectives of the experiment were (i) to measure the growth of the various parts of healthy and virus-infected plants during the development of disease; and (ii) to attempt to relate these changes to virus synthesis and changes in the amounts of certain compounds and organelles in the leaf In addition, the importance of the sub-systemic group of leaves for the growth of the plant was assessed by removing these leaves from some plants after the disease was established. At the commencement of the experiment, 146 one-month-old plants were selected for uniformity. One group of 10 were used for base time measurements (week 0, in the figures). Eighty were inoculated on two leaves with a severe white strain of TYMV and sorted into eight groups of 10 plants. The remaining 56 plants were inoculated with water and divided into groups of seven. At intervals thereafter a group of 10 virus infected and seven healthy plants were analysed for fresh weight, leaf area, leaf number, dry weight, chlorophyll, Fraction I protein, Fraction II proteins, 83 S ribosomes, 68 S ribosomes, virus and green island content. All the plants were re-potted into successively larger pots at three and five weeks after inoculation. Twenty-five days after inoculation, one group of healthy and one group of infected plants had their sub-systemic leaves removed to test the importance of these leaves for continued plant growth. The e$eectof infection on total aerial fresh weight During the 7-week experimental period the total aerial fresh weight of healthy plants increased about 68fold in approximately logarithmic fashion (Fig. 1). Increase in the weight of TYMV-infected plants was approximately linear during the period of rapid virus increase (as measured for the plant as a whole). The fresh weight per infected plant was 240 g less than for healthy plants at a time when 35 mg of TYMV per plant had been produced. Measurable green island tissue in the leaves showing mosaic disease first appeared four weeks after infection. Growth curves similar to those of Fig. 1 for healthy and infected plants were also obtained from measurements of total lamina area or total dry weight of lamina. Comparison
of various plant parts
Expressed as a percentage of corresponding healthy tissue, virus infection had similar effects on the fresh weight of root, petiole and stem. However, total leaf lamina fresh weight was less affected than other parts of the plants (Fig. 2).
392
E. S. Crosbie and R. E. F. Matthews c 320 -
Time after inoculation
(weeks)
FIG. 1. Effect of infection with a severe white strain of TYMV on aerial fresh weight of Chinese cabbage plants. Infected plants were inoculated at week 0. (O-O) Total aerial fresh weight, g/healthy plant, (0 - . - . - . 0) total aerial fresh weight, g/infected plant, (V . . . .v) total virus, mg/infected plant, (A - - - - A) dark green island tissue, g/infected plant.
Ti&
after inoculati&
(weeks)
FIG. 2. Effect of infection with a severe white strain of TYMV on the fresh weight of various parts of the Chinese cabbage plant. Fresh weight of roots, stem, petiole and lamiua from infected plants are expressed as a percentage of the corresponding healthy plants. The virus content of the infected plant is also given. Infected plants were inoculated at week 0. (A -. -. -. A) Lamina, (V - - - - - V) roots, (t-m) petiole, (O- - -0) stem, (V . . . V) virus, mg/plant.
E$ects on lam&a fresh weight for various groups of leaves The effects of TYMV infection on the four leaf groups defined under “Materials and Methods” are surnmarized in Fig. 3. Inoculated leaves of the diseased plants became stunted after the appearance of local lesions and such leaves began to senesce earlier than the corresponding healthy leaves. All inoculated leaves were dead by week 5 [Fig. 3 (a)].
393
Effects of TYMV infection
The reduced total fresh weight of the sub-systemic leaves of the diseased plants [Fig. 3(b)] was due to enhanced senescence rather than stunting.
‘*O
‘“Lw .
.
total lamina
l
00 b
9
/
,$ F.w. systemic
lqminq .
120 ml 00
(bf F.W. subsystemic
Time after inoculation FIG 3. Effect of infection for various groups of leaves (o-.-.-. 0) infected, (A diseased plants from which
lamina
(week!
with a severe white strain of TYMV on the total lamina of Chinese cabbage. Fresh weights in g/plant. (0-O) - - - A) dark green island tissue, (0 - - Cl) systemic sub-systemic leaves were removed.
fresh weight Healthy, lamina from
The fresh weight of systemic infected lamina increased in a linear fashion after week 3, in contrast to the corresponding healthy leaves, which increased in an approximately logarithmic manner [Fig. 3(c)]. Fig. 3(c) also shows the great reduction in plant growth after the removal of the sub-systemic leaves at 3.5 weeks. The fresh weights in Fig. 3 (d) largely reflect those of the systemic groups of leaves. Growth curves for two subgroups of the systemic leaves are given in Fig. 4 for the later part of experiment (these subgroups were not measured at earlier times). For the older group (1 to 6) the fresh weight of healthy leaves continued to increase as expansion more than counterbalanced senescence [Fig. 4(a)]. By contrast weight loss due to senescence of the infected 1 to 6 groups was greater than gain due to expansion [Fig. 4(b)]. This group of diseased leaves did not contain islands of dark green tissue. The younger group of leaves (7 +) grew at an increasing rate in the healthy plants and at a linear rate in the infected plants. The infected 7 + group contained green island tissue.
394
E. S. Crosbie
E$ect of infection on total chorophyll
and
R. E. F. Matthews
in the plant
Samples were taken from every third leaf on the plants, pooled and analysed for chlorophyll using unfractionated extracts (Fig. 5). The rate of increase in chlorophyll a was similar to the increase in fresh weight of the healthy plants shown in Fig. 1. There was a 57-fold increase in chlorophyll a per healthy plant over the 7-week period. In infected plants the reduction in chlorophyll compared with healthy plants was greater than the reduction in fresh weight, due of course to the combined effects of stunting and reduced chlorophyll concentration.
