The effect of chilling on the photosynthetic activity in coffee (Coffea arabica L.) seedlings: The protective action of chloroplastid pigments

Efeito do resfriamento na atividade fotossintética em plântulas de café (Coffea arabica L.): a ação protetora dos pigmentos cloroplastídicos

The effect of chilling on the photosynthetic activity in coffee (Coffea arabica L.) seedlings. The protective action of chloroplastid pigments

Jurandi Gonçalves de Oliveira^1*, Pedro Luis C.A. Alves² and Antonio Celso Magalhães³

¹ Laboratório de Melhoramento Genético Vegetal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense, CEP 28013-600, Campos dos Goytacazes, RJ, Brasil;

² Departamento de Biologia Aplicada à Agropecuária, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual de São Paulo, CEP 14870-000, Jaboticabal, SP, Brasil;

³ Departamento de Fisiologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, 13083-970, Campinas, SP, Brasil;

* Corresponding author: [e-mail] Received: 18/01/2002, Accepted: 08/08/2002

Coffea arabica is considered to be sensitive to low temperatures, being affected throughout its entire life cycle. Injury caused by chilling (low temperatures above zero degree centigrade) is characterized primarily by inhibition of the photosynthetic process. The objective of this work was to evaluate the role of photosynthetic pigments in the tolerance of coffee (C. arabica L.) seedlings to chilling. The evaluation the photosynthetic activity was made by emission of Chl a fluorescence at room temperature (25 ºC) in vivo and in situ, using a portable fluorometer. The pigment content was obtained by extraction with 80 % acetone, while estimation of membrane lipid peroxidation was determined by measuring the MDA content in leaf tissue extracts. The results indicated a generalized reduction in the quantum yield of PSII when the seedlings were maintained in the dark. The reduction occurred in the seedlings submitted to chilling treatment as well as in the control ones. This demonstrates that not only chilling acts to cause an alteration in PSII. It is possible that the tissue storage reserves had been totally exhausted, with the respiratory rate exceeding the photosynthetic rate; the later was nil, since the seedlings were kept in the dark. The efficiency in the capture, transfer and utilization of light energy in PS_II photochemical reactions requires a sequence of photochemical, biochemical and biophysical events which depend on the structural integrity of the photosynthetic apparatus. However, this efficiency was found to be related to the protective action of chloroplastid pigments, rather than to the concentration of these pigments.

Key words: chlorophyll a fluorescence, coffee, lipid peroxidation, photosynthetic pigments.

Efeito do resfriamento na atividade fotossintética em plântulas de café (Coffea arabica L.): a ação protetora dos pigmentos cloroplastídicos: A espécie Coffea arabica é considerada sensível às baixas temperaturas, podendo ser afetada em todo o seu ciclo. A injúria pelo frio (temperaturas baixas acima de zero) se caracteriza, principalmente, pela inibição do processo fotossintético. Este trabalho teve como objetivo avaliar a participação dos pigmentos fotossintéticos na tolerância de plântulas de cafeeiro (C. arabica L.) à temperatura de resfriamento. A avaliação da atividade fotossintética foi feita através da emissão de fluorescência da clorofila a à temperatura ambiente (25 ºC) in vivo e in situ, usando um fluorímetro portátil. O conteúdo dos pigmentos foi obtido a partir da extração com acetona 80 % e a peroxidação lipídica estimada a partir das medidas do conteúdo de MDA em extratos do tecido foliar. Os resultados indicaram uma redução generalizada na eficiência quântica do PSII quando as plântulas foram mantidas no escuro. A redução ocorreu tanto nas plântulas submetidas ao tratamento de resfriamento, quanto no material controle. Isso demonstra que não apenas a temperatura de resfriamento estaria atuando como causa dessas alterações na atividade do PSII. É provável que tenha ocorrido um esgotamento das reservas do tecido, com a taxa respiratória excedendo à fotossintética, já que essa era nula, uma vez que a energia de excitação esteve comprometida pela permanência das plântulas no escuro. A eficiência na captura, transferência e utilização da energia luminosa em reações fotoquímicas no PSII, requer uma seqüência de eventos fotoquímicos, bioquímicos e biofísicos intimamente dependentes da integridade estrutural do aparelho fotossintético. Sendo que essa eficiência se mostrou mais dependente da ação protetora dos pigmentos cloroplastídicos do que propriamente da concentração desses.

