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Effects of aster yellows virus infection on transport through plant stem sections
VIROLOGY
32, 570-579 (1967)
Effects of Aster
Yellows
Virus
Infection
on Transport
through
Plant Stem Sections WEN-POH Department
of Plant
Pathology,
TInTG
A. H. GOLD
AND
Unicersity
Accepted April
of California,
Berkeley,
Cali.fornia
21, 1967
Basipetal transport of various substances through half-inch stem sections of healthy and aster yellows virus-infected Xicotiana rustica I,. var. humilis was determined. The effects of infect,ion by three strains of the virus, dwarf, severe, and Tulelake were observed on the t,ransport of 1%~labeled P-indoleacetic acid, malic acid, succinic acid, stlcrose, glucose, mannose, and t)he ions sulfate, phosphate, and rubidium. Transport of all these subst,ances except phosphate ion was more rapid through diseased than through healthy stem sections. In the dark, and with supplcmentary auxin, transport of phosphate ion was also more rapid through diseased than through healt.hy stem sections. Transport of fl-indoleacetic acid and of sugars through healthy stem sections was more rapid in the dark than in the light, bllt, transport of these compounds through diseased stem sections was rrnaffected by light. No aD-
preciable chemical coIlversion of the scrbstances tested was observed during the i,rs,nsport time used.
Stem sections from t,he plant offered a con-
1NTROl)UCTIOX
The severe malformation in t)he vascular tissue observed in aster yellows virus-infect,ed plants (Rasa and Esau, 1961) suggest,ed to us that transport’ of met,abo&es might be interfered wit#h. Indeed, Grieve (1943) reported that tomato spotted wilt, virus infection reduced the rate of auxin transport in tjomato stem sections, alt#hough he indicat,ed no morphological disturbance in t,he vascular t,issue, such as is observable in aster yclloms virus infection. The present, report describes comparisons in the transport of various substjances t#hrough stem sect.ions of healthy and ast,er yellows virus-infected Nicotiawa ,rustica 1,. var. humilis
plants. MATERIALS
AND
METHODS
of plant material. Nicotiana J,. var. humilis was chosen as the test
Preparation
m&a plant for this study because the t’hree strains of aster ~~110~s virus, dwarf, severe, and Tulelake described by Freitag (1964) prosymptoms in this plant. duce distinct,ive 570
venient
system
for
investigation.
The
N.
seedlings were raised in the grecnhouse and inoculat’ed with infective leafhoppers, Jlamosteles Jascijro?7s (St,%l) when 2-3 inches tall (4-A leaf st#age). The colonies of infective leafhoppers were maintained on common plantain, PZa77tago major J,., infectcd with one of the strains of aster yellows virus. To ensure reliable infection, ‘it was necessary to transfer these colonies at frequent int,crvals to fresh plantain plants with prominent new symptoms. Each N. mstica plant was inoculated with 20 infect,ive leafhoppers. The insects were allowed t#ofeed on the N. I-ustica plant for 6 days. Sympt,oms usually appeared 10-11 days after inoculation. Infected plants were used for transport experimenOs 4 weeks after inoculat8ion. Only plant,s showing prominent symptoms were used. HeaIthy pIants of the same age grown under the same conditjions mew used as COIItrols. Detew~Lination of trampod. The relat’ive transport, described in detail later was dctcrwstica
ASTb:R
YELLOWS
mined by measuring the radioactivity of labeled compounds which passed into a receiver agar block. The compounds chosen for st,udy included 3?P-labeled phosphate (at’ about pH 6.2), auxin (/3-indoleacetic-3-‘4C acid), rJ-malic acid-14C (U),l succinic acid1 ,4-14C, sucroseJ4C (U),l u-glucose-14C (U)l l)-lna11110se-l-14C,sodium sulfate (Sa$OJ, and rubidium chloride (86RbC12). The specific activity dat)a for these compounds are shown in t,he tables. The substances chosen to be involved in plant, met,aboarc k~mvn measurements were lism. lZll transport made in the basipetal direction, because prcvious evidence (Esau et al., 1957) indicated that transport in this directJion was largely through t,he phloem. In the early experiments, transport. t,hrough he&h?- stem sections was compared directly wit’h that in stem sections infected with the various strains of aster vellows. later, as it was not8iced t’hat’ t,he diseased stem sections were consistently sinsller in diamet,er than t,he healthy stem sections, the observed transport amountIs were correct’ed for stem areas. In general, this correction tended t,o enhance, but’ not fundamentally change, the differences in transport found between diseased and healthy st,em sections. Four plants each, infected by one of t’he 3 strains of ast,er yellows virus and 4 healthy controls were used in each experiment,. All plants were placed in a dark chamber for 12 hours prior to each experiment so t’hat, the plants would have the same pretreatment. One-half-inch-long stem segments were removed between the sixth and twelft’h leaf from each of the plant’s, using mounted razor blades set, one-half inch apart. Older part)s of stems were not used because their morphology had been dctcrmined before t)he onset) of symptoms. The plants mere selected for uniformity of growth and development wit’hin each treatment, and for equal stage of sympt’om development in the case of infected plnnt,s. Since t,he experiments extended from t,he middle of March, 1966, until the middle of October, 1966, plant responses to varying day lcngt hs, light levels, and glasshouse temperat,ures made it difficult to maintain 1 Uniformly
labeled
\-IRUS
571
IKFECTION
strict,ly comparable plant material over the whole period. Undoubtjedly part of the variabilit’y observable in the data can be attributed to this. Unless indicated otherwise in t#he respective t’ables, each experiment involved 5 replications. Physical arra~rgement for transport studies. The procedure followed was similar to that of other workers Audying transport, in stem sections (Dedolph et al., 1966; Goldsmith, 1966, a, b; 1IcCreadg, 1966; Morgan and Gausman, 196G). The substance to be studied was incorporated i&o an agar block. This donor block was placed on top of the stem section. The stem section was placed Chrough a hole in a plastic cap support on a receiver block. The receiver block, in turn, was placed in a planchet (Fig. 1). Donor blocks were prepared by including the desired concentration of the material under study in 1% agar (Oxoid agar Ko. X). The melted agar containing the material was pip&ted to a prechilled brass mold. The blocks were S.5 X 11.5 X 2 mm in size. To test the hypothesis that nuxin level might influence the rate of transport of some subst’ance, 15 ppm of 3-indoleacetic acid were added to some of the donor blocks for each substrance tested. The receiver blocks were prepared in the same manner as the donor blocks, but COWtained no test substance. The block sizes and t’he concentration of material in t,he donor block were chosen so that during t,he experiment no appreciable proportion of the test substances diffused out of the donor blocks, and so that the amount of material t’ransporbed through the st’em sect,ions t)o t,he receiver blocks was conveniently measurable. Conditions 01 transpo7~t and nzeasurenaent. The transport assemblies were placed in a large Pyrex dish lined at the bottom with moist filter paper. The top of the Pyrex dish was covered with Saran wrap (Dow ChemiDonor block
FIG. 1. Diagram port studies.