90 - (a'
60-
30 0
,. .’ ,’--tL ---a vs *.__---- , I-E ,*.&HA’ I 3 5 7 o---o-
0 L-
I
Time after inoculation
(weeks)
FIG. 4. Comparative growth of older (1 to 6) and younger (7 + ) systemically infected leaves of TYMV-infected Chinese cabbage nlants and the corresponding leaves of healthy plants. Plants infected with a severe white &a$. Growth is expressed in total grams fresh weighdplant. (a) (A-A) Healthy HS/,+ leaves, (o-e) healthy H S,-, leaves. (b) (0 - - - - 0) Infected V S,-s leaves, (A- - -A) infected VS,+ leaves, (A- - -A) dark green island tissue.
As shown in Fig. 5, the green island tissue total chlorophyll of the infected plants from that only the green island tissue large enough although many very small green islands were
made a substantial contribution to the week 5 onwards. It should be noted to be measured ( > 2 mm) was recorded present in some leaves.
E$ect of infection on macromolecules The effects of infection on the total amounts of Fraction I protein, Fraction II protein, 83 S and 68 S ribosomes per plant compared with healthy plants are shown in Fig. 6. Compared with healthy plants the components found in the chloroplasts of infected plants (68 S ribosomes, Fraction I protein and chlorophyll a) were reduced very rapidly between weeks 2 to 4. These components were reduced both in conBy contrast, the components known to be centration and total amount per plant.
Effects
of TYMV
infection
90
E 60 -
.
I
I 30 ,. f
,,,/--v
/p+ L
FIG.
severe (v-----
.d
,~c ._.-.- ‘3 ‘5’
I 3 5 Time after inoculation
7 (weeks)
5. Chlorophyll a content of healthy Chinese cabbage white strain of TYMV. Total chlorophyll a expressed V) infected, (A - . - . - . A) dark green island tissue
plants and plants infected with as mg/plant. (r--v) Healthy, in virus-infected plants.
a
i I
I I 2 Time after
I l 4 inoculation
l 1 6 (weeks)
l 8
FIG. 6.
cabbage healthy.) protein,
Percentage reduction in amount of various components in TYMV-infected Chinese leaves. (Total amount for all leaves in infected plants expressed as a percentage of (O-O) 83 S ribosomes, (A- . -.- .A) 68 S ribosomes, (W - - W) Fraction I (+ - - - - +) Fraction II protein, (v----y) chlorophyll a.
associated entirely or mainly with the cytoplasm (83 S ribosomes and Fraction II protein) were reduced far less in amount over the same period, due to the reduced plant size and not to a reduced concentration of the components.
396
E. S. Crosbie
and
R. E. F. Matthews
Such reductions were very large compared with the amount of virus produced. Thus at 7 weeks, for the experiment of Fig. 6, the total dry weight per plant has been reduced from 1 l-5 to 4.5 g; Fraction I protein from 540 to 134 mg; Fraction II protein from 405 to 161 mg; 83 S ribosomes from 67 to 21 mg and 68 S ribosomes from 9.3 to 0.8 mg. There was, therefore, a total reduction in macromolecules of 660 mg/plant in the plants which contained 36 mg/plant of virus. It was not possible to establish which of the three chloroplast components was first affected by virus infection. In attempts to do this the various components were measured at daily intervals in systemically infected leaves of one age group (the second oldest systemically infected leaf) for comparison with those in healthy leaves. This was the leaf age group used to study the effects of infection on carbon fixation [I]. The results obtained (Fig. 7) showed that the Fraction I protein and chlorophyll a concentration were reduced before 68 S ribosome concentration. Cytoplasmic ribosomes were significantly increased in concentration at later times.
>&! 50I 9
;/; 10
I Time after inaculdt?an (days)
I
I
14
FIG. 7. Effect of TYMV infection on concentration of various components in a systemically infected Chinese cabbage leaf. Four sets of g-week-old plants were inoculated, two with water and two with infected sap, containing the severe white strain. Infection resulted in a range of strain symptoms. From day 9 to 14 each set was analysed for each component in duplicate. Chlorophyll a was estimated by whole extract analysis and virus, Fraction I protein, 83 S and 68 S ribosomes by analytical ultracentrifugation. Concentrations are expressed as a percentage of corresponding healthy samples, except for virus. (O-O) 83 S ribosomes, (A - . - . - * A) 68 S I protein, (v---v) chlorophyll a, (m---a) mg virus/g fresh ribosomes, (O - - - q ) Fraction weight.
Role of non-diseased leaf tissue in growth of infected plants
As the leaves of plants showing chronic disease due to infection with a severe white strain of TYMV have a markedly reduced chlorophyll content [4], such plants must mainly rely on leaf tissues not affected by the virus for the products needed for plant growth. Such tissue is of two distinct types: Dark green island tissue in leaves with mosaic symptoms. The timing of the appearance of green island tissue in relation to the growth of other tissue is shown in Figs I,3 and 4. Figure 5 shows that green island tissue makes a substantial contribution to the total chlorophyll a content of chronically diseased plants. The proportion of green island
Effects
of TYMV
391
infection
tissue (as a percentage of the fresh weight of the whole leaf) is shown for successive leaves in Fig. 8.
n Systemic leaf .number Wk.4 Wk.5 Time after inocdation
Wk.3
Wk.7
FIG. 8. The proportion of green island tissue in Chinese cabbage leaves systemically infected with TYMV. The proportion of green island tissue is expressed as a percentage of the fresh weight of the whole leaf. Groups of ten plants were measured at each sampling time.