Palavras-chave: café, pigmentos fotossintéticos, fluorescência da clorofila a, peroxidação de lipídios.

INTRODUCTION

The economic exploitation of coffee plants, Coffea arabica L., is limited to regions where the average annual temperature varies between 18 and 22 ºC (Bauer et al., 1985). This species is considered to be sensitive to low temperatures (Levitt, 1980; Bauer et al., 1985), presenting decreased growth and stunting (Smille et al., 1988); coffee plants are also prone to injuries caused by low temperatures (Levitt, 1980). Studies involving the mechanisms that modulate tolerance or susceptibility to chilling in coffee are rare (Bauer et al., 1985; Queiroz et al., 1998).

The ability of plants to tolerate chilling stress has been studied as an important ecophysiological adaptation phenomenon and is the subject of intense research in the field of plant biology with many agricultural applications (Bauer et al., 1985; Smillie et al., 1988; Alonso et al., 1997; Queiroz et al., 1998).

In general, chilling injury is characterized by inhibition of the photosynthetic process (Hodgson and Raison, 1989), by alteration of cellular functions (Graham and Patterson, 1982) and, in extreme conditions, by deterioration of these functions, which may result in plant death. The activity of certain antioxidant enzymes may be intensified in leaves acclimatized to low temperatures (Schöner and Krause, 1990). There could also be an alteration in the composition of leaf pigments conferring greater capacity to tolerate the stress effects. Some chilling injuries present evident symptoms, such as leaf chlorosis, suggesting destruction of photosynthetic pigments (Muruyama et al., 1990).

Recent studies indicate that cold stress is expressed when peroxidation of cellular membrane lipids is triggered, causing alterations in fluidity (Merzlyak, 1994; Alonso et al., 1997; Queiroz et al., 1998). The most important biochemical events associated with modifications in membrane fluidity include changes in enzyme activity and loss of cell compartments. The degradation of polyunsaturated fatty acids by peroxidation produces not only peroxide ions but also MDA. Therefore, changes in the MDA concentration have been used as lipid peroxidation indicators and, consequently, of the chloroplastidic membrane integrity (rich in polyunsaturated fatty acids) in plants submitted to stress (Dhinsda et al., 1981; Hodgson and Raison, 1991; Jouve et al., 1993; Qiu and Liang, 1995).

The photosystem II (PS_II) reaction centers are obviously well protected against adverse environmental effects (low temperature, excess light, water and nutritional deficits, etc.) and are only affected when this protection is overcome. The stress effect on photosynthetic activity is manifested in many different ways, leading to efficiency loss in the photosynthetic apparatus, such as decreased use of photons for NADPH, ATP and carbohydrate production.

Recent proposals to study the reaction of plants submitted to extreme temperature conditions consider the use of less invasive techniques that allow the extrapolation of results to natural conditions. In this research, the effects of chilling on the photosynthetic activity of coffee seedlings were studied by fluorescence induction of Clo a, in vivo, which yields information about the functioning of the photosynthetic apparatus (Schreiber, 1983; Krause and Weis, 1984, 1991; Krause, 1994; Govindjee, 1995). The use of this technique has contributed greatly to the understanding and monitoring of environmental stress effects on photosynthesis (Govindjee, 1995) or even for the selection of cultivars less susceptible to photoinhibition induced by low temperatures (Bjorkman and Demming, 1987).

The objective of this study was to evaluate the participation of photosynthetic pigments in the tolerance of coffee seedlings (C. arabica L.) to chilling.

MATERIAL AND METHODS

Plant material and treatments:C. arabica L. seedlings cv. 'Catuaí Amarelo' were used. The seeds were provided by the Agronomic Institute of Campinas-IAC. Seedlings with expanded cotyledons were transplanted into plastic pots (500 mL) and transferred to a growth chamber under low light intensity (80 mmol.m^-2.s^-1), with an 8 h photoperiod. The temperature was controlled (25 ºC / 20 ºC, day/night) until the application of the chilling treatments (3 weeks). One week after transfer, the seedlings started to accumulate chlorophyll pigments; after three weeks the leaves were completely green. During this period, the seedlings were fertilized once a week with nutrient solution (Hoagland and Arnold, 1951) and soil humidity was maintained by daily watering. The position of the plastic pots in the growth chamber was changed once a week, to allow equal light interception by the cotyledons and to minimize any differences in PPFD on the plants.