of assembly
used in the trans-
572
TINti
L4N11 (:OLl)
cal Company) to prevent drying of the agar blocks. The dishes were kept in controlled chambers at, 70°F. For t’he treatments exposed to light, the chambers w’crc illumnated with fluoresceut tubes, with an ittten&y of 550 foot-candles at the level of the Pyrex dishes. ITor the dark treatments, the same temperature and humidity conditions were maintained in the chambers. The toursport experiments were made over a period of 10 hours. At the conclusion of each experimcnt~, planchets containing the agar blocks were dried at about 80°C using a I>ubnoff metabolic-shaking incubator (Precision Scient,ific Co.). The agar blocks tnelted in the incubator, resulbing in a more even distribution of nlaterial before they were completel?dried down. The samples were then courkcd in a gas flow Det#ector (Model D47 SuclcarChicago Corporst.ion) . The transport data are expressed in terms of relative radioactivity (counts per mitnke) transported per unit, of time per square centimeter of stem area. In an at,tempt to determine whether appreciable proport,iotts of the t,est. substances undenvent chemical t~ransform:kiott during the lo-hour transport period, partition coefficients of t,he substances from the donor blocks were compared with t,hosc in the receiver blocks. In each case the substance was acidified wit,h 0.02 J/ acetic acid. The solvent, s&em chosen for p:trtit,iott coefficietttj determinkott was xmyl alcoholLw:ttw. This procedure would have dct e&cd gross changes-~ -for example, from sucrose to one of t#hc organic acids. However, because no change was observed in the p:trtit,iott CYK~cient of any test, substance during its JXLSSagC! through s:tcm sections, the procedure> will not, be dtrt ailed furt hw. RISSGLTS
Transport, of all the substances tested was affected by aster yellows virus infect’iott of the plants. Data are presented in Tables 1-3. Ratios of the relative transport in diseased and healthy stem sections are included in these tables. When t,hc experitnents were performed in the dark or in th(, presence of added auxin, phosphate tended to bc trnnsporl-ed fastjet
through infected than through healthy stein se&otts. It was apparent, t,hat the transport ratios of bot#h sulfate and rubidium ions n-we much greater than t’hose of phospl1:tt.c. (‘ompounds of similar chemical structjurc had approximat,ely similar transport ratios. A comparison of the transport ratios of the sugars, for example, indicnt ed that, the transport ratios lended to he higher than those of organic acids with thcl Tulelnl~ st’rain. Homevcr, the transport ratio of mnnnosc was lower than that of glucose OI sucrose. ;\ comparison of the transport of ntalic acid in t’hc flowering stage with k:msportJ at t)he stage normally used in the cxperimcttt (Table 2) showed a grent)l\- reducctl trattsport, ratSi0 at t,he flowering stagcP not bccause t,herc is an?- appreciable change in the rat,e of transport of nialatc in diseased stems, but, because t,he t~rattsport in he:Mty stems is grcath- increased. Ilight dccrertscd transport of all the sugars in healthy stews (Table 1). Since the transport of sugars itt diseased stems is not, :qqreciabl\: nflectcd by light., the transport r:tt’ios reflect t’his differetw in light response. Supplcmetttnr~~ ittdolc:rwtic the ~r:~IlsJ)Or~, acid dso seemed t’o aft’cct, especially- of phnsphatc~ in t hc wverc :md T~~lclal~e strains of the virus. DISCUSSION
Akhough much information exists on I he t,ransport, of various substattccs in phlocm and xylem of healthy plants (Hollard, l!KiO; Esau et al., 1957; Zimmerman, 1960), the changes which occur following virus infcclion arc only postulated. Thtts &~det~ (1950) called attent,ion t#o :L corrclntion between phloetn necrosis in nst,cr yc~llows infect,iott, and st#arch accutnulutiott in the leaves of intact plants. Although he interpreted this as due to interfcrenw \I-ith transloc:~tiot~, the int,erpret,ation was not, bawl on exlwrimentjnl evidence. The cxknsivc efforts t’o dctrrminc the mechanisms of stem t,ransport in health) plants recorded in the literature OII plant physiology have not) resulted in universally accepted concepts. The results of our Stud> ogler litt,le :~ddit,ionnl informntic~u on how transloc:~t~iott occurs in rl cmli of (4 1tc.r diseased or lK2,lth~ plants, tJ11t tll(k! (10
ASTER YELLOWS
\.IRUS
In fact, Van Overbeek (19.33) has indicated parenchymatous transport of growth substances. Our attempts to use autoradiography t’o identify the specific tissues transporting the test substances were unsucccss-
indicate some specific differences which may he useful in the study of t’he translocation mechanism. The possibilit,y of tJransportj by other tissues t,hnn phlocm has not been ruled out. TABLE BAYIPETAL
TR,4NSPoRT
OF
sUG.41~3
THROUGH
:Vicotiana
-c‘ 91.3
IKFECTION
rustica
1
HEALTHY
END
~~41~.
humilis
I>ISE.~SED
STEM
SECTIONS
OF
Transport in counts/min/cm*
I-
Healthy
Tulelake
Dwarf
Treatment
(5D-GlucoseW(U)u-b u-Glllcose‘“C (IT) -IY wGlwose“C(U) + IAAh-I, u-Glllcose“C(U) + IAA-I) _--*SucroselC(U)-I> Sucrose-
I 5
39 *
4 5,536 f
5
94 +
70 2,323 xk
5
15 +
1 5
15 zt
I
!____!