The proportion of green island tissue rapidly increased 3 to 5 weeks after inoculation. The data for week 5 show that the proportion of green island tissue increased with younger leaves. At week 5 there was very little breakdown of green island tissue, but by week 7 substantial breakdown of the green island tissue had taken place. Sub-systemic leaves not infected by the virus. The importance of the sub-systemic leaves for growth of the infected plant is shown in Fig. 3(c). Plants which had their subsystemic leaves removed about 3 weeks after inoculation showed a net loss of lamina fresh weight over the next 4 to 5 weeks. Further data on these plants are given in Table 1. TABLE Role
of sub-systemic
leaves in the growth
Aerial fresh weight Root fresh weight Chlorophyll content Green island tissue in systemic lamina
1
of Chinese cabbage plants infected with a severe white strain of TP’MV
Plants without sub-systemic leaves Percentage Weight of healthy
Plants with sub-systemic leaves Percentage Weight of healthy
25.0 g 2.0 g 8.0 mg 40%
105.0 g 4.0 g 24.0 mg 17%
7.0 8.0 8.0 -
31 31 18 -
Two groups of plants were grown in large pots of soil after inoculation. One group had their sub-systemic leaves removed 3 weeks after inoculation. The fresh weight, chlorophyll and green island tissue content of both groups of plants were analysed 7.5 weeks after inoculation.
398
E. S. Crosbie
and
R. E. F. Matthews
It is especially noteworthy that the removal of the sub-systemic sequently led to a substantial increase in the proportion of green island younger leaves. E$ect on plant growth
of mild and severe strains of TYMV
Various measurements (Table 2) were made on healthy with the pale green or severe white strains of TYMV.
Plant
plants
infected
on Chinese cabbage plants
Infected with severe white straina
part
and on plants
2
TABLE
of seuew white or pale green strains of TYMV
Effects
leaves subtissue of the
Total aerial fresh weight Fresh weight of roots Leaf number Chlorophyll a Green island tissue content of systemic lamina (%)
39 37 86 (wk 7) 22 17 (wk 7)
a Results are expressed as a percentage of healthy control measurements were made 6 weeks after inoculation.
Infected with pale green strain 45 &wk8) 29 13 (wk
plants.
8)
Except
where
stated
The total aerial fresh weight and chlorophyll content per plant were somewhat more reduced by the severe white strain but the reduction in leaf number and fresh weight of roots was similar. There was a larger proportion of green island tissue in leaves infected with the severe white strain compared with the pale green strain. The differences caused by infection with different virus strains in the distribution of chlorophyll in various parts of the plant (Table 3) were more marked than the total reduction in chlorophyll (Table 2). It should be noted that the differences recorded were greater than indicated by the data because some leaves infected with the severe white strain contain numerous very small green islands that were not Consequently background tissue for the severe possible to dissect out and measure. white strain contained a much lower proportion of the total chlorophyll and green island tissue a much higher proportion than indicated by the results given in Table 3. TABLE Distribution
3
of chlorophyll
a in Chinese cabbage plants infected with severe white and$ale green strains of TT-MV
Leaf
or tissue
group
Sub-systemic (whole leaves) Systemic (background tissue) b Systemic (green island tissue) a Figures are percentage of total chlorophyll b Background tissue is whole lamina minus
Severe
white
straina
Pale green
32 36 32 a per plant, 6 weeks after dark green island tissue.
strain
16
inoculation.
Effects
of TYMV
399
infection
The most striking difference between the strains was on the growth habit of the plant (Table 4). The size of petiole, midribs and stems was much more reduced relative to the lamina in plants infected with the severe white strain compared with plants infected with the pale green strain. TABLE
4
tissue to lamina in systemica& infected leaves of Chinese cabbage plants with a pale green or a severe white strain of TTMV
Ratio of supfiorting
Week
Plants Healthy Pale green strain Severe white strain
a Ratio
=
Fresh
weight of stem, Fresh weight
after
infected
infection
5
6
7
8
l.56a 1.19
1.62 1.43 -
1.60 1.21
1.92 1.46 -
petioles and midribs of lamina
DISCUSSION Infection with TYMV reduced the three major chloroplast components-chlorophyll a, Fraction I protein and 68 S ribosomes-at about the same time and to the same extent, when compared with healthy leaves on a per plant basis (Fig. 6). Marked reduction in the amount of components that occur primarily in the cytoplasm (Fraction II protein and 83 S ribosomes) occurred some weeks later and reflected stunting of the plant rather than reduction in concentration of the components. The results of experiments given in Fig. 7 established that a reduction in chlorophyll a and Fraction I protein concentrations preceded the fall in concentration of chloroplast ribosomes but it was not firmly established whether chlorophyll a or Fraction I protein was affected first. This was partly because of inaccuracy in the analytical procedures when differences were small and partly because, at the early times after systemic infection, different cells in the leaf are at different stages of infection, It seems likely that the key events leading to stunting of plant growth are the reduction in the production of photosynthetic pigments and Fraction I protein. Thus as the disease induced by the severe white strain progresses, plant growth becomes critically dependent on the presence of tissue containing normal amounts of chlorophyll. When the sub-systemic leaves were removed at 3 weeks after inoculation, subsequent net increase in fresh weight was eliminated for at least 4 weeks [Fig. 3(c)]. In the healthy plant the logarithmic nature of plant growth over the period studied here is based on the increasing photosynthetic capacity of the plant. The severe white strain causes virtual cessation of pigment production (until dark green islands of tissue appear). Growth of the diseased plant is almost linear, presumably because the total photosynthetic capacity of the plant increases very little over a period of weeks (Fig. 5). The diseased plant appears to be able to respond to this situation in two ways: (i) a higher proportion than normal of available material is used to provide leaf lamina (Fig. 2) ; (ii) the proportion of dark green island tissue increased in the successively younger leaves, at least for a time (Fig. 8). If sub-systemic leaves