Seedlings with one pair of cotyledons were then transferred to growth chambers (Forma Scientific Inc., model 24) previously adjusted to the treatment temperature (10 ± 3 ºC ¾ chilling treatment and 25 ± 3 ºC ¾ control). The seedlings were kept in darkness for the duration of the test (twenty days).

Chl a fluorescence kinetics, pigment content and lipid peroxidation were evaluated by sampling at two and four day intervals, respectively, for both treatments, with four replications represented by four independent assays.

Chlorophyll a fluorescence kinetics: Chl a fluorescence was determined at room temperature (25 ºC) in vivo and in situ, using a portable fluorometer PEA (Plant Efficiency Analyser, Hansatech Instruments Ltd.). Seedlings were pre-adapted in the dark for 30 min before fluorescence determination. This 30 min period was sufficient for the oxidation of the entire photosynthetic electron transport system (Oliveira, 1995). Following exposure to saturating light (2,400 mmol.m^-2.s^-1) and complete "opening" of the system, the fluorescence peak emitted was considered as maximum fluorescence (F_M). The following variables of Chl a fluorescence were evaluated: initial fluorescence (F_o), F_M, variable fluorescence (F_V), the area above the fluorescence curve between the values F_o and F_M and the ratios F_V/F_M and F_V/F_o.

A mathematical relationship with the equations of the curves was made to express, by means of curves, the effects of the chilling treatment proportionally to the control. The resulting equation expresses the response of each variable after the application of the chilling treatment as a percentage of the control. An example of the mathematical relationship used is given below.

Control treatment Þ Y_co = a_co + b_cox + c_cox² (Eq. 1)

Chilling treatment Þ Y_ch = a_ch + b_chx + c_chx² (Eq. 2)

Chilling treatment as a percentage of the control

Þ Y_% = (Y_ch / Y_co) . 100 (Eq. 3)

Equation 3 was used to obtain the points plotted in the figures expressing the response of the variables in relation to the control (% of control). The curves were appropriately adjusted from these points.

Pigment content: The Chl a and chlorophyll b (Chl b) and carotenoid (C _x+c) concentration was determined using a 50 mg (FW) sample of leaf tissue collected and placed in a test tube containing 5 mL 80 % acetone. The sample was incubated at 70 ºC for 20 min, cooled in the dark and then the volume was adjusted to the initial 5 mL. Absorbance was monitored by a B390 Micronal spectrophotometer, at wavelengths of 470, 646 and 663 nm. Pigment concentration was calculated according to the procedure described by Lichtenthaler and Wellburn (1983). The concentration of Chl a and Chl b and C _x+c was expressed in relation to the concentration of these pigments in the plant tissue at the beginning of the assay. The best fitting curves using the polynomial model were used. The rate of pigment accumulation was also calculated.

Lipid peroxidation: The membrane lipid peroxidation was estimated by measuring the malondialdehyde (MDA) content, a by-product of lipid peroxidation in leaf tissue extracts. The MDA concentration was calculated from 150 mg (FW) of leaf tissue samples, using the procedure described by Dhindsa et al. (1981), adapted from Heath and Packer (1968). The extinction coefficient (x) for this assay was 155 mM^-1.cm^-1. The MDA concentration was expressed as a function of the period of acclimatization of the treatments and of the total pigment content. For the 10 ºC treatment, the data was adjusted in two straight lines, representing two distinct stages: the first one, from the beginning until the 12^th day and the second starting on the 12^th day until the end of the assay. For the 25 ºC treatment the data were adjusted as a single straight line.

RESULTS

The plants maintained at 25 ºC initially presented an increasing quantum yield of PS_II until the third day with a subsequent decrease. This greater initial yield also resulted in an increased flow of electrons, characterized by the initial decrease in the F_o curve (data not shown). From the third day on, the quantum yield in the control treatment decreased, apparently at a constant rate, until the end of the assay.

Plants submitted to chilling temperatures maintained the same quantum yield until the 6^th day. After that, the ratio F_V/F_M decreased until the end of the experiment. The values of F_V/F_M for the 10 ºC treatment plants were similar to those for the control between the 6^th and 12^th day, whereas after day 12 the reduction of F_V/F_M in the plants at 25ºC was greater, differing from the results for the plants treated at 10ºC (figure 1A).

