Comparative Study of Jatropha curcas Accessions Control of Photoinhibition and Photoprotective Mechanisms in Senescing Leaves in a Semi-Arid Region Botswana
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Senescence in plants is the last development phase, leading ultimately to the death of organs such as leaves, sepals, petals, and fruits. During senescence, internal factors and the environment play an important role in tightly controlled alterations at the molecular, cellular, biochemical, and physiological levels. However, leaves are programmed to perform the crucial task of nutrient remobilization. Remobilization of nutrients is a life strategy to supply nutrients to plant parts, such as leaf primordia, emerging new leaves, reproductive organs, or storage organs. This study focussed on how the J. curcas accessions in Southeast Botswana compared in their control of photoinhibition and photoprotective mechanisms of their senescing leaves as a life strategy. J. curcas accessions were raised in a field located in the Department of Agricultural Research, Sebele, Botswana (25° 56′ 37′′ E 24° 3′ 40′′ S). The accessions originated from several parts of the country: Tsamaya, from the north; Tabala, from the central region; and Tlokweng, from the southeast region. One of the accessions was obtained from Ghana. Seedlings were transplanted into the 0.5 ha field with a spacing of 2 m × 2 m in December 2011. Drip irrigation supplied 5 litres of water per week. Gas exchange, chlorophyll fluorescence, photosynthetic pigments, and antioxidants were studied. The onset of senescence triggered degradation of chlorophyll and carotenoid pigments with the consequent decline of photosynthesis. Reduction in the dark adapted Fv/Fm ratio pointed to increased photoinhibition. In early senescence, carotenoid levels decreased gradually and remained functional, allowing photoprotection through their dissipation of excess energy harmlessly as heat. Increased SOD and CAT activities implied increased ROS levels. SOD and CAT activities slowed down destruction by ROS, facilitating nutrient remobilisation. In conclusion, the degradation of the photosynthetic machinery of senescing leaves increases photoinhibition and photooxidation stress. Photoinhibition was more pronounced towards the end of senescence, while photoprotection was more pronounced earlier in senescence to prevent premature death of leaves during remobilization. Ghana and Tlokweng accessions exhibited stronger photoprotection mechanisms in early senescence, allowing nutrient remobilisation compared to the Tsamaya and Tlokweng accessions. Their higher anthocyanin levels in early senescence added to the photoprotective mechanisms in early senescence.
Introduction
Senescing leaves are at the final stage of the life cycle of leaves. Through senescing leaves, the plant reclaims valuable cellular building blocks that have been deposited in the leaves and other parts of the plant during growth. This takes place through processes of degradation and mobilization which are highly controlled. The internal structures are dismantled macromolecules are broken down and reclaiming takes place as these are mobilised to other parts of the plant (leaf primordia emerging new leaves reproductive organs or storage organs) (Juvanyet al., 2013). Plants, therefore need to maintain an efficient senescence process for the survival of the plant and/or its future generations.
Chloroplasts are one of the first organelles to be targeted for disintegration once senescence is initiated. A decrease in photosynthesis, increased photoinhibition, and chlorophyll degradation are thus all signs that the photosynthetic system is being rapidly destroyed (Juvanyet al., 2013). The decrease in photosynthesis normally occurs earlier than the decrease of the maximal efficiency of photosystem II (PSII) photochemistry. The most noticeable sign of leaf senescence is yellowing, which is brought on by the breakdown of the pigment-protein complexes in the chloroplast and the transformation of the constituent chlorophylls (Chl) into catabolic derivatives that are not green (Maytaet al., 2019). Guoet al. (2021) reports that in perennial plants, such as deciduous trees, nutrients deconstructed from senescent leaves are transferred to phloem tissues, where they create bark storage proteins, which are subsequently stored during the winter and remobilized and used for shoot or flower growth during the following growing season. Therefore, it is crucial for plant fitness that leaf senescence begins and progresses in the proper ways (Uauyet al., 2006). Senescence must be efficient to maximize viability in the following generation or season. However, early senescence brought on by a variety of environmental conditions reduces crop plant productivity and fresh product quality (Hörtensteiner & Feller, 2002). Numerous studies have shown that ROS rises sharply during the initial stages of leaf senescence after chlorophyll breakdown (Smart, 1994; Buchananet al., 2003). According to Hörtensteiner and Feller (2002), this rapid ROS production results in the oxidation of proteins, pigments, and lipids, which is a necessary step in the remobilization of nutrients. Finally, leaf death is caused by oxidative processes working in concert with other mechanisms (Zimmermann & Zentgraf, 2005). Since singlet oxygen is one of the most potentially harmful chemicals generated in chloroplasts during photo-oxidative processes and is known to trigger leaf senescence, oxidative stress plays various functions in senescing leaves. As a result, ROS generation should be minimized. As the photosynthetic apparatus degrades, not only is the chlorophyll decreasing, so too are carotenoid pigments (xanthophyll cycle pigments), the photoprotective mechanism that dissipates excess energy as heat.