400
E. S. Crosbie and R. E. F. Matthews
are removed the proportion of dark green tissue in younger leaves is increased still further (Table 1). We know that the pattern of green island tissue is laid down in very young shaded leaves in the shoot apex in which average chlorophyll content is only about onetenth that of the expanded leaf [4]. Results of the experiment in which sub-systemic leaves were removed (Table 1) suggested that reducing the supply of photosynthetic products to the apical region must favour development of cells with functionally normal chloroplasts, even though at that early stage of leaf development very little photosynthesis is being carried out. The more prominent symptoms caused by the severe white strain compared with a pale green strain are due mainly to a different distribution of chlorophyll within the diseased plant (Table 3) and to a more extreme alteration in growth form (reduction of stem, petiole and midrib) (Table 4) and not to marked differences in total chlorophyll content or to differences in fresh weight. REFERENCES 1. BEDBROOK, J. R. & MATTHEWS, R. E. F. (1973). Changes in the flow of early products of photosynthetic carbon fixation associated with the replication of TYMV. Virology 53, 84-91. 2. CHALCROPT, J. P. & MIAYIXEWS, R. E. F. (1966). Cytological changes induced by turnip yellow mosaic virus in Chinese cabbage leaves. Virolou 28, 555-562. 3. CLARK, M. F., MATTHEWS, R. E. F. & RALPH, R. K. (1964). Ribosomes and polyribosomes in Brassica pekinensis. Biochimica et biophysics Acta, 91, 289-304. 4. CROSBIE, E. S. & MATTHEWS, R. E. F. (1974). Effects of TYMV infection on leaf pigments in Bra&a pekinensis, Rupr. Physiological Plant Pathology, 4, 379-387. 5. REID, M. S. & MATTHEWS, R. E. F. (1966). On the origin of the mosaic induced by turnip yellow mosaic virus. Virology 28, 563-570. 6. SMITH, K. M. (1972). A Textbook of Plant Virus Diseases, 3rd ed. Longman. 7. TAKAHASHI, T. (1972). Studies on viral pathogenesis in plant hosts. Phytopathologische
Plant Pathology
(1974)
4, 389-400
Effects of TYMV infection on growth of Brassica pekinensis Rupr. E. S. CROSBIE and R. E. F. MATTHEWS Department of Cell Biology, University of Auckland, Auckland, (Accepted for publication,
December
New
Zealand
1973)
The effect of turnip yellow mosaic virus (TYMV) infection on the growth of various parts of Chinese cabbage plants was studied in relation to virus synthesis, the production of leaf pigments and various macromolecules. Mild and severe strains of the virus affected overall growth to about the same extent, but growth form and distribution of chlorophyll were affected differently. Among the host components studied, the first to be affected by infection were chlorophyll and Fraction I protein. Late in infection the reduction of ribosomes and proteins on a weight/plant basis was about 20 times greater than the weight of virus produced. Dark green islands of tissue of the mosaic caused by a severe white strain of the virus made a significant contribution to the total chlorophyll of the diseased plant. The proportion of lamina that became dark green tissue in new leaves could be increased by removal of old expanded leaves that had escaped infection.
INTRODUCTION The symptoms of many plant virus diseases have recently been summarized [6]. Descriptions of these diseases have usually been confined to the visual symptoms, and to general statements concerning overall effects on growth. In a few studies changes in the external morphology of the plant have been observed in detail during disease development (e.g. with tobacco mosaic virus in tobacco [7]). However there is very little quantitative information concerning the development of disease in different parts of the plant in relation to virus replication, and to the effects of infection on important classes of molecules and organelles in the cell. The work described below forms part of a general study of the relationship between the replication of turnip yellow mosaic virus (TYMV) and disease production in Chinese cabbage. The stock culture of TYMV induces a typical mosaic disease in systemically infected Chinese cabbage plants. Islands of tissue in the mosaic contain different strains of the virus that affect the chloroplasts more or less severely. As the disease develops, dark green islands of tissue appear in the mosaic. These islands contain little or no virus and appear to be cytologically and metabolically normal [Z, 51. After a period of weeks these dark green islands may “break down”, a process involving the appearance of many yellow local lesion-like areas containing virus. In the work described here we have measured the effects of infection with a mild and a severe strain of TYMV on the growth of various parts of the Chinese cabbage plant during the,development of disease. We have attempted to relate these changes to the production of virus and to changes in important classes of compounds and
390
E. S. Crosbie
and
R. E. F. Matthews
organelles in the leaf. We have also assessed the significance of dark green islands in the mosaic (and of older leaves which escape infection for a time) for the survival and growth of diseased plants.
