Photosynthesis and Translocation Rate in Arachis hypogaea L. Mutants

Biochem. Physiol. Pflanzen 180, 337-343 (1985)

Photosynthesis and Translocation Rate in Arachis hypogaea L. Mutants M. L. LODHA, R. P. JOHARI, K. D. SHARMA and S. L. MEHTA Division of Biochemistry, Indian Agricultural Research Institute, New Delhi, India Key Term Index: photosynthesis, photosynthate, translocation rate, mutants; Arachis hypogaea

Summary Photosynthesis and translocation rate of photosynthate have been studied in ground nut ( Arachis hypogaea) mutants TG-1 and TG-16 and their parent Spanish Improved at different stages of growth. Photosynthetic rate per plant was significantly higher in mutants as <:ompaJed with their parent, with TG-l being the most efficient. The translocation of 14C-labelled photosynthate to nodules in mutants was also higher than the parent. The lower kernel yield in mutant TG-l, which had high rates of photosynthesis, appears to be due mainly to poor mobilization of photosynthate for kernel development. The comparison of photosynthesis and translocation in TG-l and TG-16 indicates that mobilization of food reselves to developing kernels is important for high yields.

Introduction

Carbon dioxide enrichment of soybean (HARDY and HAVELKA 1975) and groundnut (HAVELKA and HARDY 1976) crop canopy resulted in increase in shoot dry weight, seed yield per unit area, nodule weight and specific nitrogen fixation activity, suggesting that the principal limitation for symbiotic nitrogen is the availability of photosynthate. Genetic manipulation of legumes, however, offers a great potential for increasing nitrogen fixation and yield in grain legumes. Mutation breeding has been successful in inducing variability for yield and other characters in groundnut (PATIL 1971), which is an important oil seed crop. Several mutants popularly known as Trombay groundnut (TG) varieties with high yield potential have been derived. With the development of these mutants, it is possible to examine the effect of mutation on nitrogen fixation, dark CO 2 fixation and photosynthesis, and to show the coupling between photosynthesis and nitrogen fixation. Earlier studies have shown that groundnut mutants TG-l and TG-16 have higher nitrogen fixation capacity as judged by acetylene reduction and higher dark CO 2-fixation capacity of nodules compared to their parent Spanish Improved (LODHA et al. 1983). In the present study photosynthesis and translocation rate during crop growth in these mutants, and their parent Spanish Improved, have been studied. Material and Methods Plant material

Uninoculated seeds of groundnut c. v. Spanish Improved and its mutants TG-l and TG-16 developed by mutation breeding at the Biology and Agrienlturc Division of BARC, Trombay, were 22

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grown at the Institute farm during the monsoon season under norm:>1 recommended agronomic practices, exeept that the soil was not given nitrogenous fertilizer. The crop matured in approximately 115 days. Mutant TG-1 was developed after treating kernels of Spanish Improved variety with 75 KR gamma irrad iation and by repeated selection for improved kernel weight. The mutant has profuse branching habit with dark green leaves. TG-16 was derived after crossing TG-1 with virescent. This mutant has improved kernel weight as well as high yield.

Dry weight determination Dry weight of different plant parts was determined by oven drying the samples to a constant weight. Harvest index at matmity has been caleulated as pod weight X lOO/total plant weight. Eaeh value in various figures is the average of 5 determinations anll vertical bar indicates the standard error (SE) of these values. Chlorophyll estimation Chlorophyll a and b was estimated in the acetone extract from fresh leaves aeeolding to the method of A IC",01\" (1949). The results are an average of duplicates run by using a composite sample of 5 plants. 14 C0 2 feeding

This was done as described by SANTH.\ et al. (1982). A plant canopy was covered by a 22,805 cm3 perspex chamber, sealed, and then exposed for 1.0 min to 13 /lCi 14C02 , released by the addition of 1 NHCI to Na 2 14C0 3 (spec. act. 1.93 GBq/m mol). For studying photosynthetic rate, plants were harvested immediately after a 1 min label feeding, chilled over ice, separated into different plant parts and lyophilized. However, for determining translocation rate, plants were allowed to photosynthesize for 5 h in an open atmosphere after a 1 min 14C0 2 feeding and then harvested.