12
2,481 *
2 4,702 4~ 1,070 313
2,043 i
30: 4,439 3z
4,830 f
1,60(
124
4,231 +
74(
22
31(
578
G90’ 34 Gj204 zk 2,00(1 1 1
54
80’ 140 I G.703 * 1,000 428 130
450
39
3,872 zk
4201 13
1,050~ 136 ~ 8.G65 f
i--~_- I
I
5
32(
202
551 zt
15(
20
0
158 +
a0
13
2.927 zk
2i*
10
879 +
280
32
2,149 i
1,1801 131
2.G33 f
300, 1G-l 3.06G f I
1,210 875
2,154 f
1,404 f
48(
8,931 f
2,050i
30
8.313 41 1,200
20
10 +
4; 2,103 i (
5
3+
-15
295 f
1’ 2,625 i
730
244
2,430 xk
12&
G4(1 192
)I 468
-1 ---I 150~7,665 + 2.3Gi-T
10,047 + 1.6501
34
I
5
414 *
80 8>492 *
1,040:
20
3.779 zk
10,314 f 14C +
580 1.300
,12.527 f
1,NO
24
9 ‘10.32i * 1,470
13
MA-L
wRI:tlrnose-l‘Tz + IA&u
1 5 ~
1785 + 300 8,485 f
1,7901 11 ~ 7,4iO k 1,730 I
a Collcelltration: 16.5 X 10-a &/block (specific activity: ‘) Concetltration: 16.5 X 1V ,&/block (specific activity: c Concentration: 19.5 X 10-S&/block (specific activity: d Expressed as mean ratte =I=standard error. e III the light. f III the dark. 0 Uniformly ‘4C labeled. h IA.% at. 15 ppm.
65.7 mC/mmole). 185 mC/mmole). 24.2 mC/mmole).
acid-D
p -Indoleacetic-2-14C
acid-14C(U)
acid- 14C(U)+IAA1-L
acid-14C(U)+lX
L-Malic
L-MaXc
L-bIalic
-D
acid-14C(U)j-L
L-Malic
d
-D
acid-L
p-Indoleacetic-2-14C
c
acid-D
C acid-L
o-Indoleacetic-2-14C
p-Indoleacetic-2-
14
2-14C acid-D1
P-Indoleacetic
b
2-14C acid-Lk
P-Indoleacetic
a,*
Treatment
5
5
5
5
5
5
5
5
2
2
Number of Replications
Basipetal
4 30
1072 351 +
66 -I_ 20
9
6,202 + 1,070
70
6,866 +
940
70
60
120
30
542
2
-
-
5,673 + 2,410
3,509
447 +
529 2
693 +
263 +
230
311
Mean rate
Dwarf
94.0
127.0
62.0
27.0
1.0
5.0
2.0
1.0
0.8
2.0
var.
-
-
80
50
4,C~19 2
4,242 i-
5,380 -i
5,039 +
3,585 )
870
730
940
980
160
2,063 2 1,140
376 2
1-77 +
192
435
Mean rate
Severe
2,753 + _
70.0
390
42.0
40.0
2,159 t
78.0
450
65.0
1.0
5,950 I 1,380
90
3.0
65.0
465 2
10.0
30
4.0
2.0
50.0
344 2
lg.0
670
180
12.0
14.0
6,389 + 1,040
1,392 +
1.0
-
-
39.0
303 2
3,310
0.7
1.0
1,804
Ratio Tulelake/ Healthy
Tulelake Mean Rate
3.0
Ratio Severe/ Healthy
ana Diseased
humilis
Healthy
in counts/min/cm2
Ratio Dwarf/ Healthy
Transport
rustica
Acids through
of Nicotiana
of Organic
92 5 40
ao
70
377 +
128 -+
20
-
269
184 +
-
127
Mean rate"'
Healthy
Stem Sections
Transport
TABLE 2
g t
4 G 2 > %
ASTER
YELLOWS
VIRUS
IKFECTION
0000 . 4;
if% -I
+I
+I
4 3
+I
.
. G
+I
-+I
+I
4; 4
P,
8 ri
2
+I
+I
+I
+I
+I co 2
4 cn
n
+I w % n N
fl 2
cd
+I
+I sj n cu o\
+I
2
2
H232P 0; f xAAf-L
H23% 04 f xAAf-Dh
-
3,465
2
H2321? “I; -t IfA
- D
-
2,040
2
H232P 0; + IAA - L
-
1,915
2
H232P Oi - D
-
3,920
2
H232P 0;; - L
C
2
H232P 04 - D -
2
3,764
-
4,431
-
-
5,674
5,423
”
5,984
Mean rate
H232P 0;; - L
b
2
a,* H23*P 0; - LB
Treatment
Number of Replications
NCSlthy
4,139
2,819
5,622
2,449
4,110
4,083
9,207
4,041
3,061
Mean rate
-
-
-
-
-
-
-
-
-
Dwarf
1.0
1.0
3.0
0.6
1.0
0.7
2.0
0.7
0.5
Ratio Dwarf/ Healthy
Rates of transport
-
-
5,146 3,272
-
-
-
-
-
-
8,026
1,524
10,083
42-
%,3x9
7,249
2,319
Mean rate
Severe
and
1.0
2.0
4.0
0.4
3.0
-
1.9
1.3
0.4
Ratio Severe/ Healthy
humilis
Healthy
in co~nts/min/cm2
var.
through
rustica
Substance
of Nicotiana
of Inorganic
Stem Sections
Transport
Diseased
Basipetnl.
