Revista Brasileira de FisiologiaVegetal, 10(1):13-18, 1998

PHOTOSYNTHETIC POTENTIAL OF FIVE GENOTYPES OF COFFEA CANEPHORA PIERRE1

Eliemar Campostrini2 and Moacyr Maestri3
Departamento de Biologia Vegetal, Universidade Federal de Viosa, Viosa, MG, 36571-000, Brazil.

ABSTRACT - Twelve-month-old potted plants of Coffea Canephora Pierre, grown under field conditions, were used. Five genotypes were obtained from selected matrices for low, average and high productivity. The five genotypes were found to have similar quantum yield. The average productivity clones showed higher maximum net photosynthetic potential which was unrelated to productivity. No relation between leaf physiological characteristics and productivity was observed. All genotypes presented the same efficiency of the photosynthetic apparatus, as evaluated by fluorescence emission parameters. Additional index terms: coffe, fluorescence, photosynthesis, productivity

POTENCIAL FOTOSSINTTICO DE CINCO GENTIPOS DE COFFEA CANEPHORA PIERRE

RESUMO - Empregaram-se plantas de Coffea canephora Pierre de 12 meses de idade crescidas em condies de campo. Utilizaram-se cinco gentipos, escolhidos de matrizes com baixa (clones 12 e 25), mdia (clones 49 e 123) e alta produtividade (clone 128). Verificou-se que os cinco clones apresentaram o mesmo comportamento quanto ao rendimento quntico. Houve uma tendncia de os clones 49 e 123 apresentarem maiores valores para a fotossntese lquida potencial mxima (Pnmax), mas sem relao direta com a produtividade. No foi encontrada nenhuma relao entre as caractersticas fisiolgicas da folha com as produtividades. Todos os gentipos apresentaram a mesma eficincia do aparelho fotossinttico, avaliada pela emisso de fluorescncia. Termos adicionais para indexao: caf, fluorescncia, fotossntese, produtividade

1 2

Received 03/11/1997 and accepted 15/05/1998 Eng. Agr., D.Sc. Universidade Estadual do Norte Fluminense, CCTA, Campos dos Goytacazes, RJ, 28015620, Brasil, e-mail: mazinho@uenf.br. Eng. Agr., M.Sc., PhD., Prof. Titular, Universidade Federal de Viosa, Viosa, MG, 36571-000

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14 INTRODUCTION

Campostrini & Maestri The leaf area was determined by a leaf area meter (Model Delta-T). The specific leaf area was obtained by dividing the leaf area by its dry matter. All leaf measurements were made using one or both leaves of the third completely expanded leaf pair on a plagiotropic branch counting from the apex.

Productivity is one of the targets in coffea breeding programs. Yield variation among cultivars can be associated with the plant morphological characteristics (architecture), growth pattern (growth rates and assimilate partitionning), photosynthetic efficiency and reproductive activity. Knowledge about such characteristics is a necessary step towards establishing useful indices and criteria for predicting the yielding potential of new genotypes. The coffee plant is a perennial crop, so the use of traditional methods for its breeding demands time and does not always achieve satisfactory results. Therefore, it is necessary to develop a reliable methodology to predict the yield capability of coffee genotypes at the initial stages of plant development. This kind of research would save substantial amounts of time, space and resources, making it possible to reject inadequate plant material at a much earlier stage, instead of taking the ten to twelve years that are spent in the traditional practices (Antunes & Carvalho, 1957; Carvalho et al., 1984). So, information on the causes of the differential behavior of genotypes is essential for prediction of yield potential, or ecological adaptation of Coffea spp. Research relating coffee plant productivity to mor phological, biochemical or photosynthetic leaf characteristics in either no-existent or rare. The work reported here sought to determine the possible relationships between the productivity of five Coffea canephora genotypes and some physiological characteristics associated with the photosynthetic capacity of vegetatively propagated young plants.