The relative quantum yield (% relative to control) of seedlings submitted to chilling temperatures, after an initial decrease, increased from the fourth day on, surpassing the control after the 9^th day (figure 1B). With a decrease in the quantum yield of the control material, the relative yield of the plants treated at 10 ºC increased, surpassing, at the end of the assay, the yield of the control plants by more than 6% (figure 1B).

In relation to the F_o signal, it was observed that the increase in F_o was greater in the seedlings treated at 10ºC during the first 16 days of the assay, reaching the maximum around the 8^th day (figure 2A). Thereafter, the F_o increase in the seedlings submitted to the chilling temperature was lower (in relation to the increase in the control) and was exceeded by the control levels after the 16^th day.

F_M, in relation to the control (figure 2B), decreased until the 3^rd day, and then increased, surpassing the control values after the 6^th day and reaching a maximum around the 16^th day. The control treatment (25 ºC) results, for the first three days, differed from the chilling treated plants in that there was an increase in F_M. This response pattern was similar to that observed for the F_o variable.

Completing these observations, the plastoquinone pool response (represented by the area traced between the signals by F_o and F_M), was analyzed as a function of exposure time to the treatments (data not shown). It was determined that the significant difference between the data for the chilling and that for the control treatment, was due the plastoquinone pool which at 10 ºC remained low, while in the control treatment it increased, practically doubling the initial values. Considering the relative values (% of the control) of the plastoquinone pool (figure 2C), a constant decrease in these values was observed during the treatment at 10 ºC.

The F_V/F_o ratio showed a response pattern similar to the F_V/F_M ratio but with significantly higher values. At the end of the assay (19^th day), the F_V/F_o ratio for the control treatment decreased to approximately 20% of its maximum value; which was a much steeper decrease in comparison with the F_V/F_M ratio (approximately 6%). The chilling temperature treatment caused a decrease in the F_V/F_o ratio throughout the entire period of the study, with a smaller decrease in the F_V/F_M ratio, than the control treatment. Both ratios presented a high correlation regardless of the treatment (figure 2D).

For the seedlings maintained at 25 ºC, the accumulation in the content of all pigments presented a similar pattern. The pigment content as a function of time was mathematically adjusted using a 3^rd degree polynomial, describing three distinct phases. At the beginning of the treatment, the increase in the pigment content occurred very rapidly until the 7^th day, approximately three fold for the chlorophylls or even four fold for the carotenoids, in comparison with their initial contents. In the second phase, between the 7^th and the 15^th day, there were small increases in the contents. The third phase of the greening process occurred from the 15^th day until the end of assay (19^th day) (figures 3A, 3B and 3C). The increase rates in the pigment content in this 3^rd phase were greater when compared to the second phase but lower than the values observed during the first phase (table 1). During this period, the seedlings accumulated chlorophyll a and b as well as carotenoids, which were about 2/3 of the total content.

The concentration of carotenoids, which include carotenes and xanthophylls (figure 3C), presented the same pattern as the green pigments but with greater accumulation rates, especially during the 3^rd phase of the process (table 1).

The accumulation pattern for the chilling treatment was similar when the pigments were compared, describing an asymptotic curve, and stabilized on the 8^th day (figures 3A, 3B and 3C). The pigment content during this treatment showed an intense increase in the first 8 days (with average rates around 35 % per day for the chlorophylls and 33 % for the carotenoids), and then stabilized. At the end of this phase, the increase in pigment content was around 3.7 fold the observed values at the beginning of the treatment and close to that observed for the control treatment during the same period. The second phase began after the 8^th day, with reduced rates (rates of approximately 2 %), while the accumulation continued in the control seedlings, especially by the 19^th day (figures 3A, 3B and 3C).

A decrease in MDA was observed in the control treatment, with decreasing rates practically throughout the entire period of the experiment. The chilling treatment resulted in a less intense decrease. In spite of the stress conditions (10 ºC) to which the plants were subjected, the level of lipid peroxidation decreased during the treatment period.

The plants submitted to chilling stress presented, basically, two phases in the lipid peroxidation level (figures 4A, 4B). The first phase ended on the 12^th day of treatment. During this period, the MDA concentration was practically unaltered, which corresponded to the stabilization in the levels of chloroplastidic membrane degradation. A second phase began after the 12^th day, lasting until the end of the assay. In this phase, the MDA concentration in the tissues decreased; however, with higher values than the plants treated at 25ºC during same period (figure 4A).