MATERIALS
AND
METHQDS
Growth of Chinese cabbage plants (Bras&a pekinensis Rupr. var. Wong of TYMV and pigment analyses were as described previously [4]. Analysis
of Fraction
I protein
and Fraction
Ilproteins,
TTMV,
Bok)
cultures
83 S and 68 S ribosomes
Fresh leaf lamina (0.5 to 0.8 g) was mixed with twice its weight of extraction medium (modified from [3] : 0.01 M-Tris-HCl buffer, pH 7.2, 0.02 M-MgC1, and 0.003 M-mercaptoethanol. The mixture was ground in a small pestle and mortar until there were no visible pieces of leaf remaining. The extract was then subjected to a low speed centrifugation and the resulting supernatant fluid was examined in a Model E analytical ultracentrifuge using Schlieren optics. Photographs showing the ribosomes and virus boundaries were recorded after centrifugation at 40 000 rev/min for 8 min. Fraction I and Fraction II components were recorded after a further 16 min at 52 000 rev/min. From a photographic record of the schlieren pattern the concentrations of the components were estimated by planimetry, with correction for radial dilution. The area units were converted to absolute concentrations by comparison with values for a known concentration. Measurement
of areas
Total leaf lamina area was measured by weighing paper facsimile cutouts of leaves using paper of known weight per unit area. Green islands were outlined on the paper and estimated in the same way. Only islands larger than about 2 mm diam were measured. Fresh weights Leaves, petioles plus midrib and stems were excised, grouped as described below and weighed immediately. Root fresh weight was obtained after flushing the soil from the roots with water. Excess water was removed by placing the roots on an absorbent surface for half an hour before they were weighed. Definition
of leaf groups used
In growth experiments it was not practicable to consider every leaf separately. Leaves were pooled in groups as follows. Inoculated leaves. Two well-expanded leaves of each plant were inoculated mechanically. Two or three much smaller and older leaves were removed from each plant at the time of inoculation and discarded. Sub-systemic leaves. Under our conditions of growth the group of about three leaves immediately above the inoculated leaves did not become infected when the virus moved into the younger leaves. Some leaves of this group remained uninfected for a period of weeks, or sometimes until they senesced. Others became infected by a slow
Effects
of TYMV
infection
391
invasion of the virus from the base of the leaf, beginning after systemic infection of younger leaves was well advanced. Systemic leaves. These are the leaves in which the characteristic mosaic pattern of TYMV infection develops. They were numbered from the oldest (i.e. S, is the oldest systemically infected leaf on the plant). For sampling the S leaves were usually taken in two groups:-S,-, that do not develop green islands and S,, that do.
RESULTS
After preliminary trials a major growth experiment was carried out using four sets of Chinese cabbage plants. The objectives of the experiment were (i) to measure the growth of the various parts of healthy and virus-infected plants during the development of disease; and (ii) to attempt to relate these changes to virus synthesis and changes in the amounts of certain compounds and organelles in the leaf In addition, the importance of the sub-systemic group of leaves for the growth of the plant was assessed by removing these leaves from some plants after the disease was established. At the commencement of the experiment, 146 one-month-old plants were selected for uniformity. One group of 10 were used for base time measurements (week 0, in the figures). Eighty were inoculated on two leaves with a severe white strain of TYMV and sorted into eight groups of 10 plants. The remaining 56 plants were inoculated with water and divided into groups of seven. At intervals thereafter a group of 10 virus infected and seven healthy plants were analysed for fresh weight, leaf area, leaf number, dry weight, chlorophyll, Fraction I protein, Fraction II proteins, 83 S ribosomes, 68 S ribosomes, virus and green island content. All the plants were re-potted into successively larger pots at three and five weeks after inoculation. Twenty-five days after inoculation, one group of healthy and one group of infected plants had their sub-systemic leaves removed to test the importance of these leaves for continued plant growth. The e$eectof infection on total aerial fresh weight During the 7-week experimental period the total aerial fresh weight of healthy plants increased about 68fold in approximately logarithmic fashion (Fig. 1). Increase in the weight of TYMV-infected plants was approximately linear during the period of rapid virus increase (as measured for the plant as a whole). The fresh weight per infected plant was 240 g less than for healthy plants at a time when 35 mg of TYMV per plant had been produced. Measurable green island tissue in the leaves showing mosaic disease first appeared four weeks after infection. Growth curves similar to those of Fig. 1 for healthy and infected plants were also obtained from measurements of total lamina area or total dry weight of lamina. Comparison
of various plant parts
Expressed as a percentage of corresponding healthy tissue, virus infection had similar effects on the fresh weight of root, petiole and stem. However, total leaf lamina fresh weight was less affected than other parts of the plants (Fig. 2).
392
E. S. Crosbie and R. E. F. Matthews c 320 -
Time after inoculation
(weeks)
FIG. 1. Effect of infection with a severe white strain of TYMV on aerial fresh weight of Chinese cabbage plants. Infected plants were inoculated at week 0. (O-O) Total aerial fresh weight, g/healthy plant, (0 - . - . - . 0) total aerial fresh weight, g/infected plant, (V . . . .v) total virus, mg/infected plant, (A - - - - A) dark green island tissue, g/infected plant.
Ti&
after inoculati&
(weeks)
FIG. 2. Effect of infection with a severe white strain of TYMV on the fresh weight of various parts of the Chinese cabbage plant. Fresh weight of roots, stem, petiole and lamiua from infected plants are expressed as a percentage of the corresponding healthy plants. The virus content of the infected plant is also given. Infected plants were inoculated at week 0. (A -. -. -. A) Lamina, (V - - - - - V) roots, (t-m) petiole, (O- - -0) stem, (V . . . V) virus, mg/plant.