This study focussed on how the J. curcas accessions in the South-East Botswana compared in their control of photoinhibition and photoprotective mechanisms of their senescing leaves as a life strategy.
Materials and Methods
Study SITE
The growth measurements were carried out at an agricultural field located in the Department of Agricultural Research, Sebele, Botswana (Tominagaet al., 2014). It is a semi-arid area with a wide range of diurnal temperatures throughout the year. The average precipitation in this area is below 490 mm annually (Tominagaet al., 2014). Precipitation occurring from October to March accounts for almost 100% of the annual rainfall (Tominagaet al., 2014). Summer temperatures range from 15 °C in the morning to over 40 °C at midday and winter temperatures range from 3 °C early morning to 25 °C in the afternoons (Tominagaet al., 2014). The soils are reddish brown and are of the Rendzic Leptosol type. They are poor soils with high aluminium and iron content consisting of silt and clay (Tominagaet al., 2014).
The field was established in 2011 from seedlings planted from the seed of various J. curcas accessions collected from different areas of Botswana (Tsamaya from the north, Tabala from the central region and Tlokweng from the South East region). One of the accessions was obtained from Ghana. The experiment was conducted in April 2015 to May 2016. The plants were grown in an area of 0.5 ha with a plant spacing of 2 m × 2 m between the plants.
Experimental Design and Treatments
The field experiment was laid out in a randomized block design with five replications. The treatments were four J. curcas accessions, namely, Tsamaya, Thabala, Tlokweng, and Ghana. These accessions were randomly selected from several parts of the country, and the accession from Ghana was regarded as the control since it has been widely studied.
In earlier observations of these studies, the leaves of J. curcas accessions reached maturity (ceased to increase in size) at around 35 days. These observations agreed with Massaoudouet al. (2020). After reaching maturity at around 35 days the photosynthetic rate of the J. curcas accessions began to decline constantly. Day seven is, therefore, defined as early senescence when the photosynthetic rate would have been consistently declining for 7 days. The onset of senescence is, therefore, the day photosynthesis begins to decline. Measurements were subsequently determined at 7-day intervals till cell death or for 28 days.
Photosynthetic Rate Measurements
Photosynthetic rate (µmol CO2 m−2s−1) measurements were taken using a portable photosynthesis system (LICOR 6400XT equipped with a LED 2 cm × 3 cm leaf chamber, LICOR USA). Diurnal measurements were first carried out at the onset of senescence (day 0) and continued to be taken every seven days up to day 28/leaf death. Each day, the measurements were taken once a day around midday.
Chlorophyll Fluorescence Measurements
The measurements of the maximum photochemical efficiency of PSII (estimated from dark-adapted Fv/Fm ratio) were determined after the leaves were dark-adapted for 30 minutes (Maxwell, 2000), using a fluorimeter (Hansatech Instruments Ltd, Norfolk, UK). The dark adapted Fv/Fm ratio measurements were taken once a day around midday. These dark adapted Fv/Fm ratio measurements were first carried out on leaves at the onset of senescence (day 0) and continued to be taken at day 7, day 14, day 21 and day 28/leaf death.