Foliar pigments assay

The chlorophyll concentration was determined according to Harbone (1991) using three leaf discs. The extractions were carried out in a porcelain mortar with 80% ketone to which calcium carbonate was added. The extract was filtered for separation and the final volume was adjusted to 100 ml. The ketone extract was measured spectrometrically at 646 and 663 nm. The absorbances were used to determine the contents of chlorophyll a, b and total. The carotenoids were extracted and quantified according to the methodology described by Duke & Kenyon (1986) and modified by Argenta (1990). Two leaf discs each were homogenized with 12 ml of potassium hydroxide in methanol (6% mass/volume). The homogenate was centrifuged at 5000 x g for five minutes, resuspended twice, and the supernatant liquid was transferred to a test tube. The separation of the pigments was achieved by partitioning the extract with 14 ml of petroleum ether under vigorous shaking. The epiphase was collected with an automatic pipette and the extract was partitioned twice. After adjusting the final volume of the ether phase, the spectrophotometric absorbance was determined at 450 nm. The carotenoid content was quantified, using the coefficients of the molar absorption according to Goodwin (1976). Total leaf nitrogen was determined according to the method described by Umbreit et al. (1972), in which an acid digestion with oxygen peroxide was used. Nessler reagent was added to the extract and the spectrophotometric absorbance was determined at 420 nm. The standard curve was obtained using ammonium sulphate, in the range 10 to 50 g of nitrogen. The total protein content was determined by multiplying the nitrogen values obtained by a factor of 6.25.

MATERIALS AND METHODS

Plant material and growth conditions
Five genotypes of coffee (Coffea canephora Pierre) were used: clones 12 and 25, 49 and 123, and 128, of low ( 2,272 kg ha-1), average (3,300 kg ha-1) and high (4,159 kg ha-1) productivity, respectively. The clones were obtained from Empresa Capixaba de Pesquisa Agropecuria (EMCAPA), selected competition experiments on the basis of average yields over four years, in Marilndia-ES, Brasil. Plants of approximately 12 months were obtained by vegetative propagation, and grown in black polythene bags (0.32 x 0.60 m), filled with 15 L of soil mixture (soil and manure in a ratio of 70% to 30%). The soil was fertilized and the pH corrected according to Comisso de Fertilidade do Solo do Estado de Minas Gerais (1989). Nitrogen fertilizers were added as a side dressing and a soluble micronutrient fertilizer was sprayed every 45 days throughout the experiment. The nitrogen sources were urea and ammonium sulphate alternately. A mixture of zinc sulphate 0,2%, potassium chloride 0,2%, boric acid 0,2%, copper oxychloride and Agril-320 0,02% (wetting agent), was used as the micronutrient solution. In the dry period, the soil moisture was maintained by regular irrigation.

Measurement of photosynthetic oxygen evolution

The evolution of photosynthetic oxygen (potential photosynthesis) of each leaf disc was determined using a gaseous phase Clark electrode (Model Leaflab 2, Hansatech, England), connected to the sample chamber LD2 also used to measure fluorescence. The measurements were carried out at 35C under a halogen lamp bulb of 250 W with a photon flux density up to 1130 mol m-2 s-1 at the leaf disc, measured with a photometer (LI-1000, LI-COR, USA). The rates of oxygen evolution were obtained after the methodology described by Delieu & Walker (1983). The potential photosynthesis rate versus the photon flux densitity was obtained by changing the distance between the sample chamber and the light source. The values of potential net photosynthesis rate were adjusted according to the model.

Specific leaf area

Pn = Pbmax [1 - e-( I Pbmax)] - Rd

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Photosynthetic potential where Pn is the potential net photosynthesis rate; I is the photon flux density; is the initial slope of the curve Pn x I; Pbmax is the asymptotic value of Pb (maximum potential photosynthetic rate) when I tends to infinite and Rd is the dark respiration rate (Thornley, 1976).