A highly significant correlation (r = 97.1 %) was observed between the accumulation of total pigment accumulation and the MDA concentration for the control treatment (figure 4B). High pigment concentrations were seen with the lower lipid peroxidation levels (lower MDA concentration). Two phases were observed in this correlation in the chilling treatment: the first showed a smaller decrease (r = 85.8 %), followed by the second (r = 86.3 %), calculated from the values obtained from the 12^th day on (second phase of the peroxidation activity), showing a greater decrease. The results indicate that chilling also causes (due to the reduction of the metabolic activity) a reduction in the peroxidation activity, although in this treatment (10 ºC) the correlation between peroxidative activity and pigment concentration was lower (86.0 %) than that seen in the control treatment (97.0 %) (figure 4B).

DISCUSSION

The results of this study show a reduction in the PS_II quantum yield, signaled by the F_V/F_M ratio response. This reduction occurred in the plants submitted to chilling as well as in the controls. This decrease in the quantum yield may have been caused by the reduced flow of electrons through the PS_II, observed by an increase in F_o for both treatments. Bilger et al. (1984), Ducruet and Lemoine (1985) and Buknhov et al. (1990) described the increase in F_o as a consequence of the electron flow inhibition from Q_A to Q_B. Rosenqvist et al. (1991) attribute the increase in F_o to functional damage of P₆₈₀ in the center of the reaction of PS_II. F_o signals a route of energy dissipation in the form of fluorescence through Chl a from the antenna complex, after capturing excitation energy, when P₆₈₀ is in the oxidized state (Strasser et al., 1995).

According to Oquist (1983), low temperatures drastically affects the photosynthetic apparatus in cold sensitive plants. Coffee seedlings, which are cold sensitive (Levitt, 1980; Bauer et al., 1985), were also affected by cold temperatures. Low temperatures generally cause a decrease in the entire metabolism and also in the photochemical steps of photosynthesis, which are interdependent on the biochemical phase and, as expected, more directly influenced by low temperatures (Wang, 1982; Oquist, 1983). Creencia and Bramlage (1971) considered that although the first consequence may be a reduction in the F_V/F_M ratio, the damage caused by low temperature is not restricted to the photosynthetic apparatus. A relevant aspect, regarding the effect by low temperatures on the photosynthetic activity, is the physical alteration of membranes. These, distributed in organized manner as a network all over the chloroplast, support the photosynthetic electron transport (Hall and Rao, 1995). A decrease in temperature induces an increase in the predominance of the lipid domains in gel state, in the chloroplastid membranes, causing a greater rigidity of these membranes (Lyons, 1973; Murata and Fork, 1975). As a consequence, there is a decrease in the lateral diffusion of PQ, which reflects in the PQ pool and in the rate of PQ reduction (Bowyer and Leegood, 1997).

In this study on coffee seedlings, the amount of PQ decreased drastically with the chilling temperature. If there was less plastoquinone (smaller pool) to respond after its reduction, via P₆₈₀ re-oxidization, and furtherl, a decrease in the electron flow through PS_II, it is probable that, as a consequence, there was less Q_A^- (quinone in the reduced state). The decrease in the Q_A reduction rate in the coffee seedlings could have been the cause of the systematic reduction in the F_M signal (data not shown). This reduction was more intense than the control treatment and was reflected in a relative increase of F_M in the 10 ºC treatment (figure 2B).

According to Schreiber (1983) and Xu et al. (1989), the redox state of the primary Q_A electron acceptor and the H⁺ inter-membrane gradient are the main factors that control the PS_II photochemical reactions. The efficiency in the capture, transfer and use of light energy in PS_II photochemical reactions requires a sequence of photochemical, biochemical and biophysical events (Nobel, 1991). These, however, are energy dependent, whether for the synthesis of constitutive (proteins), metabolic or structural compounds, such as thylacoid membranes. The maintenance of this photosynthetic apparatus structure in a typical and functional arrangement is also energy dependent, being directly affected by the metabolism decrease caused by chilling (Lyons, 1973; Wang, 1982).