TABLE 3
4,699
4,918
2,084
1,508
4,017
2,714
9,393
9,676
557
Mean rate
-
-
-
-
-
-
-
-
-
Tulelake
1.0
2.0
1.0
0.4
1.0
0.5
2.0
1.7
0.1
Ratio Tulel&e/ Healthy
EE;
8
5
3
_( 2
ASTER. YELLOWS
h
VIRUS
INFECTIOK
577
57s
TING AND GOLD
ful. This failure to localize the subst,ances may have resulted from their relatively great solubility in the solvents used for histological preparat,ioiis. Certainly the experimental results indicate active and selective translocation, rather than simple diffusion. Although t,he different substances tested might be expected to diffuse at different rates, it’ is difficult to reconcile relatively low diseased: healthy diffusion rat)ios in the case of phosphate ion with very high diseased : healthy diffusion ratios for one of the sugars or organic acids. In addition we have shown, using L-malic acid-% and half-inch agar (1% Oxoid agar Ko. 3) cylinders instead of st(em sect’ions, t’hat under the same experimental conditions the amount transported through agar cylinders is only one-third of that through diseased stem sections, but about seventeen t’imes t#hat through healthy st’em sections. Thus diffusion may be more rapid than translocation of certain substances in healthy stem sections. The relative rate of diffusion of a substance depends on t’he nat#urc of the solvent, the size of the molecule, its chemical st’ructure, and a number of other factors, but in any comparison between diseased and healthy stems most of these factors are similar in diseased and healthy plants. Thus water content of diseased and healthy tissues is similar wit,hin 1 or 2%. %Ioreover, diffusion occurs from a region of high concentraGon to one of low concent,ration of a molecular species. The molecular concent’rat’ion of most’ of the substances tested is much lower in the donor blocks than in t’he stem tissue. On the basis of mass action, one would expect 1iMle movement of t’he t,est#edsubst,ances out of the donor blocks and through the stem sections. The substantial t’ranslocat’ion act,ually observed t,hus would require the expenditure of metabolic energy. The format#ion of callose at the cut ends cannot be overlooked. Currier (1957) has reported that callose format’ion due to wounding is relatively slow in dormant tissues, and rapid in active tissues. Older tissues respond more slowly than young tissues. Histological st,udies carried out by us have shown that stem sect’ions bet’ween
leaves of the same stage of development have more secondary vascular elements in the aster yellows-diseased stems than in healthy stems. It has also been observed, however, t,hat flowering was delayed if not akoget’hcr absent in diseased plants. On the basis of one criterion, aster yellows virus-infected plant,s would be considered more mature, whereas in another criterion, it is more juvenile. Preliminary studies conducted by us have indicated that kansport in both healthy and diseased stem sectlions is essentially linear up to S hours, and may still actually increase in rate between S and 10 hours. Moreover, the transport ratios of diseased: healthy stem sections remained essentially similar over this period. Therefore, it seems unlikely that differential callose formation would have greatly influenced the results. The differences between strains arc not sufficiently consistent to draw any detailed conclusions. The Tulelake and severe strains tend to be more closely relat,ed in their transport behavior. Whereas t,he st’em sections of dwarf strain appear to be closer to healthy stem sections in its transport behavior than t,o t’he severe and Tulelake st’em sections. ACKNOWLEDGMENTS The authors are indebted to Dr. J. H. Freitag, Department of Entomology University of California, Berkeley, for his cooperation and supply of infective leafhoppers. This investigation was supported in part by Institutional Grant Funds of the American Cancer Society. The senior author wishes to thank the Rockefeller Foundation, which supported him on a scholarship during the study. REFERENCES F. C. (1950). “Plant Viruses and Vir\ls Diseases,” 3rd ed., p. 53. Chronica Botanica, Waltham, Massachusetts. BOLLARD, E. G. (1960). Transport in t,he xylem. Ann. Rev. Plant Phgsiol. 11, 141-166. CURRIER, H. B. (19.57).Callose substance in plant cells. Am. J. Botany 44, 478-488. DEDOLPH, R.R., NAQVI, S.M.,and GORDAN, S. A. (1966). Role of indole3-acetic acid in modification of geot,ropic responses in clinostat roBAWDEN,
tated Avena seedlings.
Planf
Physiol.
41, 89i-
902.
K., CCRRIEK, II. B., and CHEADLE, I-. I. (1957). Physiology of phloem. iInn. Rev. Plant Physiol. 8, 349-374. FKEITAG, J. H. (1964). Interaction and mutual suppression among three strains of aster yellou-s virus. Virology 24, 401-413. GOLDSMITH, M II. 11. (1966a). Movement of indoleacetic acid in coleoptiles of Avena sativa L. II. Suspension of polarity by total inhibition of basipetal transport. Plant Physiol. 41, 15-27. GOLDSMITH, 1~1.II. M. (1966b). Maintenance of polarity of auxin movement by basipetal transport. Plant Physiol. 41, 749-731. GRIEVE, B. J. (1943). Studies in the physiology of host-parasite relations. 4. Some effects of toI&AU,
mato spotted Exptl.
Viol.
wilt, on growth.
;lusttdian
J.
Med. Sci. 21, 89-101.
C. C. (1966). Translocation of growt,h reg‘rdators. Ann. Rev. Plant Ph!ysioZ. 17, 283-
MCCKE.WY,
291. ~UORG.~, 1’. W., and GALSMAN,
fects of ethylene Physiol.
on auxin
W. (1966). Eftrausport. Plant
II.
41, l.ihX.
ILLS.\, E. A., and Es.irr, K. (1961). Anatomic effects of cllrly t,op and aster yellows viruses on tomato. Hilgartbia 30, 469-51.5. Vas OVERBEEK, J. (1936). Absorption and translocation of plant regulators. Ann. IZev. Plant Physiol.
7, 3.55-372.