Photosynthetic Pigments Quantification
Chlorophyll and carotenoid contents were determined according to Lichtenthaler and Welburn (1983). 0.2 g of leaf tissue was macerated using a mortar and pestle. The macerated tissue was placed in 10 ml of 80% acetone for total pigment extraction. The crude extract was centrifuged at 3000 rpm for 5 minutes, and the pellet was discarded. The supernatant was used to determine absorbance by a UV mini 1240UV-VIS spectrophotometer (Shimadzu, Tokyo, Japan) at 470, 649, 530, and 470 nm. The chlorophylls and carotenoids were calculated according to Lichtenthaler and Welburn (1983) and Sims and Gamon (2002) using the following equations:
Chla(μmol ml−1)=(0.01373A−0.000897B−0.003046C)Weight of sample(Volume of sample)where A, B, and C are absorbances at 663, 573, and 646 nm, respectively.
Chl b(μmol ml−1)=(0.02405A−0.004305B−0.0.005507C)Weight of sample(Volume of sample)where A, B, and C are absorbances at 647, 537, and 663 nm, respectively.
Carotenoids(μmol ml−1)=[A470−(17.1×(chl a+chl b)−9.479×anthocyanin)]119.26(VW)where A470 is the absorbance at a wavelength of 470 nm, V is the volume of the sample (ml 80% acetone), and W is the weight of the sample in g.
Determination of Antioxidants
Determination of Superoxide Dismutase (SOD)
SOD was measured/determined as previously described by Giannopolitis and Ries (1977). 0.2 g of leaf tissue was homogenized using chilled glass mortar and pestle in a medium consisting of 50 mM phosphate buffer (pH 7.8) and 100 mg of polyvinyl polypyrolidone as phenolic binder. The homogenate was centrifuged at 16000× g for 15 minutes in a refrigerated centrifuge at 4 °C. The supernatant was collected for the assay. Different sets of the assay system were prepared separately: one for the assay, one for the dark control, and one for the light control. The reaction mixture consisted of 0.1 ml of 1.5 M sodium carbonate, 0.3 ml of 0.13 M methionine, 0.3 ml of 10 µM EDTA, 0.3 ml of 13 µM riboflavin, 0.3 ml of 0.63 mM nitroblue tetrazolium. The nitroblue tetrazolium was withheld in the light control system. To the reaction system, 0.1 ml of enzyme extract was added to the blank and to the light-control system. The reaction mixture was made up to 3.0 ml using 50 mM phosphate buffer (pH 7.8). The tubes with dark control samples (blank: all reagents and sample added) were kept in the dark chamber, and the tubes of the light control (all reagents except nitroblue tetrazolium with sample were added just before reading absorbance) and the assay systems (all reagents and sample) were kept under a fluorescent lamp. After 30 minutes of incubation, the optical densities of the solutions of the assay, the dark control, and the light control systems were measured at 560 nm using a spectrophotometer (UV mini-1240 UV-VIS, Shimadzu Tokyo Japan). One unit of SOD was defined as enzyme activity that inhibited the reduction of nitro-blue tetrazolium to blue formazin by 50% or the amount of enzyme, which reduced the absorbance reading to 50% in comparison to tubes lacking enzyme.
SOD was expressed in Units per milligram (units/mg):
SOD Activity=A0−A1A0÷50%×System VolumeSample Volumewhere A0 is the absorbance in the absence of enzyme extract. A1 is the absorbance in the presence of enzyme extract. The enzymatic activity that causes 50% inhibition in the system is defined as one unit (Zhanget al., 2016).
Determination of Catalase Activity
Catalase activity was measured according to Kar and Mishra (1976). Enzyme extract was prepared by grinding 0.2 g leaf tissue in a chilled mortar and pestle by adding 4 ml 50 Mm phosphate buffer pH 7 mixed with 100 mg polyvinyl polypyrolidone as phenolic binder and centrifuged for 15 minutes at 4000 rpm (4 °C). The assay consisted of 1.0 ml 3% hydrogen peroxide. The phosphate buffer and enzyme extract were pipetted out and mixed well in a test tube. To this 3% hydrogen peroxide was added to initiate enzyme activity. Immediately after the addition of hydrogen peroxide, enzyme activity was measured at 240 nm for 180 seconds at 15-second intervals using Spectrophotometer, and 1 ml 50 mM phosphate buffer (pH 7) was used as blank.