The decrease in the Q_A reduction rate and electron flow, as well as the reduction in the PS_II quantum yield, were observed in the coffee seedlings in both treatments. This demonstrates that not only chilling was acting as the cause of these alterations in the electron flow. It is possible that a decrease in all the energy transfer steps might have occurred, since the excitation energy was hindered due to the fact that the seedlings were kept in darkness. Probably, there was drainage of the storage tissue, with the respiratory rate exceeding the photosynthetic rate, since the latter was nil. As the metabolism was repressed in the seedlings submitted to chilling, the respiration rate was lower than in the control treatment, leading to a slower drainage of the reserves in these seedlings. This would explain the more drastic decrease in the PS_II quantum yield in the seedlings treated at 25 ºC, compared to the ones treated at 10 ºC, as observed after the 10^th day of treatment.

The variation in the F_V/F_o ratio, throughout the entire period of acclimatization of the seedlings in this experiment, was shown to be similar to the F_V/F_M ratio, which reflects the PS_II quantum yield, but with greater amplitude of values. However, the use of the latter ratio (F_V/F_M), allows a better discrimination of the small differences in the PS_II quantum yield, which can be used to select ecotypes more adapted to low temperatures. For Schindler and Lichtenthatler (1994), the F_V/F_o ratio also presented values with greater amplitude than F_V/F_M in studies of photoinhibition.

The results related to the alterations in the content of photosynthetic pigments showed a tendency to a sharp increase in the concentration of the accessory pigments (Chl b and carotenoids) when compared to Chl a, probably in order to increase the photon capture, essential to seedling survival. The accessory pigments are responsible for the capture and transfer of photons to the reaction center (Lichtenthaler and Calvin, 1964; Hall and Rao, 1995).

A significant increase in the carotenoid content in the seedlings was not observed, even with the low temperature stress. These pigments are involved in protection strategies against photoinhibition (Hall and Rao, 1995). Plants maintained in the dark were not subjected to the photoinhibitory process and all its damaging consequences to the photosynthetic apparatus. Without the risk of photoinhibition, there was no need for greater carotenoid accumulation in the coffee seedlings treated at 10 ºC. The carotenoid group of pigments are present in the PS_I and PS_II antennae complex. They can be found more abundantly in the lutheine, b-carotene, neoxanthine, violaxanthene, anteroxanthene and zeanxanthine forms in PS_II (Bowyer and Leegood, 1997). The xanthophyll group plays an important role in the plant protection mechanism. Through the xanthophyll cycle, these pigments allow for the non-photochemical dissipation of the energy excess, protecting PS_II against damage caused by the photoinhibitory process in plants sensitive to certain environmental stresses, such as low temperature (Sarry et al., 1994; Thiele and Krause, 1994). Under these conditions, there is a significant decrease in the photosynthetic capacity of the plant tissue (Rosenqvist et al., 1991).

Similarly to protein degradation, lipid peroxidation also has a triggering agent, the action of free radicals. These, through a cascade reaction, lead to the formation of reactive species of oxigen (superoxide anion radical, hydroxyl radical, etc.) that are highly damaging to the integrity of the thylacoids (Barber and Anderson, 1992). However, to generate free radicals, light energy is required (Barényi and Krause, 1985; Richter et al., 1990). Since this experiment was conducted in the absence of light, the formation of these reactive species was not stimulated.

During the chilling treatment, the level of chloroplastid membrane degradation was, at the first stage, stable. However, at the second stage a sharper reduction in the peroxidative activity was observed, possibly due to the decreasing production of reactive oxygen species in response to darkness, and the greater concentration of chloroplastidic pigments.

The results demonstrated that pigment accumulation during the chilling treatment could be one of the factors responsible for the decrease in lipid peroxidative activity. Since the correlation between this activity (peroxidative) and the pigment concentration was lower (86%) in the 10 ºC treatment than in the control treatment (97 %), it indicated that the chilling temperature also influences (due to the decrease in metabolic activity) the peroxidative activity. The synthesis of pigments (especially xanthophylls, from the carotenoid group) may act as a protective mechanism (Schindler and Lichtenthaler, 1994; Thiele and Krause, 1994), possibly against the formation of free radicals (Young 1991), inhibiting the peroxidation of constitutive thylacoid membrane lipids (Sarry et al., 1994). This protection against the "attack" on the integrity of the chloroplast membrane is very important for the maintenance of the photosynthetic activity as a whole (Oquist, 1983; Dwivedi et al., 1995). This was observed here in the first six days, when the PS_II quantum yield (F_V/F_M) of the 10 ºC treatment was kept above 0.800 ¾ a high yield.