XI. H. (1960). Transport in the phloem. Ann. Rev. Plant Physiol. 11, 167-190.
ZIMMEILMANN,
32, 570-579 (1967)
Effects of Aster
Yellows
Virus
Infection
on Transport
through
Plant Stem Sections WEN-POH Department
of Plant
Pathology,
TInTG
A. H. GOLD
AND
Unicersity
Accepted April
of California,
Berkeley,
Cali.fornia
21, 1967
Basipetal transport of various substances through half-inch stem sections of healthy and aster yellows virus-infected Xicotiana rustica I,. var. humilis was determined. The effects of infect,ion by three strains of the virus, dwarf, severe, and Tulelake were observed on the t,ransport of 1%~labeled P-indoleacetic acid, malic acid, succinic acid, stlcrose, glucose, mannose, and t)he ions sulfate, phosphate, and rubidium. Transport of all these subst,ances except phosphate ion was more rapid through diseased than through healthy stem sections. In the dark, and with supplcmentary auxin, transport of phosphate ion was also more rapid through diseased than through healt.hy stem sections. Transport of fl-indoleacetic acid and of sugars through healthy stem sections was more rapid in the dark than in the light, bllt, transport of these compounds through diseased stem sections was rrnaffected by light. No aD-
preciable chemical coIlversion of the scrbstances tested was observed during the i,rs,nsport time used.
Stem sections from t,he plant offered a con-
1NTROl)UCTIOX
The severe malformation in t)he vascular tissue observed in aster yellows virus-infect,ed plants (Rasa and Esau, 1961) suggest,ed to us that transport’ of met,abo&es might be interfered wit#h. Indeed, Grieve (1943) reported that tomato spotted wilt, virus infection reduced the rate of auxin transport in tjomato stem sections, alt#hough he indicat,ed no morphological disturbance in t,he vascular t,issue, such as is observable in aster yclloms virus infection. The present, report describes comparisons in the transport of various substjances t#hrough stem sect.ions of healthy and ast,er yellows virus-infected Nicotiawa ,rustica 1,. var. humilis
plants. MATERIALS
AND
METHODS
of plant material. Nicotiana J,. var. humilis was chosen as the test
Preparation
m&a plant for this study because the t’hree strains of aster ~~110~s virus, dwarf, severe, and Tulelake described by Freitag (1964) prosymptoms in this plant. duce distinct,ive 570
venient
system
for
investigation.
The
N.
seedlings were raised in the grecnhouse and inoculat’ed with infective leafhoppers, Jlamosteles Jascijro?7s (St,%l) when 2-3 inches tall (4-A leaf st#age). The colonies of infective leafhoppers were maintained on common plantain, PZa77tago major J,., infectcd with one of the strains of aster yellows virus. To ensure reliable infection, ‘it was necessary to transfer these colonies at frequent int,crvals to fresh plantain plants with prominent new symptoms. Each N. mstica plant was inoculated with 20 infect,ive leafhoppers. The insects were allowed t#ofeed on the N. I-ustica plant for 6 days. Sympt,oms usually appeared 10-11 days after inoculation. Infected plants were used for transport experimenOs 4 weeks after inoculat8ion. Only plant,s showing prominent symptoms were used. HeaIthy pIants of the same age grown under the same conditjions mew used as COIItrols. Detew~Lination of trampod. The relat’ive transport, described in detail later was dctcrwstica
ASTb:R
YELLOWS
mined by measuring the radioactivity of labeled compounds which passed into a receiver agar block. The compounds chosen for st,udy included 3?P-labeled phosphate (at’ about pH 6.2), auxin (/3-indoleacetic-3-‘4C acid), rJ-malic acid-14C (U),l succinic acid1 ,4-14C, sucroseJ4C (U),l u-glucose-14C (U)l l)-lna11110se-l-14C,sodium sulfate (Sa$OJ, and rubidium chloride (86RbC12). The specific activity dat)a for these compounds are shown in t,he tables. The substances chosen to be involved in plant, met,aboarc k~mvn measurements were lism. lZll transport made in the basipetal direction, because prcvious evidence (Esau et al., 1957) indicated that transport in this directJion was largely through t,he phloem. In the early experiments, transport. t,hrough he&h?- stem sections was compared directly wit’h that in stem sections infected with the various strains of aster vellows. later, as it was not8iced t’hat’ t,he diseased stem sections were consistently sinsller in diamet,er than t,he healthy stem sections, the observed transport amountIs were correct’ed for stem areas. In general, this correction tended t,o enhance, but’ not fundamentally change, the differences in transport found between diseased and healthy st,em sections. Four plants each, infected by one of t’he 3 strains of ast,er yellows virus and 4 healthy controls were used in each experiment,. All plants were placed in a dark chamber for 12 hours prior to each experiment so t’hat, the plants would have the same pretreatment. One-half-inch-long stem segments were removed between the sixth and twelft’h leaf from each of the plant’s, using mounted razor blades set, one-half inch apart. Older part)s of stems were not used because their morphology had been dctcrmined before t)he onset) of symptoms. The plants mere selected for uniformity of growth and development wit’hin each treatment, and for equal stage of sympt’om development in the case of infected plnnt,s. Since t,he experiments extended from t,he middle of March, 1966, until the middle of October, 1966, plant responses to varying day lcngt hs, light levels, and glasshouse temperat,ures made it difficult to maintain 1 Uniformly
labeled
\-IRUS
571
IKFECTION
strict,ly comparable plant material over the whole period. Undoubtjedly part of the variabilit’y observable in the data can be attributed to this. Unless indicated otherwise in t#he respective t’ables, each experiment involved 5 replications. Physical arra~rgement for transport studies. The procedure followed was similar to that of other workers Audying transport, in stem sections (Dedolph et al., 1966; Goldsmith, 1966, a, b; 1IcCreadg, 1966; Morgan and Gausman, 196G). The substance to be studied was incorporated i&o an agar block. This donor block was placed on top of the stem section. The stem section was placed Chrough a hole in a plastic cap support on a receiver block. The receiver block, in turn, was placed in a planchet (Fig. 1). Donor blocks were prepared by including the desired concentration of the material under study in 1% agar (Oxoid agar Ko. X). The melted agar containing the material was pip&ted to a prechilled brass mold. The blocks were S.5 X 11.5 X 2 mm in size. To test the hypothesis that nuxin level might influence the rate of transport of some subst’ance, 15 ppm of 3-indoleacetic acid were added to some of the donor blocks for each substrance tested. The receiver blocks were prepared in the same manner as the donor blocks, but COWtained no test substance. The block sizes and t’he concentration of material in t,he donor block were chosen so that during t,he experiment no appreciable proportion of the test substances diffused out of the donor blocks, and so that the amount of material t’ransporbed through the st’em sect,ions t)o t,he receiver blocks was conveniently measurable. Conditions 01 transpo7~t and nzeasurenaent. The transport assemblies were placed in a large Pyrex dish lined at the bottom with moist filter paper. The top of the Pyrex dish was covered with Saran wrap (Dow ChemiDonor block
FIG. 1. Diagram port studies.
