Indian J. Physiol., Vol. XXVII No.1 pp. 64-73 (January 1984)

PARAQUAT - INDUCED SUPPRESSION OF CRASSULACEAN ACID METABOLISM CHARACTERISTICS IN FIVE WOODY WEEDS*

I. MADHtTSUDANA RAO", P.M. SWAMY AND V.S. RAMA DAS

Department of Botany, Sri Venkateswara University. Tirupati 517502, Andbra Pradesb,

(Revised: February 22,1983)

SUMMARY

Five nonsucculent semi-arid scrub species, . Maha huxifolia. Flocourtia sepiaria, Gymnosporia emorginata and viscosa exhibi.ted scotoactive stomatal opening, nocturnal carbon assimila­ tion and dark acidification while day time CO. uptake was predominant in Chomelia asiatica. Foliar application of 100 mg 1-1 paraquat (I, 1'­ dimethlyl-4,4'-bipyridinium ion) completely inhibited the dark uptake of CO.. Phosphoenolypyruvate (PEP) carboxylase activity was suppres­ sed in the treated while Ribulose bisphosphate (RuBP) carboxy­ lase was relatively unaffected. The results indicated that tbe process of dark CO. uptake was sensitive to paraquat.

INTRODUCTION

Crassulacean acid metabolism (CAM) has been described in 17 families, (13 dicotyledon and 4 monocotyledon families) 1 Gymnos­ perm (Welwitchia) and in two ferns (Kluge and Ting, 1978; Ting and Gibbs, 1982). Earlier workers had noticed the occurrence of CAM in some nonsuccu­ lent (Mc William, 1970; Kluge et. al., 1973). We observed the occurrence of CAM characteristics in five nonsucculent scrub species, belonging to five taxonomically diverse dicotyledon famHies. Apocynacea, Ebenacae, Flacourti­ aceae Celastraceae and Sapindacea (Rao et. al., 1979). These are unproductive woody weeds, growing in a semi-arid climate.

We observed earlier that paraquat treatment could bring the reversal of the scotoactive stomatal opening, exhibited by these plants, into photoactive

·The results form a part of the Ph.D. thesis of I.M.R. approved by Sri Venkateswara Univer. sity, Tirupati. ·*Present address: Department of Plant &. Soil Biology, University of California, 108, Hilgard Hall, Berkeley, CA, 94720 USA. SUPPRESSION OF CAM BY PARAQUAT 65 resulting in rapid desiccation and death of the foliage (Rao et. al., 1977). The above investigation prompted us to undertake a detailed study on the action of paraquat on the photosynthetic metabolism of these woody weeds.

MATERIALS AND METHODS

The scrub formation investigated, is situated in the Tirumala foot hills, near Tirupati, India. The scrub comprises of the following non-succulent woody weeds in a decreasing order of dominance: Carisso spinarum, L; Maba buxifolia, CI; Flacourtia sepiaria, Roxb; Chomelia aSiatica, O. Kze; Gymnosporia emarginata, Laws; and Dodonaea viscosa, L. Paraquat (1. l' - dimethyl-4,4' -bipyridinium ion) at 100 mg 1-1 was applied as foliar spray at 9 a.m.

The width of the stomatal apertures was measured by obtaining silicone rubber and nail polish impressions on the abaxial surfaces (Rao et. al.,1977). The titratable acidity of leaves was determined following Ting and Dugger (1968). Malic acid content was estimated by recording the reduction of NAD+ in the presence of malate dehydrogenase (photometrically) at 340 nm (Hohorst.

1970). The dark and light CO2 uptake was determined by using a HCOz feed­ ing technique ( et. al., 1970). PEP carboxylase and RuBP carboxylase enzymes were assayed according to the methods followed by Raghavendra and Das (1977).

RESULTS

The morphological and physical features of leaves of six weed species, growing under natural habitat are given in Table I. A striking result of the study of the diurnal rhythms of the size of the stomatal aperture was the occure­ nce of pronounced nocturnal opening (scotoactivity) of stomata in five of the six weed species investigated namely C. spinarum, M. buxifolia, F. sepiaria, D viscosa and G. emarginata. Foliar application of paraquat caused an appreci­ able reversal of the night opening behaviour of stomata into day opening- This was more pronounced in M. buxifolia, F. sepiaria and D. viscosa (Table 1).