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Measurement of fluorescence emission

The chlorophyll fluorescence emission was monitored with a Hansatech fluorescence (Model Leaflab 2, Hansatech, England) detector coupled to an LD2 leaf chamber, which was illuminated with a high light flux density lamp (3000 mol m-2 s-1 over the spectrum of 640680 nm and emission peak at 660 nm). An interference filter of 740 10 nm was placed between the leaf disc and the fluorescence detector to allow the partial passage of the fluorescence signal while excluding the excitation light. Leaves from the third completely expanded pair, counting from the apex on a plagiotropic branch, were detached and placed into black plastic bags kept in the dark for 30 minutes so that all reaction centers would be in the open state (non-reduced quencher). For the signal detection a leaf disc of 1,000 mm2 was cut out from such leaves and placed into the sample chamber at 35C. Afterwards, it was subjected to actinic excitation light and the signal was recorded at a speed of 100 mm.min-1. At that speed maximum (Fp) and terminal (Ft) fluorescence were detected. From the Fp and Ft values, the ratio Fp/Ft (Siffel et al., 1988), the time (t1/2) to reach the level (Fp-Ft)/2, the ratio of fluorescence decrease (Rfd = (Fp-Ft)/Ft) (Epron and Dreyer, 1990), the extinction potential (Fq = Fp-Ft) and the maximum rates of fluorescence decrease after the peaks P (r2) and M(r3) (Hetherington and Smille, 1982), were calculated.

sis values since it was not possible to obtain uniform photon flux densities for all replicates. The equation coefficient (quantum yield), dark respiration (DR), compensation irradiance (CI) and oxygen evolution asymptotic value at light flow density tending to infinite (Pnmax) were compared using the t test of Student. Statical analysis of all data were performed with the Sistema de Anlises Estatsticas e Genticas (SAEG/ Universidade Federal de Viosa/Brasil).

RESULTS AND DISCUSSION

Specific leaf area and photosynthetic pigments
The most productive clone (128) showed a specific leaf area smaller than the others (Table 1) but the group of medium productivity (49 and 123) presented the highest values, differing significantly from the less productive ones (12 and 25) so that the pattern of observed values were not consistent. The protein content was not significantly different among the genotypes studied (Table 1). The concentration of the pigments, in the leaves, in general, decreased from the less productive clone to the most productive. The concentration of carotenoid increased as the concentration of chlorophyll increased (Table 2). Maximum values of total chlorophyll concentration (Table 2) expressed per unit of area for clones 25 and 12 were higher than those reported by Goldberg et al. (1988). Similar results were reported by Gabrielsen (1948) who observed that the chlorophyll concentration reaches 0.4-0.5 mg m-2, and an increase in concentration beyond this threshold does not change the rate of photosynthesis. So almost certainly the chlorophyll concentration was not a limiting factor for the coffee genotypes studied (Tables 2 and 3). An increase of chlorophyll concentration from 0.17 to 0.7 mg m -2 increased both the incident quantum yield and the leaf absorption

Experimental design
A randomized complete block design was used with six replicates, with individual plants being an experimental unit. Analysis of variance with group comparisons based on ortogonal contrasts was done for the following groups: very productive (clone 128), median productivity (clone 49 and clone 123) and low productivity (clones 25 and 12). The quantum yield values and maximum photosynthesis were obtained by adjusting a median curve (Pb x I). The curves were drawn by using the average values of irradiance replicates and of net photosynthe-

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Campostrini & Maestri

coefficient by 28% in Hedera canariensis, but a change from 0.5 to 0.7 mg m-2 did not (Bjrkman & Demming, 1987). For H. canariensis values of chlorophyll content above 0.6 mg m-2 had a small interference in the optical leaf properties with little influence on the quantum yield value. In the present study total chlorophyll was higher than 0.6 mg m-2 for all five clones (Table 2). Table 3 shows the concentrations of chlorophyll a and b in the selected clones, expressed as either mg m-2 and mg kg-1 DM. The results show that there is no strong correlation between concentration of chlorophyll and productivity.

Potential net photosynthesis in coffee leaf discs in relation to irradiance.