of assembly
used in the trans-
572
TINti
L4N11 (:OLl)
cal Company) to prevent drying of the agar blocks. The dishes were kept in controlled chambers at, 70°F. For t’he treatments exposed to light, the chambers w’crc illumnated with fluoresceut tubes, with an ittten&y of 550 foot-candles at the level of the Pyrex dishes. ITor the dark treatments, the same temperature and humidity conditions were maintained in the chambers. The toursport experiments were made over a period of 10 hours. At the conclusion of each experimcnt~, planchets containing the agar blocks were dried at about 80°C using a I>ubnoff metabolic-shaking incubator (Precision Scient,ific Co.). The agar blocks tnelted in the incubator, resulbing in a more even distribution of nlaterial before they were completel?dried down. The samples were then courkcd in a gas flow Det#ector (Model D47 SuclcarChicago Corporst.ion) . The transport data are expressed in terms of relative radioactivity (counts per mitnke) transported per unit, of time per square centimeter of stem area. In an at,tempt to determine whether appreciable proport,iotts of the t,est. substances undenvent chemical t~ransform:kiott during the lo-hour transport period, partition coefficients of t,he substances from the donor blocks were compared with t,hosc in the receiver blocks. In each case the substance was acidified wit,h 0.02 J/ acetic acid. The solvent, s&em chosen for p:trtit,iott coefficietttj determinkott was xmyl alcoholLw:ttw. This procedure would have dct e&cd gross changes-~ -for example, from sucrose to one of t#hc organic acids. However, because no change was observed in the p:trtit,iott CYK~cient of any test, substance during its JXLSSagC! through s:tcm sections, the procedure> will not, be dtrt ailed furt hw. RISSGLTS
Transport, of all the substances tested was affected by aster yellows virus infect’iott of the plants. Data are presented in Tables 1-3. Ratios of the relative transport in diseased and healthy stem sections are included in these tables. When t,hc experitnents were performed in the dark or in th(, presence of added auxin, phosphate tended to bc trnnsporl-ed fastjet
through infected than through healthy stein se&otts. It was apparent, t,hat the transport ratios of bot#h sulfate and rubidium ions n-we much greater than t’hose of phospl1:tt.c. (‘ompounds of similar chemical structjurc had approximat,ely similar transport ratios. A comparison of the transport ratios of the sugars, for example, indicnt ed that, the transport ratios lended to he higher than those of organic acids with thcl Tulelnl~ st’rain. Homevcr, the transport ratio of mnnnosc was lower than that of glucose OI sucrose. ;\ comparison of the transport of ntalic acid in t’hc flowering stage with k:msportJ at t)he stage normally used in the cxperimcttt (Table 2) showed a grent)l\- reducctl trattsport, ratSi0 at t,he flowering stagcP not bccause t,herc is an?- appreciable change in the rat,e of transport of nialatc in diseased stems, but, because t,he t~rattsport in he:Mty stems is grcath- increased. Ilight dccrertscd transport of all the sugars in healthy stews (Table 1). Since the transport of sugars itt diseased stems is not, :qqreciabl\: nflectcd by light., the transport r:tt’ios reflect t’his differetw in light response. Supplcmetttnr~~ ittdolc:rwtic the ~r:~IlsJ)Or~, acid dso seemed t’o aft’cct, especially- of phnsphatc~ in t hc wverc :md T~~lclal~e strains of the virus. DISCUSSION
Akhough much information exists on I he t,ransport, of various substattccs in phlocm and xylem of healthy plants (Hollard, l!KiO; Esau et al., 1957; Zimmerman, 1960), the changes which occur following virus infcclion arc only postulated. Thtts &~det~ (1950) called attent,ion t#o :L corrclntion between phloetn necrosis in nst,cr yc~llows infect,iott, and st#arch accutnulutiott in the leaves of intact plants. Although he interpreted this as due to interfcrenw \I-ith transloc:~tiot~, the int,erpret,ation was not, bawl on exlwrimentjnl evidence. The cxknsivc efforts t’o dctrrminc the mechanisms of stem t,ransport in health) plants recorded in the literature OII plant physiology have not) resulted in universally accepted concepts. The results of our Stud> ogler litt,le :~ddit,ionnl informntic~u on how transloc:~t~iott occurs in rl cmli of (4 1tc.r diseased or lK2,lth~ plants, tJ11t tll(k! (10
ASTER YELLOWS
\.IRUS
In fact, Van Overbeek (19.33) has indicated parenchymatous transport of growth substances. Our attempts to use autoradiography t’o identify the specific tissues transporting the test substances were unsucccss-
indicate some specific differences which may he useful in the study of t’he translocation mechanism. The possibilit,y of tJransportj by other tissues t,hnn phlocm has not been ruled out. TABLE BAYIPETAL
TR,4NSPoRT
OF
sUG.41~3
THROUGH
:Vicotiana
-c‘ 91.3
IKFECTION
rustica
1
HEALTHY
END
~~41~.
humilis
I>ISE.~SED
STEM
SECTIONS
OF
Transport in counts/min/cm*
I-
Healthy
Tulelake
Dwarf
Treatment
(5D-GlucoseW(U)u-b u-Glllcose‘“C (IT) -IY wGlwose“C(U) + IAAh-I, u-Glllcose“C(U) + IAA-I) _--*SucroselC(U)-I> Sucrose-
I 5
39 *
4 5,536 f
5
94 +
70 2,323 xk
5
15 +
1 5
15 zt
I
!____!