Our results suggest that the maintenance of the integrity of the thylacoid membranes in coffee seedlings is crucial for the conservation of high PS_II quantum yield, whereas this efficiency was shown to be more dependent upon the protecting action of the chloroplastidic pigments than the actual concentration of these pigments. The use of the F_V/Fo ratio in detriment to the F_V/F_M ratio allows for better discrimination of small differences in the PS_II quantum yield which can be used for the selection of ecotypes more adapted to low temperatures.

Acknowledgements: The authors are indebted to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Grant and Dr. Richard Ian Samuels (UENF-CCTA) for reviewing the manuscript.

References

• Alonso A, Queiroz CS, Magalhães AC (1997) Chilling stress leads to increased cell membrane rigidity in roots of coffee (Coffea arabica L.) seedlings. Biochim. Biophys. Acta 1323:75-84.
• Barber J, Andersson B (1992) Too much of a good thing: light can be bad for photosynthesis. TIBS 17:61-66.
• Barényi B, Krause GH (1985) Inhibition of photosynthetic reactions by light. A study with isolated spinach chloroplasts. Planta 163:218-226.
• Bauer H, Wierer R, Hatheway WH, Larcher W (1985) Photosynthesis of Coffea arabica after chilling. Physiol. Plant. 64:449-454.
• Bilger HW, Schreiber U, Lange OL (1984) Determination of leaf heat resistance: comparative investigation of chlorophyll fluorescence changes and tissue necrosis methods. Oecologia 63:256-262.
• Björkman O, Demmig B (1987) Photon yield of O₂ evolution and chlorophyll fluorescence characteristics at 77k among vascular plants of diverse origins. Planta 170:489-504.
• Bowyer JB, Leegood RC (1997) Photosynthesis. In: Dey PM, Harborne JB (eds), Plant Biochemistry, pp. 49-110. Academic Press, San Diego.
• Bukhov NG, Sabat SC, Mohanty P (1990) Analysis of chlorophyll a fluorescence changes in weak light in heat-treated Amaranthus chloroplasts. Photosynth. Res. 23:81-87.
• Creencia RP, Bramlage WJ (1971) Reversibility of chilling injury to corn seedlings. Plant Physiol. 47:389-392.
• Dhindsa RS, Plumb-Dhindsa P, Thorpe TA (1981) Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32:93-101.
• Ducruet JM, Lemoine Y (1985) Increased heat sensitivity of the photosynthetic apparatus in triazine-resistant biotypes from different plant species. Plant Cell Physiol. 26:419-429.
• Dwivedi U, Sharma M, Bhardwaj R (1995) Down regulation of photosynthesis in Artabotrys hexapetatus by high light. Photosynth. Res. 46:393-397.
• Govindjee (1995) Sixty-three years since Kautsky: chlorophyll a fluorescence. Aust. J. Plant Physiol.. 22:131-160.
• Graham D, Patterson BD (1982) Responses of plants to low, nonfreezing temperatures: proteins, metabolism, and acclimation. Annu. Rev. Plant Physiol. 33:347-372.
• Hall DO, Rao KK (1995) Photosynthesis. 5^th edn. Cambridge University Press, Cambridge.
• Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125:189-198.
• Hoagland DR, Arnold DI (1951) The water culture method for growing plants without soil. California Agricultural Experimental Station Bulletin 347.
• Hodgson RAJ, Raison JK (1989) Inhibition of photosynthesis by chilling in moderate light: a comparison of plants sensitive and insensitive to chilling. Planta 178:545-552.
• Jouve L, Engelmann F, Noirot M, Charrier A (1993) Evaluation of biochemical markers (sugar, proline, malonedialdehyde and ethylene) for cold sensitivity in microcuttings of two coffee species. Plant Sci. 91:109-116.
• Krause GH (1994) Photoinhibition Induced by Low Temperatures. In: Baker NR, Bowyer JR (eds), Photoinhibition of Photosynthesis - from Molecular Mechanisms to the Field, pp.331-363. Bios Scientific Publishers, Oxford.
• Krause GH, Weis E (1984) Chlorophyll fluorescence as a tool in plant physiology. ii interpretation of fluorescence signals. Photosynth. Res. 5:139-157.