Catalase Activity=ΔAbs×Vt/ε240×d×Vs×Ct×0.001where ΔAbs is the difference between the initial and final absorbance, Vt is total volume of reaction, Ɛ240 is the molar extinction coefficient for H2O2 at OD240 (34.9 mol−1 cm−1), d is optical path length of cuvette (1 cm), Vs is volume of sample, Ct is the total protein concentration in the sample and 0.001 is absorbance change caused by 1 U of enzyme per minute at 240 nm OD.
Results
Physiological Data on Senescing Leaves
Photosynthetic Rates of Senescing Leaves of J. curcas Accessions from the Day 0 of Senescence to Day 28
At the onset of senescence (day 0) the photosynthetic rates of the accessions were higher than at all the other stages (Fig. 1). Though the photosynthetic rates of the accessions continued to decline during senescence the Ghana and Tlokweng accessions displayed higher photosynthetic rates compared to the Tsamaya and Tabala accessions (Fig. 1). At each senescence stage the photosynthetic rates of the accessions differed significantly (P ≤ 0.05).
Maximum Photochemical Efficiency of PSII, Estimated from Dark-Adapted Fv/Fm Ratios of Senescing Leaves of Various J. curcas Accessions
At the onset of senescence (day 0) the dark adapted Fv/Fm ratio of the accessions were higher than at all the other stages (Fig. 2). Though the dark adapted Fv/Fm ratio of the accessions continued to decline during senescence those of the Ghana and Tlokweng accessions displayed higher dark adapted Fv/Fm ratios compared to the Tsamaya and Tabala accessions (Fig. 2). At each senescence stage the dark adapted Fv/Fm ratios of the accessions differed significantly (P ≤ 0.05).
Biochemical Assessments of J. curcas Accessions of Senescing Leaves
Photosynthetic Pigments of J. curcas Accessions of the Senescing Leaf Stages
At the onset of senescence (day 0), all accessions appeared to exhibit higher chlorophyll content (Fig. 3) and carotenoid content (Fig. 4). The Ghana and Tlokweng accessions exhibited higher chlorophyll content throughout the senescing period compared to the Tsamaya and Tabala accessions (Fig. 3). Though the carotenoids decrease with progressing senescence relative to chlorophyll, their levels are higher. However, the Ghana accession at day 0- and day 7 stages displayed the highest carotenoids contents, while the Tabala accession exhibited the lowest (Fig. 4). Thereafter, as senescence progressed the Ghana and Tlokweng accessions displayed the highest carotenoids compared to Tabala and Tsamaya accessions.
Comparisons of Chlorophyll and Carotenoid Degradation of J. curcas Accessions During Senescence
At the onset of senescence chlorophyll and carotenoid pigments did not differ significantly from each other (Figs. 5 and 6). At day 14 stage of senescence the percentage degradation of the pigments was at its lowest with chlorophyl degrading much faster than the carotenoids (Figs. 5 and 6). However, as senescence progressed, percentage degradation increased concomitantly at each stage. The Ghana and Tlokweng accessions generally displayed lower percentage degradations than the Tsamaya and Tabala accessions as senescence progressed (Figs. 5 and 6).
Antioxidants Contents (SOD and CAT) of J. curcas Accessions from Onset of Senescence till end of Senescence
All accessioned appeared to display the lowest SOD contents (Fig. 7) and CAT contents (Fig. 8) at the onset of senescence. Throughout the 28-day period, the SOD contents (Fig. 7) and CAT contents (Fig. 8) of the accessions differed significantly (P ≤ 0.05). At almost each stage of senescence, the Ghana and Tlokweng accessions displayed significantly higher SOD levels than the Tsamaya and Tabala accessions (Figs. 7 and 8).