Extraction and Coullting Lyophilized plant parts (leaf and stem, 100 mg each; root, kernel and pod wa,lI, 50 mg each; nodule, 10 mg) wero extracted after grinding in 80 % ethyl alcohol at 60 °C for 10 min. The extract wa.s centrifuged at, 5,000 . g for 10 min, the supernatant collected and the residue re-extracted. The supernatants were pooled and made upto 10 ml. For radioactive counting, 2 ml sample was taken in a vial, 1-2 drops H 2 0 2 added where required, and evaporated to near dryness. The counting was done in a Packard Liquid Scintillation Spectrometer using a dioxan based seintillator. Each value represents the mean of two separate experiments.

Results

Dry weight The dry weight per plant of root, nodule, leaf, stem a.nd pod for mutants TG-l and TG-16 and parent Spanish Improved, at different stages of growth, is shown in Fig. 1. The root weight in TG-l was higher than its parent and TG-16. TG-16 also had a higher root weight than Spanish Improved up to day 69, and at later stages the differences were not significant (Fig. lA). Nodule weight in TG-l at 44 days after sowing (DAS), was more than twice that of Spanish Improved and TG-16. During later stages both TG-l and TG-16 had higher nodule weight than Spani8h Improved. However, at maturity the nodule weight in TG-16 was substantially higher than TG-l and Spanish Improved (Fig. 1 B).

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Throughout the developmental period TG-l had a substantially higher leaf weight (1.5-2fold) than either TG-l6 or Spanish Improved. The leaf weight of TG-l6 did not differ significantly from that of Spanish Improved (Fig. le). Stem weight per plant of TG-l, TG-l6 and Spanish Improved did not differ from each other significantly during development except at 69 DAS TG-l6 had a lower and at maturity TG-l had a higher stem weight than the other two (Fig. 1 D). During development, pod yield per plant was generally higher in both TG-l and TG-l6 than Spanish Improved. At maturity, the pod yield in TG-l6 and TG-l was, respectively, 2- and l.5fold greater than Spanish Improved (Fig. 1E). The harvest index for Spanish Improved, TG-l and TG-16 was 38.3, 39.8 and 53.3%, respectively. Dry weight of a plant as a whole in TG-16 and Spanish Improved was comparable but substantially lesser than TG-l up to 97 DAS. However, at maturity dry weight of

Fig. 1. Dry ll'eight of different plaltt organs. (A) root; (B) nodule; (C) leaf; (D) stem; (E) pod and (F) total plant of gronndnut mutants TG-l (-0 - 0-) and TG-16 (-i',.-i',.-) and parent Spanish Improved (-e-e-) at different stages of growth. Vertical bars represent the standard error (SE). 22'"

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Table 1. Chlorophyll a and b contents in leaves of groundnut mutants and parent at different stages of growth Days after sowing

Spanish Improved

38 47 66 101

Chlorophyll a (mg/g leaf fresh wt.) 0.70 1.10 0.79 1.11 1.35 1.76 0.93 1.02

38 47 66 101

Chlorophyll b (mg/g leaf fresh wt.) 0.20 0.25 0.25 0.36 0043 0.60 0.29 0.33

Chlorophyll a Chlorophyll b -

TG-1

TG-16 0.73 0.80 1.22 0.92 0.18 0.24 0040

0.29

Critical difference (C.D.) for stages at 5 %, 0.19 and at 1 %, 0.29 C.D. for varities at 5 %, 0.17 and at 1 %, 0.25 C.D. for stages at 5 %, 0.07 and at 1 %, 0.11 C.D. for varieties at 5 %, 0.06 and at 1 %, 0.10.

Table 2. Leaf photosynthetic rate in groundnut mutants and parent Days after sowing

Spanish Improved

TG-1

TG-16

45 61 82

mg CO 2 /g dry wt./h 29.5 16.0 3.6

3G.2 18.2 3.7

36.1 19.5 8.1

45 61 82

mg/C0 2 /plant leaf/h 216.5 305.5 99.9

564.3 633.1 160.9

298.9 435.8 211.3

mg CO 2 /g dry wt. mg CO 2 /plant leaf -

Critical difference (C. D.) for stages and varieties at 5 %, 3040 and at 1 % 4.9. C. D. for stages and varieties at 5 %, 57.7 and at 1 %, 82.9.

TG-1 and TG-16 plant was comparable and substantially higher than Spanish Improved (Fig.1F).

Chlorophyll content Chlorophyll a and b content in the leaves of TG-1, TG-16 and Spanish Improved increased significantly at 66 DAS and decreased thereafter at 101 DAS (Table 1). The content of both chlorophyll a and b was significantly higher in TG-1 than TG-16 and Spanish Improved at all the stages of growth except at 101 DAS. Chlorophyll content of Spanish Improved and TG-16 was similar during development.