12
2,481 *
2 4,702 4~ 1,070 313
2,043 i
30: 4,439 3z
4,830 f
1,60(
124
4,231 +
74(
22
31(
578
G90’ 34 Gj204 zk 2,00(1 1 1
54
80’ 140 I G.703 * 1,000 428 130
450
39
3,872 zk
4201 13
1,050~ 136 ~ 8.G65 f
i--~_- I
I
5
32(
202
551 zt
15(
20
0
158 +
a0
13
2.927 zk
2i*
10
879 +
280
32
2,149 i
1,1801 131
2.G33 f
300, 1G-l 3.06G f I
1,210 875
2,154 f
1,404 f
48(
8,931 f
2,050i
30
8.313 41 1,200
20
10 +
4; 2,103 i (
5
3+
-15
295 f
1’ 2,625 i
730
244
2,430 xk
12&
G4(1 192
)I 468
-1 ---I 150~7,665 + 2.3Gi-T
10,047 + 1.6501
34
I
5
414 *
80 8>492 *
1,040:
20
3.779 zk
10,314 f 14C +
580 1.300
,12.527 f
1,NO
24
9 ‘10.32i * 1,470
13
MA-L
wRI:tlrnose-l‘Tz + IA&u
1 5 ~
1785 + 300 8,485 f
1,7901 11 ~ 7,4iO k 1,730 I
a Collcelltration: 16.5 X 10-a &/block (specific activity: ‘) Concetltration: 16.5 X 1V ,&/block (specific activity: c Concentration: 19.5 X 10-S&/block (specific activity: d Expressed as mean ratte =I=standard error. e III the light. f III the dark. 0 Uniformly ‘4C labeled. h IA.% at. 15 ppm.
65.7 mC/mmole). 185 mC/mmole). 24.2 mC/mmole).
acid-D
p -Indoleacetic-2-14C
acid-14C(U)
acid- 14C(U)+IAA1-L
acid-14C(U)+lX
L-Malic
L-MaXc
L-bIalic
-D
acid-14C(U)j-L
L-Malic
d
-D
acid-L
p-Indoleacetic-2-14C
c
acid-D
C acid-L
o-Indoleacetic-2-14C
p-Indoleacetic-2-
14
2-14C acid-D1
P-Indoleacetic
b
2-14C acid-Lk
P-Indoleacetic
a,*
Treatment
5
5
5
5
5
5
5
5
2
2
Number of Replications
Basipetal
4 30
1072 351 +
66 -I_ 20
9
6,202 + 1,070
70
6,866 +
940
70
60
120
30
542
2
-
-
5,673 + 2,410
3,509
447 +
529 2
693 +
263 +
230
311
Mean rate
Dwarf
94.0
127.0
62.0
27.0
1.0
5.0
2.0
1.0
0.8
2.0
var.
-
-
80
50
4,C~19 2
4,242 i-
5,380 -i
5,039 +
3,585 )
870
730
940
980
160
2,063 2 1,140
376 2
1-77 +
192
435
Mean rate
Severe
2,753 + _
70.0
390
42.0
40.0
2,159 t
78.0
450
65.0
1.0
5,950 I 1,380
90
3.0
65.0
465 2
10.0
30
4.0
2.0
50.0
344 2
lg.0
670
180
12.0
14.0
6,389 + 1,040
1,392 +
1.0
-
-
39.0
303 2
3,310
0.7
1.0
1,804
Ratio Tulelake/ Healthy
Tulelake Mean Rate
3.0
Ratio Severe/ Healthy
ana Diseased
humilis
Healthy
in counts/min/cm2
Ratio Dwarf/ Healthy
Transport
rustica
Acids through
of Nicotiana
of Organic
92 5 40
ao
70
377 +
128 -+
20
-
269
184 +
-
127
Mean rate"'
Healthy
Stem Sections
Transport
TABLE 2
g t
4 G 2 > %
ASTER
YELLOWS
VIRUS
IKFECTION
0000 . 4;
if% -I
+I
+I
4 3
+I
.
. G
+I
-+I
+I
4; 4
P,
8 ri
2
+I
+I
+I
+I
+I co 2
4 cn
n
+I w % n N
fl 2
cd
+I
+I sj n cu o\
+I
2
2
H232P 0; f xAAf-L
H23% 04 f xAAf-Dh
-
3,465
2
H2321? “I; -t IfA
- D
-
2,040
2
H232P 0; + IAA - L
-
1,915
2
H232P Oi - D
-
3,920
2
H232P 0;; - L
C
2
H232P 04 - D -
2
3,764
-
4,431
-
-
5,674
5,423
”
5,984
Mean rate
H232P 0;; - L
b
2
a,* H23*P 0; - LB
Treatment
Number of Replications
NCSlthy
4,139
2,819
5,622
2,449
4,110
4,083
9,207
4,041
3,061
Mean rate
-
-
-
-
-
-
-
-
-
Dwarf
1.0
1.0
3.0
0.6
1.0
0.7
2.0
0.7
0.5
Ratio Dwarf/ Healthy
Rates of transport
-
-
5,146 3,272
-
-
-
-
-
-
8,026
1,524
10,083
42-
%,3x9
7,249
2,319
Mean rate
Severe
and
1.0
2.0
4.0
0.4
3.0
-
1.9
1.3
0.4
Ratio Severe/ Healthy
humilis
Healthy
in co~nts/min/cm2
var.
through
rustica
Substance
of Nicotiana
of Inorganic
Stem Sections
Transport
Diseased
Basipetnl.