E$ects on lam&a fresh weight for various groups of leaves The effects of TYMV infection on the four leaf groups defined under “Materials and Methods” are surnmarized in Fig. 3. Inoculated leaves of the diseased plants became stunted after the appearance of local lesions and such leaves began to senesce earlier than the corresponding healthy leaves. All inoculated leaves were dead by week 5 [Fig. 3 (a)].
393
Effects of TYMV infection
The reduced total fresh weight of the sub-systemic leaves of the diseased plants [Fig. 3(b)] was due to enhanced senescence rather than stunting.
‘*O
‘“Lw .
.
total lamina
l
00 b
9
/
,$ F.w. systemic
lqminq .
120 ml 00
(bf F.W. subsystemic
Time after inoculation FIG 3. Effect of infection for various groups of leaves (o-.-.-. 0) infected, (A diseased plants from which
lamina
(week!
with a severe white strain of TYMV on the total lamina of Chinese cabbage. Fresh weights in g/plant. (0-O) - - - A) dark green island tissue, (0 - - Cl) systemic sub-systemic leaves were removed.
fresh weight Healthy, lamina from
The fresh weight of systemic infected lamina increased in a linear fashion after week 3, in contrast to the corresponding healthy leaves, which increased in an approximately logarithmic manner [Fig. 3(c)]. Fig. 3(c) also shows the great reduction in plant growth after the removal of the sub-systemic leaves at 3.5 weeks. The fresh weights in Fig. 3 (d) largely reflect those of the systemic groups of leaves. Growth curves for two subgroups of the systemic leaves are given in Fig. 4 for the later part of experiment (these subgroups were not measured at earlier times). For the older group (1 to 6) the fresh weight of healthy leaves continued to increase as expansion more than counterbalanced senescence [Fig. 4(a)]. By contrast weight loss due to senescence of the infected 1 to 6 groups was greater than gain due to expansion [Fig. 4(b)]. This group of diseased leaves did not contain islands of dark green tissue. The younger group of leaves (7 +) grew at an increasing rate in the healthy plants and at a linear rate in the infected plants. The infected 7 + group contained green island tissue.
394
E. S. Crosbie
E$ect of infection on total chorophyll
and
R. E. F. Matthews
in the plant
Samples were taken from every third leaf on the plants, pooled and analysed for chlorophyll using unfractionated extracts (Fig. 5). The rate of increase in chlorophyll a was similar to the increase in fresh weight of the healthy plants shown in Fig. 1. There was a 57-fold increase in chlorophyll a per healthy plant over the 7-week period. In infected plants the reduction in chlorophyll compared with healthy plants was greater than the reduction in fresh weight, due of course to the combined effects of stunting and reduced chlorophyll concentration.
90 - (a'
60-
30 0
,. .’ ,’--tL ---a vs *.__---- , I-E ,*.&HA’ I 3 5 7 o---o-
0 L-
I
Time after inoculation
(weeks)
FIG. 4. Comparative growth of older (1 to 6) and younger (7 + ) systemically infected leaves of TYMV-infected Chinese cabbage nlants and the corresponding leaves of healthy plants. Plants infected with a severe white &a$. Growth is expressed in total grams fresh weighdplant. (a) (A-A) Healthy HS/,+ leaves, (o-e) healthy H S,-, leaves. (b) (0 - - - - 0) Infected V S,-s leaves, (A- - -A) infected VS,+ leaves, (A- - -A) dark green island tissue.
As shown in Fig. 5, the green island tissue total chlorophyll of the infected plants from that only the green island tissue large enough although many very small green islands were
made a substantial contribution to the week 5 onwards. It should be noted to be measured ( > 2 mm) was recorded present in some leaves.
E$ect of infection on macromolecules The effects of infection on the total amounts of Fraction I protein, Fraction II protein, 83 S and 68 S ribosomes per plant compared with healthy plants are shown in Fig. 6. Compared with healthy plants the components found in the chloroplasts of infected plants (68 S ribosomes, Fraction I protein and chlorophyll a) were reduced very rapidly between weeks 2 to 4. These components were reduced both in conBy contrast, the components known to be centration and total amount per plant.
Effects
of TYMV
infection
90
E 60 -
.
I
I 30 ,. f
,,,/--v
/p+ L
FIG.
severe (v-----
.d
,~c ._.-.- ‘3 ‘5’
I 3 5 Time after inoculation
7 (weeks)
5. Chlorophyll a content of healthy Chinese cabbage white strain of TYMV. Total chlorophyll a expressed V) infected, (A - . - . - . A) dark green island tissue
plants and plants infected with as mg/plant. (r--v) Healthy, in virus-infected plants.
a
i I
I I 2 Time after
I l 4 inoculation
l 1 6 (weeks)
l 8
FIG. 6.
cabbage healthy.) protein,
Percentage reduction in amount of various components in TYMV-infected Chinese leaves. (Total amount for all leaves in infected plants expressed as a percentage of (O-O) 83 S ribosomes, (A- . -.- .A) 68 S ribosomes, (W - - W) Fraction I (+ - - - - +) Fraction II protein, (v----y) chlorophyll a.
associated entirely or mainly with the cytoplasm (83 S ribosomes and Fraction II protein) were reduced far less in amount over the same period, due to the reduced plant size and not to a reduced concentration of the components.