Studies on the diurnal variations in the titratable acidity and malic acid indicated high degree of dark acidification in five of the six weed species which exhibited scotoactive stomatal opening (Figs. I and 2). Consistent with the dark acidification, malic acid also accumulated in large quantities only during night (Fig. 2). Paraquat strongly inhibited the degree of dark acidification in all the five weed species. Paraquat caused an increase in the titrable acidity and malic acid content during day in D. viscosa and G. emarginala (Figs. 1 and 2). ~~c - :;-,--­ I •. "'11

66 J. MADHUSUDANA, P.M. SWAMY AND V.S. llAMA DAS

Table I : Morphological and physical features of leaves under natural habitat

Plant Species Average Stomatal Leaf Ratio of Dry wei&ht leaf area frequency Thickness dry weight to percml (cml) (No. per mm') (mm) fresh weight (mg)

Apocynaceae: Carissa spinarum 3.10 652 0.45 0.70 17.20 Ebenaceae:

Maba buxijolill O.g() 147 0.34 0.74 31.00 Flacourtiaceae:

F[llcourtia sepiarill 1.82 716 0.20 0.86 20.12 CeJastraceae: Gymnosporill emarignala 1.35 450 0.48 0.78 n.2() : Dodonaea viscosa 7.10 410 0.20 0.81 14.34 Rubiaceae: Chomelill asiatica 9.11 504 0.28 0.86 57.20

The occurrence of nocturnal carbon assimilation in addition to normal day time COl! uptake was noticed in five weed species which exhibited dark acidification while there was only day time COli uptake in C. asiatica. Paraquat remarkably inhibited COli uptake both during day time and also in darkness (Fig. 3). Studies on the carbon assimilation of leaves up to an extended period of 3 days after chemicaftreatment indicated a strong inhibition of carbon dioxide uptake with paraquat treatment in all the weed species tested (Table 2).

The levels of PEP carboxylase activity were high in F. sepiaria, M. buxifolia and G. emarginata. Paraquat suppressed the PEP carboxylase activity in all the weed species up to 72 h after treatment, whereas RuBP carboxylase was relatively unaffected (Table 3 and 4).

DISCUSSION

The present investigation holds an important implication of the occurr­ ence ofsome CAM characteristics in five non-succulent scrub species belonging to five taxonomically diverse dicotyledon families of higher plants. The occurr­ ~.

Table II: Effect. or"paraquat on stomatal diurnal rhythm upto 24 hours after treatment (Stomatal openin, in microns (p.m) ) .

Plant Ipecies Treatment 12 Noon 3 p.m. 6 p.m. 9 p.m. 12 Midnight 3a.m 6a.m 9 a.m.

Carissa spinaru", Control 2.0 4.8 5.8 6.0 8.0 4.0 5.4 5.8 paraquat 7.4 7.6 9.4 9.8 11.2 8.8 5.6 6.8

Maba buxifolia Control 2.4 4.8 1.6 8.0 8.2 6.0 2.8 3.0 Ul ~ paraquat 8.6 8.6 9.2 9.2 9.2 7.8 2.8 8.2 ." ~ EJ Flacourtia Control 1.8 4.6 0.8. 2.0 6.8 1.2 3.0 3.2 5"" sepiarie paraquat 6.2 9.2 8.4 4.~ 4.2 2.6 2.6 5.4 Z g GymnDsporia Control 0.8 1.8 2.8 4.0 4.2 3.1l 3.0 2.4 ("l > 'marz/nata paraquat 4.2 5.2 4.6 4.2 3.0 3.2 3.0 3.8 .ii:

2.6 -<= Dodonaea Control 3.4 1.8 2.8 6.0 2.4 2.2 3.5 ." > viscosa paraquat 9.8 9.6 3.8 3.8 3.6 2.8 2.2 2.8 )1;1 ~ Chomelia Control 2.4 3.0 1.9 0.0 0.0 0.0 0.8 2.2 >~ o-j asiatica paraquat 3.6 4.2 3.0 0.0 0.0 0.0 1.0 2.8

0­ -.....I

.~ 1 ~. " 68 I. MADHUSUDANA. P.M. SWAMY ANDV.S.RAMA DAS

O·itO C.SPINAfWM 0·60 F' - SE PIARIA

0.30 ... ::>"" 0 -'" (lJ ~ '­ 0-20 >- 0.4-0 :::> "!: ..... VI 0 c: ... (lJ .2! 0. 0-10 .5 E 0·20 (lJ .s;;; ~ -Cl' :::; I 0 0 M·BUXIFOLIA 0·4-0­ D.VISCOSA 0·50 ...... s;;; 01 -0:; 0.30 ~ .r:. '"(lJ L. .'t­ -01 cT (lJ E 0

0·4-0 EMARGINATA • 0·12

0·04

3a.1II 9a.m Sam lp.m la.m TIlliE OF THE DAY Fig. 1. Influence of paraquat on the diurnal rhythm of titratable acidity of leaves (0-0 control; /::,.-/::,. paraquat). The diurnal change in light intensity (0-0) and temperature

360 420 , C. SPINA RUM F. SEPIARIA

~o 100

420 ~. BUXIFO,LIA

340

ao ~ ..,.:x iii '3: 180 :x 111 w ~ 100 -,- .. ~oo G. Et.4ARGINATA 220 III... 0s ::a.. no 160

60L-__~~______~

fa.m. 1 p.m. 9 p.m. 3a.rn U.m. 9a.m. lp.m. 9p.m. la.lII. 9p.rn TIM. E 0 F T 4 E 1")'4 Y Fig. 2. Influence of paraquat on the diurnal variation in malic acid content of leaves (0-0 control; f:::.-f:::. paraquat). list of nonsucculent CAM plants already known (Me Williams. 1970; Kluge et. ai., 1973). Further this expanding list of nonsueerilent CAM plants s.upports the views of Kluge et. 01. (1973) who treats CAM as cellular succulence rather than the succulence of whole plant or plant organ. 70 I. MADHUSUDANA, P.M. SWAMY AND V.S. KAMA DAS