• Krause GH, Weis E (1991) Chlorophyll fluorescence and photosynthesis: The basics. Ann. Rev. Plant Physiol. Plant Mol. Biol. 42:313-349.
• Levitt J (1980) Responses of plants to environmental stress. 2^nd edn. Academic Press, New York.
• Lichtenthaler HK, Calvin M (1964) Quinone and pigment composition of chloroplast and quantasome aggregates from Spinacia oleracea Biochim. Biophys. Acta 79:30-40.
• Lichtenthaler HK, Wellburn AR (1983) Determination of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Transactions 11:591-592.
• Lyons JM (1973) Chilling injury in plants. Ann. Rev. Plant. Physiol. 24:445-466.
• Merzlyak MN (1994) Free radical oxidative reactions on lipids of plant membranes. Free Rad. Biol. Med. 16:9-10.
• Murata N, Fork DC (1975) Temperature dependence of chlorophyll a fluorescence in relation to the physical phase of membrane lipids in algae and higher plants. Plant Physiol. 56:791-796.
• Muruyama S, Yatomi M, Nakamura Y (1990) Response of rice leaves to low temperatures. I. Changes in basic biochemical parameters. Plant Cell Physiol. 31:303-309.
• Nobel PS (1991) Physicochemical and Environmental Plant Physiology, Academic Press, San Diego.
• Oliveira, J.G. (1995) Evaluation of net photosynthesis and of chlorophyll a fluorescence emission kinetics of coffee crop (Coffea arabica L.) submitted to a suspension cycle and re-establishment of irrigation. Viçosa, Universidade Federal de Viçosa, Tese de Mestrado.
• Öquist G (1983) Effects of low temperature on photosynthesis. Plant Cell Environ. 6:281-300.
• Qiu Q-S, Liang H-G (1995) Lipid peroxidation caused by redox system of plasma membranes from wheat roots. J. Plant Physiol. 145:261-265.
• Queiroz CGS, Alonso A, Mares-Guia M, Magalhães AC (1998) Chilling-induced changes in membrane fluidity and antioxidant enzyme activities in Coffea arabica L. roots. Biol. Plant. 41:403-413.
• Richter M, Rühle W, Wild A (1990) Studies on the mechanism of photosystem II photoinhibition. II. The involvement of toxic oxygen species. Photosynth. Res. 24:237-243.
• Rosenqvist E, Wingsle G, Ögren E (1991) Photoinhibition of photosynthesis in intact willow leaves in response to moderate changes in light and temperature. Physiol. Plant. 83:390-396.
• Sarry J-E, Montillet J-L, Sauvaire Y, Havaux M (1994) The protective function of the xanthophyll cycle in photosynthesis. FEBS Lett. 353:147-150.
• Schindler C, Lichtenthaler, HK (1994) Is there a correlation between light-induced zeaxanthin accumulation and quenching of variable chlorophyll a fluorescence? Plant Physiol. Biochem. 32:813-823.
• Schreiber U (1983) Chlorophyll fluorescence yield changes as a tool in plant physiology. I. The measuring system. Photosynth. Res. 4:361-373.
• Schöner S, Krause GH (1990) Protective systems against active oxygen species in spinach. response to cold acclimation in excess light. Planta 180:383-389.
• Smillie RM, Hetherington SE, He J, Nott R (1988) Photoinhibition at chilling temperatures. Aust. J. Plant Physiol. 15:207-222.
• Strasser RJ, Srivastava A, Govindjee (1995) Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria. Photochem. Photobiol. 61:32-42.
• Thiele A, Krause GH (1994) Xanthophyll cycle and thermal energy dissipation in photosystem II: relationship between zeaxanthin formation, energy-dependent fluorescence quenching and photoinhibition. J. Plant Physiol. 144:324-332.
• Wang CY (1982) Physiological and biochemical responses of plants to chilling stress. Hortscience 17:173-186.
• Xu C, Rogers SM, Goldsteing G, Widholm JM, Govindjee (1989) Fluorescence characteristics of photoautotrophic soybean cells. Photosynth. Res. 21:93-106.
• Young AJ (1991) The photoprotective role of carotenoids in higher plants. Physiol. Plant. 83:702-708.

Publication Dates

• Publication in this collection
  16 Dec 2002
• Date of issue
  Aug 2002

History

• Accepted
  08 Aug 2002
• Received
  18 Jan 2002