396
E. S. Crosbie
and
R. E. F. Matthews
Such reductions were very large compared with the amount of virus produced. Thus at 7 weeks, for the experiment of Fig. 6, the total dry weight per plant has been reduced from 1 l-5 to 4.5 g; Fraction I protein from 540 to 134 mg; Fraction II protein from 405 to 161 mg; 83 S ribosomes from 67 to 21 mg and 68 S ribosomes from 9.3 to 0.8 mg. There was, therefore, a total reduction in macromolecules of 660 mg/plant in the plants which contained 36 mg/plant of virus. It was not possible to establish which of the three chloroplast components was first affected by virus infection. In attempts to do this the various components were measured at daily intervals in systemically infected leaves of one age group (the second oldest systemically infected leaf) for comparison with those in healthy leaves. This was the leaf age group used to study the effects of infection on carbon fixation [I]. The results obtained (Fig. 7) showed that the Fraction I protein and chlorophyll a concentration were reduced before 68 S ribosome concentration. Cytoplasmic ribosomes were significantly increased in concentration at later times.
>&! 50I 9
;/; 10
I Time after inaculdt?an (days)
I
I
14
FIG. 7. Effect of TYMV infection on concentration of various components in a systemically infected Chinese cabbage leaf. Four sets of g-week-old plants were inoculated, two with water and two with infected sap, containing the severe white strain. Infection resulted in a range of strain symptoms. From day 9 to 14 each set was analysed for each component in duplicate. Chlorophyll a was estimated by whole extract analysis and virus, Fraction I protein, 83 S and 68 S ribosomes by analytical ultracentrifugation. Concentrations are expressed as a percentage of corresponding healthy samples, except for virus. (O-O) 83 S ribosomes, (A - . - . - * A) 68 S I protein, (v---v) chlorophyll a, (m---a) mg virus/g fresh ribosomes, (O - - - q ) Fraction weight.
Role of non-diseased leaf tissue in growth of infected plants
As the leaves of plants showing chronic disease due to infection with a severe white strain of TYMV have a markedly reduced chlorophyll content [4], such plants must mainly rely on leaf tissues not affected by the virus for the products needed for plant growth. Such tissue is of two distinct types: Dark green island tissue in leaves with mosaic symptoms. The timing of the appearance of green island tissue in relation to the growth of other tissue is shown in Figs I,3 and 4. Figure 5 shows that green island tissue makes a substantial contribution to the total chlorophyll a content of chronically diseased plants. The proportion of green island
Effects
of TYMV
391
infection
tissue (as a percentage of the fresh weight of the whole leaf) is shown for successive leaves in Fig. 8.
n Systemic leaf .number Wk.4 Wk.5 Time after inocdation
Wk.3
Wk.7
FIG. 8. The proportion of green island tissue in Chinese cabbage leaves systemically infected with TYMV. The proportion of green island tissue is expressed as a percentage of the fresh weight of the whole leaf. Groups of ten plants were measured at each sampling time.
The proportion of green island tissue rapidly increased 3 to 5 weeks after inoculation. The data for week 5 show that the proportion of green island tissue increased with younger leaves. At week 5 there was very little breakdown of green island tissue, but by week 7 substantial breakdown of the green island tissue had taken place. Sub-systemic leaves not infected by the virus. The importance of the sub-systemic leaves for growth of the infected plant is shown in Fig. 3(c). Plants which had their subsystemic leaves removed about 3 weeks after inoculation showed a net loss of lamina fresh weight over the next 4 to 5 weeks. Further data on these plants are given in Table 1. TABLE Role
of sub-systemic
leaves in the growth
Aerial fresh weight Root fresh weight Chlorophyll content Green island tissue in systemic lamina
1
of Chinese cabbage plants infected with a severe white strain of TP’MV
Plants without sub-systemic leaves Percentage Weight of healthy
Plants with sub-systemic leaves Percentage Weight of healthy
25.0 g 2.0 g 8.0 mg 40%
105.0 g 4.0 g 24.0 mg 17%
7.0 8.0 8.0 -
31 31 18 -
Two groups of plants were grown in large pots of soil after inoculation. One group had their sub-systemic leaves removed 3 weeks after inoculation. The fresh weight, chlorophyll and green island tissue content of both groups of plants were analysed 7.5 weeks after inoculation.
398
E. S. Crosbie
and
R. E. F. Matthews
It is especially noteworthy that the removal of the sub-systemic sequently led to a substantial increase in the proportion of green island younger leaves. E$ect on plant growth
of mild and severe strains of TYMV
Various measurements (Table 2) were made on healthy with the pale green or severe white strains of TYMV.
Plant
plants
infected
on Chinese cabbage plants
Infected with severe white straina
part
and on plants
2
TABLE
of seuew white or pale green strains of TYMV
Effects
leaves subtissue of the
Total aerial fresh weight Fresh weight of roots Leaf number Chlorophyll a Green island tissue content of systemic lamina (%)
39 37 86 (wk 7) 22 17 (wk 7)
a Results are expressed as a percentage of healthy control measurements were made 6 weeks after inoculation.
Infected with pale green strain 45 &wk8) 29 13 (wk
plants.