TABLE 3
4,699
4,918
2,084
1,508
4,017
2,714
9,393
9,676
557
Mean rate
-
-
-
-
-
-
-
-
-
Tulelake
1.0
2.0
1.0
0.4
1.0
0.5
2.0
1.7
0.1
Ratio Tulel&e/ Healthy
EE;
8
5
3
_( 2
ASTER. YELLOWS
h
VIRUS
INFECTIOK
577
57s
TING AND GOLD
ful. This failure to localize the subst,ances may have resulted from their relatively great solubility in the solvents used for histological preparat,ioiis. Certainly the experimental results indicate active and selective translocation, rather than simple diffusion. Although t,he different substances tested might be expected to diffuse at different rates, it’ is difficult to reconcile relatively low diseased: healthy diffusion rat)ios in the case of phosphate ion with very high diseased : healthy diffusion ratios for one of the sugars or organic acids. In addition we have shown, using L-malic acid-% and half-inch agar (1% Oxoid agar Ko. 3) cylinders instead of st(em sect’ions, t’hat under the same experimental conditions the amount transported through agar cylinders is only one-third of that through diseased stem sections, but about seventeen t’imes t#hat through healthy st’em sections. Thus diffusion may be more rapid than translocation of certain substances in healthy stem sections. The relative rate of diffusion of a substance depends on t’he nat#urc of the solvent, the size of the molecule, its chemical st’ructure, and a number of other factors, but in any comparison between diseased and healthy stems most of these factors are similar in diseased and healthy plants. Thus water content of diseased and healthy tissues is similar wit,hin 1 or 2%. %Ioreover, diffusion occurs from a region of high concentraGon to one of low concent,ration of a molecular species. The molecular concent’rat’ion of most’ of the substances tested is much lower in the donor blocks than in t’he stem tissue. On the basis of mass action, one would expect 1iMle movement of t’he t,est#edsubst,ances out of the donor blocks and through the stem sections. The substantial t’ranslocat’ion act,ually observed t,hus would require the expenditure of metabolic energy. The format#ion of callose at the cut ends cannot be overlooked. Currier (1957) has reported that callose format’ion due to wounding is relatively slow in dormant tissues, and rapid in active tissues. Older tissues respond more slowly than young tissues. Histological st,udies carried out by us have shown that stem sect’ions bet’ween
leaves of the same stage of development have more secondary vascular elements in the aster yellows-diseased stems than in healthy stems. It has also been observed, however, t,hat flowering was delayed if not akoget’hcr absent in diseased plants. On the basis of one criterion, aster yellows virus-infected plant,s would be considered more mature, whereas in another criterion, it is more juvenile. Preliminary studies conducted by us have indicated that kansport in both healthy and diseased stem sectlions is essentially linear up to S hours, and may still actually increase in rate between S and 10 hours. Moreover, the transport ratios of diseased: healthy stem sections remained essentially similar over this period. Therefore, it seems unlikely that differential callose formation would have greatly influenced the results. The differences between strains arc not sufficiently consistent to draw any detailed conclusions. The Tulelake and severe strains tend to be more closely relat,ed in their transport behavior. Whereas t,he st’em sections of dwarf strain appear to be closer to healthy stem sections in its transport behavior than t,o t’he severe and Tulelake st’em sections. ACKNOWLEDGMENTS The authors are indebted to Dr. J. H. Freitag, Department of Entomology University of California, Berkeley, for his cooperation and supply of infective leafhoppers. This investigation was supported in part by Institutional Grant Funds of the American Cancer Society. The senior author wishes to thank the Rockefeller Foundation, which supported him on a scholarship during the study. REFERENCES F. C. (1950). “Plant Viruses and Vir\ls Diseases,” 3rd ed., p. 53. Chronica Botanica, Waltham, Massachusetts. BOLLARD, E. G. (1960). Transport in t,he xylem. Ann. Rev. Plant Phgsiol. 11, 141-166. CURRIER, H. B. (19.57).Callose substance in plant cells. Am. J. Botany 44, 478-488. DEDOLPH, R.R., NAQVI, S.M.,and GORDAN, S. A. (1966). Role of indole3-acetic acid in modification of geot,ropic responses in clinostat roBAWDEN,
tated Avena seedlings.
Planf
Physiol.
41, 89i-
902.
K., CCRRIEK, II. B., and CHEADLE, I-. I. (1957). Physiology of phloem. iInn. Rev. Plant Physiol. 8, 349-374. FKEITAG, J. H. (1964). Interaction and mutual suppression among three strains of aster yellou-s virus. Virology 24, 401-413. GOLDSMITH, M II. 11. (1966a). Movement of indoleacetic acid in coleoptiles of Avena sativa L. II. Suspension of polarity by total inhibition of basipetal transport. Plant Physiol. 41, 15-27. GOLDSMITH, 1~1.II. M. (1966b). Maintenance of polarity of auxin movement by basipetal transport. Plant Physiol. 41, 749-731. GRIEVE, B. J. (1943). Studies in the physiology of host-parasite relations. 4. Some effects of toI&AU,
mato spotted Exptl.
Viol.
wilt, on growth.
;lusttdian
J.
Med. Sci. 21, 89-101.
C. C. (1966). Translocation of growt,h reg‘rdators. Ann. Rev. Plant Ph!ysioZ. 17, 283-
MCCKE.WY,
291. ~UORG.~, 1’. W., and GALSMAN,
fects of ethylene Physiol.
on auxin
W. (1966). Eftrausport. Plant
II.
41, l.ihX.
ILLS.\, E. A., and Es.irr, K. (1961). Anatomic effects of cllrly t,op and aster yellows viruses on tomato. Hilgartbia 30, 469-51.5. Vas OVERBEEK, J. (1936). Absorption and translocation of plant regulators. Ann. IZev. Plant Physiol.
7, 3.55-372.
XI. H. (1960). Transport in the phloem. Ann. Rev. Plant Physiol. 11, 167-190.
ZIMMEILMANN,