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Table 3. Influence of age on per cellt distribution of He in various plallt parts of the groundnut plant 5 h after exposure to 14C02 Days after sowing

Plant part

Spanish Improved

TG-1

TG-16

45

Leaf Stem Pod* Root Nodule

42.00 21.78 28.56 3.28 4.38

59.51 9.44 19.97 3.25 7.83

59.43 9.31 20.74 4.47 6.05

Leaf Stem Kernel Pod wall Root Nodule

65.51 10.52 :2.37 20.56 0.12 0.92

65.02 8.09 2.58 21.35 0.29 2.67

65.59 7.92 2.18 20.62 0.70 2.99

Leaf Stem Kernel Pod wall Root

48.32 15.73 8.08 26.09 0.90 0.38

44.76 18.82 11.08 23.09 0.96 1.29

49.46 12.00 16.92 19.84 0.85 0.93

61

8:2

~odule

* Pods did not differentiate into wall and kernel.

Photosynthetic rate Results presented in Table 2 show photosynthetic rate (mg CO:: fixed per g dry weight or per plant part per h) of leaf in parent Spanish Improved and mutants TG-1 and TG-16. During early growth the photosynthetic rate (per g dry weight) was significantly higher in both the mutants as compared to their parent, but by day 61 the differences in photosynthetic rate were much less. However, at 82 DAS the photosynthetic rate in Spanish Improved and TG-1 was similar but significantly lower than TG-16. At 115 DAS photosynthetic rate in all the 3 varieties was negligible (data not shown). Thc total photosynthetic capacity is judged by comparing CO 2 fixed per plant leaf and is a better estimate of photosynthetic efficiency of a given variety than the photosynthetic rate per unit weight. The results in Table 2 indicate differences in photosynthetic capacity of the mutants vs. the parent. Thoughout the development, both the mutants had a significantly higher capacity for CO2 fixation. At 45 and 61 DAS TG-l had a more than 2-fold higher rate of CO 2 fixation per plant compared to Spanish Improved. TG-1 also had significantly higher CO2 fixation per plant than TG-16 at both 45 and 61 DAS, while at 82 DAS the trend reversed and TG-16 had higher CO 2 fixation than TG-l.

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Translocation of photosynthate In order to measure the translocation of photosynthate from leaf to other plant parts, plants were harvested 5 h after 14C0 2 feeding and the distribution of label determined. The results presented in Table 3 show differences in translocation rate of photosynthate to nodules in parent and mutants. At all the stages, the proportion of label in nodules of mutants TG-1 and TG-16 was substantially higher (1.4~3.4fold) as compared to their parent. During early growth a higher proportion of label appeared in nodules than at later stages. The proportion of label in root during development varied and was considerably less during later stages than at 45 DAS. The proportion of label in pod (kernel + pod wall) did not vary much among the three varieties at 61 and 82 DAS, while at 45 DAS Spanish Improved had higher label than both the mutants. However, at 82 DAS the mobilization of photosynthate for kernel development in TG-16, was almost twice than in Spanish Improved and 50% higher than in TG-1. Discussion

As a result of y-irradiation variability has been induced in groundnut c. v. Spanish Improved with respect to yield (PATIL 1971), oil quality (SHARMA et al. 1981) and dark CO2 fixation and nitrogen fixation (LODHA et al. 1983). Mutant TG-16 has substantially higher yield than parent Spanish Improved, while TG-1 and TG-16 have much bolder kernels than the parent. The dry matter production rate in TG-1 was substantially higher than TG-16 and Spanish Improved. The higher dry matter production in TG-1 was due to higher photosynthetic rate during early crop growth. Although the photosynthetic rate (C0 2 fixed per g leaf per h) in TG-16 was comparable to that of TG-1, the dry matter production was low due to lower total capacity for photosynthesis as judged by CO 2 fixed per plant. The primary reaction responsible for higher rate of CO 2 fixation in the mutants is not known. However, one of the mutants (TG-1) also showed higher chlorophyll content, which was observed from the early stages of germination. Although the total dry matter production in TG-1 was substantially higher, its harvest index was substantially lower than that of TG-16. This appears to be due mainly to poor mobilization of photosynthate towards kernel development at later stages and also to greater partitioning of photosynthate for vegetative growth. In addition, the significantly higher total photosynthetic capacity of TG-16 compared with TG-1 and Spanish Improved at 82 DAS, when kernel development is at a peak, could be the main contributory factor to higher kernel yield. The mobilization of photosynthate to nodules was also higher in both TG-1 and TG-16 than Spanish Improved, but the differences between the mutants did not follow a particular trend. The higher nitrogen fixation rate in both TG-1 and TG-16 (LODHA et al. 1983) is thus supported by a greater mobilization of photosynthate to nodules. However, the total proportion of the photosynthate translocated to nodules to sustain nitrogen fixation was less than 8 % at a stage when maximum nitrogen fixation occurred. This indieates that a very small proportion of photosynthate is utilized for meeting energy demands for nitrogen fixation. Probably mobilization of nitrogen reserves from senescing