8)
Except
where
stated
The total aerial fresh weight and chlorophyll content per plant were somewhat more reduced by the severe white strain but the reduction in leaf number and fresh weight of roots was similar. There was a larger proportion of green island tissue in leaves infected with the severe white strain compared with the pale green strain. The differences caused by infection with different virus strains in the distribution of chlorophyll in various parts of the plant (Table 3) were more marked than the total reduction in chlorophyll (Table 2). It should be noted that the differences recorded were greater than indicated by the data because some leaves infected with the severe white strain contain numerous very small green islands that were not Consequently background tissue for the severe possible to dissect out and measure. white strain contained a much lower proportion of the total chlorophyll and green island tissue a much higher proportion than indicated by the results given in Table 3. TABLE Distribution
3
of chlorophyll
a in Chinese cabbage plants infected with severe white and$ale green strains of TT-MV
Leaf
or tissue
group
Sub-systemic (whole leaves) Systemic (background tissue) b Systemic (green island tissue) a Figures are percentage of total chlorophyll b Background tissue is whole lamina minus
Severe
white
straina
Pale green
32 36 32 a per plant, 6 weeks after dark green island tissue.
strain
16
inoculation.
Effects
of TYMV
399
infection
The most striking difference between the strains was on the growth habit of the plant (Table 4). The size of petiole, midribs and stems was much more reduced relative to the lamina in plants infected with the severe white strain compared with plants infected with the pale green strain. TABLE
4
tissue to lamina in systemica& infected leaves of Chinese cabbage plants with a pale green or a severe white strain of TTMV
Ratio of supfiorting
Week
Plants Healthy Pale green strain Severe white strain
a Ratio
=
Fresh
weight of stem, Fresh weight
after
infected
infection
5
6
7
8
l.56a 1.19
1.62 1.43 -
1.60 1.21
1.92 1.46 -
petioles and midribs of lamina
DISCUSSION Infection with TYMV reduced the three major chloroplast components-chlorophyll a, Fraction I protein and 68 S ribosomes-at about the same time and to the same extent, when compared with healthy leaves on a per plant basis (Fig. 6). Marked reduction in the amount of components that occur primarily in the cytoplasm (Fraction II protein and 83 S ribosomes) occurred some weeks later and reflected stunting of the plant rather than reduction in concentration of the components. The results of experiments given in Fig. 7 established that a reduction in chlorophyll a and Fraction I protein concentrations preceded the fall in concentration of chloroplast ribosomes but it was not firmly established whether chlorophyll a or Fraction I protein was affected first. This was partly because of inaccuracy in the analytical procedures when differences were small and partly because, at the early times after systemic infection, different cells in the leaf are at different stages of infection, It seems likely that the key events leading to stunting of plant growth are the reduction in the production of photosynthetic pigments and Fraction I protein. Thus as the disease induced by the severe white strain progresses, plant growth becomes critically dependent on the presence of tissue containing normal amounts of chlorophyll. When the sub-systemic leaves were removed at 3 weeks after inoculation, subsequent net increase in fresh weight was eliminated for at least 4 weeks [Fig. 3(c)]. In the healthy plant the logarithmic nature of plant growth over the period studied here is based on the increasing photosynthetic capacity of the plant. The severe white strain causes virtual cessation of pigment production (until dark green islands of tissue appear). Growth of the diseased plant is almost linear, presumably because the total photosynthetic capacity of the plant increases very little over a period of weeks (Fig. 5). The diseased plant appears to be able to respond to this situation in two ways: (i) a higher proportion than normal of available material is used to provide leaf lamina (Fig. 2) ; (ii) the proportion of dark green island tissue increased in the successively younger leaves, at least for a time (Fig. 8). If sub-systemic leaves
400
E. S. Crosbie and R. E. F. Matthews
are removed the proportion of dark green tissue in younger leaves is increased still further (Table 1). We know that the pattern of green island tissue is laid down in very young shaded leaves in the shoot apex in which average chlorophyll content is only about onetenth that of the expanded leaf [4]. Results of the experiment in which sub-systemic leaves were removed (Table 1) suggested that reducing the supply of photosynthetic products to the apical region must favour development of cells with functionally normal chloroplasts, even though at that early stage of leaf development very little photosynthesis is being carried out. The more prominent symptoms caused by the severe white strain compared with a pale green strain are due mainly to a different distribution of chlorophyll within the diseased plant (Table 3) and to a more extreme alteration in growth form (reduction of stem, petiole and midrib) (Table 4) and not to marked differences in total chlorophyll content or to differences in fresh weight. REFERENCES 1. BEDBROOK, J. R. & MATTHEWS, R. E. F. (1973). Changes in the flow of early products of photosynthetic carbon fixation associated with the replication of TYMV. Virology 53, 84-91. 2. CHALCROPT, J. P. & MIAYIXEWS, R. E. F. (1966). Cytological changes induced by turnip yellow mosaic virus in Chinese cabbage leaves. Virolou 28, 555-562. 3. CLARK, M. F., MATTHEWS, R. E. F. & RALPH, R. K. (1964). Ribosomes and polyribosomes in Brassica pekinensis. Biochimica et biophysics Acta, 91, 289-304. 4. CROSBIE, E. S. & MATTHEWS, R. E. F. (1974). Effects of TYMV infection on leaf pigments in Bra&a pekinensis, Rupr. Physiological Plant Pathology, 4, 379-387. 5. REID, M. S. & MATTHEWS, R. E. F. (1966). On the origin of the mosaic induced by turnip yellow mosaic virus. Virology 28, 563-570. 6. SMITH, K. M. (1972). A Textbook of Plant Virus Diseases, 3rd ed. Longman. 7. TAKAHASHI, T. (1972). Studies on viral pathogenesis in plant hosts. Phytopathologische