Analysis of potential net photosynthesis curves (Pn) as a function of the photon flux density (Figura 1, as an example) based on the relevant parameters summarized on Table 4, showed differences among the five genotypes. Clones 49 and 123 presented maximum potential net photosynthesis values higher than those of the other clones. However, the value for clone 49 was not significantly different to that of clone 12. Clone 25 had the lowest Pnmax value. The highest values of irradiance compensation and dark respiration were those observed for clones 12 and 123, whereas clone 25 tended to have the lowest value. Photosynthetic quantum yield () can be defined as the molar quantity of O2 evolved or CO2 fixed, per mol of photons absorbed by the photosynthetic apparatus. At low photon flux density, the quantum yield is constant and maximum and is a measure of the efficiency of light conversion to stable products (Bjrkman & Demming, 1987).

Considering that at least eight photons are required for the yield of an oxygen molecule, the maximum theoretical quantum yield is 0.125. The values determined in this experiment were 74, 66, 64, 68 and 71% of the theoretical value for clones 25, 12, 123, 49, 128, respectively (Table 4). These results are similar to those of Bjrkman & Demming (1987), who found in 37 C3 species an average quantum yield of 0.089. Our results demonstrated that the genotypes studied may have the same capability of light conversion to stable products. For the genotypes evaluated in this paper the dark respiration ranged from 5.5 to 8.0% of maximum poten-

R. Bras. Fisiol. Veg., 10(1):13-18, 1998.

Photosynthetic potential

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tial net photosynthesis (Pnmax). According to Ceulemans & Saugier (1991) dark respiration accounts for to 7-10% of the potential net maximum photosynthesis. Coffee plants have shown rates of dark respiration that represented about 40% of the potential net maximum photosynthesis at 16-19 days, 15% at 40 days and 10%, at 55-60 days after emergence (Frischknecht et al., 1982). The values observed, therefore, are in agreement with such reports. Clone 25 showed values of DR and CI lower than those of the others clones tested. However, the values of the all the characteristics measure here did not show a consistent relationship to the productivity of the clones.

Fluorescence emission and maximum photosynthetic rate

Regarding to the fluorescence induction kinetics only maximum fluorescence (Fp) values did shoe significant differences among the genotypes studied (Tables 5 and 6). The most productive clone differed significantly from the others in maximum fluorescence (Fp) (Table 5). According to Siffel et al . (1988), the F p level of the fluorescence indution curve shows that the primary electron acceptor in PSII (Qa) is totally reduced with a minimum consumption of the radiant energy absorbed. The Fp level results from a faster reduction of Qa rather than from its reoxidation (Baker & Bradbury, 1981). Changes at the Fp level can be caused by decreased Qa reoxidation (increase in Fp) or low oxidation rate of the water molecule (decrease in Fp) (Toivonen & Vidaver, 1988). Therefore the 128 clone would be somewhat defficient when compared to the others considering its

water molecule oxidation rate, characterized by the low Fp value, which is not consistent with its maximum potential photosynthetic rate (Table 5). Possibly the low Fp value is due to the high Qa reoxidation rate efficiency in PSI activity, ATP generation (photophosphorilation) and NADPH+ and/or CO2 assimilation reactions, such factors being responsible for the regulation of Qa reoxidation state (Stitt, 1986; von Caemmerer & Farquhar, 1981; Walker, 1981). Another factor that is likely to reduce the Fp value is the length of the dark period before the fluorescence deter mination (Ireland et al ., 1984). Nevertheless this is not the case in this article since genotypes were maintained in the dark for equal periods of time. According to Miranda et al. (1981), the Fp value is proportional to the amount of chlorophyll molecules present in the leaf tissue. Data in Table 2, concerning to the total chlorophyll concentration expressed in units of dry matter, are in agreement with that statement. The ratio of Fp/Ft did not show significant differences among the genotypes studied and all genotypes presented quite high values (Table 5), similar to the results of Almeida (1993), who used high yield genotypes of Coffea arabica. As the ratio Fp/Ft is considered as a measure of the use of the radiant energy absorbed in photosynthesis and the high values obtained for the ratio in mature leaves mean that there had been an improvement in the use of the radiant energy absorbed and a leveling up of the reactions in Calvin cycle (Siffel et al. 1988). Therefore the ratio of Fp/Ft in these genotypes probably does not act to limit these processes. The r2 and r3 rates are associated with the electron transfer taking place between photosystem II and I