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leaves (LODHA et al. 1982) contributed more towards kernel development in TG-16 at the later stages when there was a greater demand of photosynthate for kernel development and nitrogen fixation rate was low. The close relationship of photosynthesis and translocation of photosynthate with nitrogen fixation as observed in the present study has also been observed in soybeans (LAW1.\' and BRUN 1974) and pea (LAWRIE and WHEELER 1974 and BETHLENFALVAY and PHILLIPS 1978). The rcsults presented in the present investigation indicate the superiority of mutant TG-l in terms of photosynthetic capacity over TG-16 and Spanish Improved, and offer scope for further increases in yield by improvements in harvest index through genetic manipulation. The study also suggest that besides the total photosynthetic capacity of plant, the higher mobilization of photosynthate and nitrogen reserves to developing kernels is important for obtaining higher yield. References AR:<"ON, D. 1.: Copper enzymes in isolated chloroplasts - polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). BETJlLElI"FAL"AY, G. J. , a.nd PIIlLLIPS, D. A.: InteJaction between symbiotic nitrogen fixation, combined N application and photosynthesis in Pisuln sativuln. Physiol. Plant. 42, 119-123 (1978). HARDY, R. W. F., and HAVELKA, U. D.: Nitrogen fixation research: A key to world food? Science, 188, 633-643 (1975). HAVELKA, U. D., and HARDY, R. W. F.: N2 (C 2H 2) fixation, growth and yield response of field grown peanut (Arachis hypogaea L.) when grown under ambient and 1,500 ppm CO 2 in the foliar canopy. Agron. Abstr. p. 72 (1976). LAWN, R. J., and BRUN, W. A.: Symbiotic nitrogen fixation in soybeans. L Effect of photosynthetic source - sink manipulations. Crop Sci., 14, 11-16 (1974). LAWRIF. , A. C., and WHEELER, C. T.: The effects of flowering and fruit formation on the supply of photosynthetic. assimilates to the nodules of Pisum. salivum L. in relation to the fixation of nitrogen. New Phytol. 73, 1119-1127 (1974). LODHA, 1\1.1., SHARMA, N. D., JORARI, R. P., MEliTA, S. L., and NAIK, 1\'1. S.: Nitrogen fixation and hydrogen metabolism in the developing nodules of groundnut mutants. Proc. Symp. Biologienl Nitrogen Fixation, IARI, New Delhi -12 (Feb. 25- 27, 1982), pp. 216-228. LODlf.\, M. 1.., JOIURI, R. P., SHARMA, N. D., and 1ItmTA, S. L.: Nitrogen fixation in relation to dark CO 2 fixation in developing nodules of Arachis hypogaea L. mutants. Ind. J. expt. BioI. 21, 629632 (1983). PATIL, S. H.: Use of induced mutations in breeding for quantitative characters of groundnut. Proc. Int. Symp. on use of Isotopes and Radiation in Agricnlture and Animal Husbandry Research, NRL, IARI, New Delhi - 12 (Nov. 30- Dec. 2, 1971), pp. 154- 163. SAl\TII .\ , I. ~L, MEHTA, S. 1., KOUNDAL, K. R., and SINHA, S. K.: Photosynthesis and translocation nlte in lIigh lysine mutant barley. Phytochemistry. 21, 1183-1187 (1982). SII .\R~fA, N. D., MEHTA, S. L., PATIL, S. H., and EGGUM, 13. 0.: Oil and protein quality of groundnut mutants. Qual. Plant. 31, 85-90 (1981).

Receired August 20, 1984, revised form accepted December 12, 1984 Author's address: 1\1. L. LODIIA, Division of Biochemistry, Indian Agrieultural Research Institute, New Delhi-110012, India.