C. SPIHARUM

2·0

0

'·2 .2..VISCOSA

i.4

-I .s::. N 1· 6 I E '0

N 0 0·' u en E 0

3· 2 ~. E~A~GIN~T~ C. ASIATICA

~-

o ~~:::;;;;A;;;;;;~~::J 'a.m. 3p.m. 9p.m 31.111. U.m. 9a.m 3p.m. 9p.m. Ja.J\. tut. TIME OF THE DAY Fig. 3. JnDuence 01 paraquat on the diurnal course 01 carbon dioxide uptake in leaves. (0-0 control; A-I::" paraquat).

All the succulents in which CAM reported are either xerophytes or halo­ phytes and succulence is their ecophysio]ogical adaptation to the xeric environ­ ment (Osmond, 1918; Kluge and Ting. 1918;). Similarly, these nonsucculents, SUPPRESSION OF CAM BY PARA;QUAT 71

Table III : Influence of paraquat on the carbon assimilation of leaves up to an a 1 extended period of 3 days (mg CO2 dm- h- )

species Control· Hours after treatment 24 48 72

Carissa spinarum 4.21 0.58 0.31 0.12 Maba ba:rij'o/ia 2.18 0.84 0.66 0.24 Flacourtia sepiaria 2.86 0.21 0.12 0.04 Gymnosporio emarginata 1.32 0.21 0.17 0.09 Dodonaea viscosa 1.64 0.46 0.21 0.02 Chomelia asiatica 2.93 0.14 0.09 0.05

• at 9 a.m.

Tahle IV: Influence of paraquat on the phosphoenol pyruvate carboxylase activity of leaves (p. mole mgl (chlorophy1I) h-1)

Plant species Control· Hours after treatmect 24 48 73

Carissa spinarum 489 478 264 184 Mabo buxi/olia 1648 1482 430 360 Flacourtia sepiaria 1866 1260 636 456 Gymnosporia emarginato 1560 826 320 294 Dodonaea viscosa 614 780 • 148 128 Chomelia asiatica 112 84 36 N.D.

• at 3 p.m. N.D.: not detectable

Table V: Effect of paraquat on the RuDP carboxylase activity ofJeaves (}' mole mg-1 (chlorophylJ) h-1)

Plant secies Control· Hours after treatment 24 48 72

Carissa spinarnm 185 196 188 178 Maba buxi/olia 420 468 466 432 Flacourtia sepiaria 180 228 246 210 G;ymnosporia emarginata 200 240 232 210 Dodonaea viscosa 600 466 324 240 Chomelia asiatica 338 400 387 366

• at 3 p.m. 72 I. MADHUSUDANA, P.M. SWAMY AND V.S. RAMA DAS which exhibit some CAM characteristics are growing in semi-arid environment. Our observations (Figs. 1 and 2) confirm that the high titrable acid and malic acid synchronised with the cold and most humid part at night and minimum during the hot and dry period of the day (Neales, 1975; Kluge, 1976;). These data are in confirmity with the previous findings of Bharucha and Joshi (1958) and Milburn et. 01. (1968). Paraquat is known to inhibit photosynthetic carbon dioxide fixation of leaves in light (Dodge, 1975). The inhibition of dark COs fixation in leaves by paraquat is hitherto unnoticed. Sands and Bachelard (1973) have already shown that cpicloram enters the leaf through the stomata. Since paraquat has enhanced the stomatal opening during day, an enhanced entry of herbicide into the leaf is possible. This milhi be the reason tor the inhibition of dark COs uptake and dark acidification observed in five weed species. However, paraquat also inhibited COz uptake in C. asiatica during day.

The selective suppression of PEP carboxylase activity in paraquat treated plants, while RuBP carbox if lase was unaffected, indicated that the process of dark COs uptake observed in these plants was far more sensitive to paraquat than the light dependent carbon fixation during day. The inhibition of day time COl uptake by paraquat could result from the inhibition of ligbt dependent formation of NADPH in chloroplasts (Rao et. 01., 1980).

The occurrence of CAM in these weed species can help as an ecophysio­ logical adaptation to semiarid environments for water conservation (Neales, 1975; Kluge and Ting, 1978). The suppression of this adaptive variant of the photosynthetic pathway of carbon assimilation with paraquat would help to control these unproductive woody weeds. Thus, the present investigation pro­ vided a physiological explanation for the paraquat-suppressed CAM characteri­ stics in some non-succulent scrub species.

ACKNOWLEDGEMENT

This investigation has been supported by a grant from United States Department of Agriculture under USPL-480 Project FG-IN-544 (IN-ARS-27). The authors express their sincere appreciation to Dr. Herbert M. Hull, USDA­ ARS Western Region, Tuson, Arizona for his interest and many useful com­ ments on this work.

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