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emission from photosystems I and II. Biochemistry Biophysic Acta, 653: 542-51, 1981. BJRKMAN, O. & DEMMIG, B. Photon Yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta, 170: 489-504, 1987. CARVALHO, A.; MONACO, L.C.; COSTA, W.M. de & MEDINA FILHO, H. P. Variabilidade na produo em prognie do cafeeiro Mundo Novo. Bragantia, 43: 509-17, 1984. CEULEMANS, R. J. and SAUGIER, B. Photosynthesis. In: (RAGHAVENDRA, A. S. ed. Physiology of trees). New York, John Wiley, 1991. p. 21-50. COMISSO DE FERTILIDADE DO SOLO DO ESTADO DE MINAS GERAIS. Recomendaes para o uso de corretivos e fertilizantes em Minas Gerais, 4a aproximao, Lavras. 1989. DELIEU, T. J. & WALKER, D. A. Simultaneous measurement of oxygen evolution and chlorophyll fluorescence from leaf pieces. Plant Physiology , 73:534-41, 1983. DUKE, S. O. & W. H. KANYON. Effects of dimethazone (FMC 57020) on chloroplast development. II. Pigment synthesis and photosynthetic functions of cowpea (Vigna unguiculata L.) primary leaves. Pesticid Biochemistry Physiology., 1:11-16. 1986. EPRON, D. & E. DREYER. Stomatal and non stomatal limitation of photosynthesis by leaf water deficits in three oak species. A comparison of gas exchange and chlorophyll a fluorescence data. Annals Science of Forestry, 47:435-50, 1990. FRISCHKNECHT, P. M., ELLER, B. M. & BAUMANN, T. W. Purine alkaloid formation and CO 2 gas exchange in dependence of development and environmental factors in leaves of Coffea arabica. L. Planta, 156:295-301, 1982. GABRIELSEN, E. K. Effects of different chlorophyll concentrations on photosynthesis in foliage leaves. Physiologia Plantarum, 1:5-37, 1948. GOODWIN, T. W. Chemistry and biochemistry of plant pigments , London, Academic Press, 1976. GOLBERG, A. D.; RENARD, C.; LANNOYE, R. & LEDENT, J. F. Effects and after-effects of water stress on chlorophyll fluorescence transients in Coffea canephora Pierre and Coffea arabusta Capot and Ak Assi. Caf Cacao Th, 32:11-16, 1988. HARBORNE, J. B. Phytochemical methods - A guide to modern techniques of plant analysis, London, Chapman & Hall, 1991. HETHERINGTON, S. E. & SMILLIE, R. M. Humidity-sensitive degreening and regreening of leaves of Borya nitida Labill as followed by changes in chlorophyll fluorescence. Australia Journal of Plant Physiology, 9:587-99, 1982. IRELAND, C. R., LONG, S. P. & BAKER, N. R. The relationship between carbon dioxide fixation and chlorophyll a fluorescence during induction of photosynthesis in maize leaves at different temperatures and carbon dioxide concentrations. Planta, 160: 550-58, 1984. MIRANDA, V.; BAKER, N. R. & LONG, S. P. Limitations of photosynthesis in different regions of the Zea mays leaf. New Phytologist., 89: 179-90, 1981. SIFFEL, P.; DURCHAN, M.; KUETON, J.; ONDREJ, M. & SESTAK, Z.. Photosynthesis in regenerants of tobacco transformed by plasmids of Agrobacterium. II. Fluorescence emission spectra and age induced changes in slow fluorescence induction. Photosynthetica , 22: 214-20, 1988. STITT, M. Limitation of photosynthesis by carbon metabolism. I. Evidence for excess electron transport capacity in leaves carrying out photosynthesis in saturating light and CO2. Plant Physiology , 81: 1115-22, 1986. THORNLEY, J. H. M. Mathematical models in plant physiology, London, Academic Press, 1976. TOIVONEN, P. & VIDAVER, W. Variable chlorophyll a fluorescence and CO2 uptake in water-stressed white spruce seedlings. Plant Physilogy, 86: 744-48, 1988. UMBREIT, W. W.; BURRIS, R. H. & STAUFFER, J. F. Manometric and biochemical techniques, Minnesota, Burgess Publishing, 1972. VON CAEMMERER, S. & FARQUHAR, G. D. Some relationships between the biochemistry of photosynthesis and the biochemistry of leaves. Planta, 153:376-78, 1981. WALKER, D. A. Secondary fluorescence kinetics of spinach leaves in relation to the onset of photosynthetic carbon assimilation. Planta, 153: 273-78, 1981.