Anthocyanin Contents of J. curcas Accessions from the Onset of Senescence till the End of Senescence
Fig. 9 shows that anthocyanin contents were highest at the onset of senescence (day 0) and thereafter declined. From day 0 up to day 21, the anthocyanin contents of the accessions differed significantly (P ≤ 0.05). At almost every stage of senescence, the Ghana and Tlokweng accessions displayed significantly higher anthocyanin levels than the Tsamaya and Tabala accessions.
Diurnal Photosynthetic Active Radiation (PAR) over the Three Years of the Study
Fig. 10 shows the diurnal trend of the levels of the photosynthetic active radiation (PAR) over the three years covered in the study. Regardless of season, the midday readings were always higher than the morning and later afternoon. The range of PAR in this particular was from around 500–1500 µmol photons m−2s−1.
Figs. 11A–11C show the maximum photochemical efficiency of PSII (estimated from dark adapted Fv/Fm ratio), quantum yield of PSII electron transport (ɸPSII), and the efficiency of excitation by open PSII reaction centres (estimated from light adapted F′v/F′m ratio) of J. curcas accessions under field conditions in Botswana. In this study, J. curcas accession reached maturity after approximately 35 days (when they ceased to increase in size-leaf area). In the figure below day zero to day 35 denote the mature stage of the leaves. Day 35 denotes the onset of senescence. The onset of senescence was determined after the leaves consistently decreased in their photosynthetic performance. At day 35, the dark adapted Fv/Fm ratio, the quantum yield of PSII electron transport (ɸPSII), and the efficiency of excitation capture by open reaction centres (estimated from light adapted F′v/F′m ratio) began to decrease. This was the general trend for all the four accessions used in the study.
Fig. 12 shows the midday depression and a significant degree of recovery by late afternoon of the J. curcas accessions, senescing leaves diurnal variations. The Ghana and Tlokweng are exhibiting higher Fv/Fm reading than the Tsamaya and Tabala accessions.
Figs. 13A and 13B show changing levels of carotenoids in the morning and at noondays after maturity and how the levels increased from the onset of senescence (approximately at 35 days). These increases are in comparison to chlorophyll, which degrades faster than carotenoids. The Tasamaya and Tabala accessions exhibit higher carotenoid levels than the Ghana accessions and Tlokweng accessions.
Discussions
At the onset of senescence (day 0), the photosynthetic rates of the accessions were higher than at all the other stages (Fig. 1). The higher photosynthetic rate exhibited by the leaves at day 0 of senescence can be attributed to both the higher chlorophyll content (Fig. 3) and the higher Fv/Fm ratio at this early stage. However, as senescence progresses, the chlorophyll content begins to decline. These results are consistent with those of Luet al. (2001) and Wingleret al. (2006), who pointed out that the onset of senescence is accompanied by a decline in photosynthetic pigments. As senescence progressed, the decline of photosynthetic pigments continued, though at different rates. Figs. 5 and 6 show that chlorophyll degraded faster than carotenoids. The faster degradation of chlorophyll relative to the carotenoids (Figs. 5 and 6) was indicative of the increased importance of the photoprotective role of the carotenoids and not the light absorption role of chlorophyll. These results are consistent with Yooet al. (2003), Luet al. (2001), and Luet al. (2003). From the onset of senescence, as the photosynthetic rate declined, the Ghana accession displayed the highest photosynthetic rates, followed by the Tsamaya Tlokweng and Tabala accessions (Fig. 1). However, the Ghana and Tlokweng accessions generally exhibited higher chlorophyll contents than the Tsamaya and Tabala accessions.