(Hetherington & Smille, 1982). A decrease in the electron flux through PSI due to the reduction in the transfer system on CO 2 fixation can cause a drop in the reoxidation rate of PSII acceptors as show by decrease in r2 and r3 and increase in the yield of variable fluorescence at the steady state (Hetherington & Smille, 1982). Since there are no significant difference in the values for r2 and r3 (Table 6), which were relatively high and similar to those reported by Almeida (1993), it is likely that the electron flux through PSI, the transfer to CO2 and the receptors reoxidation of receptors are under nonlimiting conditions. There were no significative differences among the clones for values of Rfd and t1/2 (Table 6), revealing stability in the extinction mechanisms which is indicative of stability in the electron flux for the primary acceptors and in the building up of the transthylacoidal electrochemical gradient. The clone 128 differed from the others by presenting a lower value for the fluorescence extintion capability Fq. Once Fq=Fp-Ft and the rates r2 and r3 remained constant, is quite likely that the low Fq value observed is due to the low Fp value which, according to Miranda et al. (1981), reflects the amount of chlorophyll in the tissue. Clone 128 was the only one that showed the lowest chlorophyll content on the basis of dry weight (Table 2). In conclusion, yield potential of C. canephora was not related to content of photosynthetic pigments, and photosynthetic capacity either associated to biochemical or photochemical reactions. The operation of the photosynthetic machinery at its full potential did not seem to control yield, the genotypical differences being due to other regulating conditions, such as diffusive resistance, plant architecture and light interception, and developmental processes.

ACKNOWLEDGMENTS
The authors gratefully acknowledge to Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES) for a Master fellowship to the first author and to Conselho Nacional de Pesquisa Cientifica e Tecnolgica (CNPq) for a fellowship to the second author. To Empresa Capixaba de Pesquisa Agropecuria (EMCAPA) for providing the plant material and for financial support. To Fundo de Amparo Pesquisa do Estado de Minas Gerais (FAPEMIG) for research grant.

REFERENCES
ALMEIDA, A. A. F. de Emisso de fluorescncia e evoluo de oxignio fotossinttico em Coffea arabica L. durante um ciclo de desidratao/reidratao., Viosa, Universidade Federal de Viosa, 1993. Tese de Doutorado ANTUNES, H. P. & CARVALHO, A. Melhoramento do cafeeiro. XI Anlise de produo de prognie e hbridos de Bourbon Vermelho. Bragantia, 16: 175-95, 1957. ARGENTA, L. C. (1990). Efeito do clomazone sobre alguns aspectos bioqumicos e fisiolgicos em plantas. Viosa, Universidade Federal de Viosa, 1990. Tese de Mestrado. BAKER, N. R. & BRADBURY, M.. Analysis of the slow phases of the in vivo chlorophyll fluorescence induction curve. Changes in the redox state of photosystem II electron acceptors and fluorescence

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