Fig. 11A shows that in mature leaves the Fv/Fm ratio was high and declined at the onset of senescence (day 35). With the onset of senescence, the dark adapted Fv/Fm ratio began to decline significantly. Diurnally, the dark-adapted Fv/Fm ratio displayed a midday depression (Fig. 12), which recovered by late afternoon. The midday depression increased as senescence progressed. The quantum yield of PSII electron transport (ɸPSII) displayed similar results, with both the morning and midday readings decreasing significantly as senescence progressed (Fig. 11B) and the midday depression recovering by late afternoon. These results are consistent with Luet al. (2001), who pointed out that in their work, the plants recovered almost to the same level as the morning. In this study, the plants recovered to 0.625 from 0.78 (Ghana accession) (Fig. 12). The recovery of the dark adapted Fv/Fm ratio and the ɸPSII can be ascribed to a down regulation of PSII, a means of photo-protecting the photosynthetic apparatus from damage (Luet al., 2001). The down regulation of PSII could result from the decrease in the efficiency of excitation capture by open PSII reaction centres (estimated from light adapted F′v/F′m ratio) (Fig. 11C). Both the morning and noon light adapted F′v/F′m ratios display significant decreases (Fig. 11C), yet the decrease of dark adapted Fv/Fm ratio (Fig. 11A) was significantly lower in comparison. Luet al. (2001) explain that this decrease in the light-adapted F′v/F′m ratio could be linked to the dissipation of excess light harmlessly as heat by the xanthophyll cycle pigments. The diurnal photosynthetic active radiation was higher at midday than in the morning (Fig. 10), and concomitant carotenoid levels were higher at midday than in the morning (Figs. 13A and 13B). The results are consistent with Mattila et al. (2021), Juvanyet al. (2013), and Luet al. (2001), who report that the higher carotenoid (xanthophyll cycle pigments) levels at midday may indicate that carotenoids dissipate excess light energy harmlessly as heat. They report that the xanthophyll pigments zeaxanthin and antheraxanthin increase relative to violaxanthin under highlight, dissipating excess light as heat. In this study however only carotenoid levels were determined (Figs. 13A and 13B) and exhibited higher readings at midday than in the morning.
As senescence progressed, the dark-adapted Fv/Fm ratios exhibited by the Ghana and Tlokweng accessions were significantly higher (P ≤ 0.05) than those of the Tsamaya and Tabala accessions. The Ghana and Tlokweng accessions can, therefore, be said to be equipped with stronger photoprotective mechanisms (Fig. 2).
The gradual chlorophyll degradation in early senescence, which rapidly increases towards the end of the senescing period (Fig. 4), leads to incomplete photosynthetic machinery. Concomitant with chlorophyll degradation was the increase in antioxidant (SOD and CAT) contents (Figs. 7 and 8). This increase in antioxidants signifies an increase in ROS, which are known to trigger pigment, protein, and lipid oxidation. These oxidation processes are necessary as they facilitate nutrient remobilization. Juvanyet al. (2013) noted that while ROS production was necessary for leaf senescence to bring about degeneration, some ROS were too reactive hindering the remobilization process. Photoprotection is therefore necessary at this early stage to slow down degeneration and to allow remobilization. However, when photo assimilate and nutrient remobilization are accomplished, severe photoinhibition occurs as a result of the failure of photoprotection mechanisms. This suggests that photoprotection of carotenoids (Figs. 13A and 13B) is needed in periods of nutrient remobilization, while it decreases during the terminal phase when photoprotection is no longer needed (Fig. 6). As senescence progressed, the Ghana and Tlokweng accessions exhibited higher SOD and CAT contents than the Tsamaya and Tlokweng accessions.
At the onset of senescence, results show higher levels of anthocyanins, which decreased as senescence progressed (Fig. 9). The results are consistent with Renner and Zohner (2019), Neill and Gould (2003) and Hochet al. (2001) who reported that the central function of anthocyanins is photoprotective. This photoprotection role allowed for the timely progressive degradation of the chloroplasts as nutrients were remobilised. Anthocyanins also scavenge ROS and reduce their generation by decreasing light as senescing leaves degrade their photosynthetic system. As senescence progressed the Ghana and Tlokweng accessions exhibited higher anthocyanins contents than the Tsamaya and Tlokweng accessions.
In conclusion, the degradation of the photosynthetic machinery of senescing leaves increases photoinhibition and photooxidation stress. Photo inhibition was more pronounced towards the end of senescence, while photoprotection was more pronounced earlier in senescence to prevent premature death of leaves during remobilization (Juvanyet al., 2013). As senescence progressed, photoprotection declined with the increase of photooxidation, leading to the death of the leaves. Ghana and Tlokweng exhibited stronger photoprotection mechanisms in early senescence allowing nutrient re mobilisation compared to the Tsamaya and Tlokweng accessions.
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