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Plant Physiology and Biochemistry 52 (2012) 66e76
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Plant Physiology and Biochemistry
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Research article
Photosynthetic performance of Jatropha curcas fruits
Sanjay Ranjan, Ruchi Singh, Devendra K. Soni, Uday V. Pathre, Pramod A. Shirke*
Plant Physiology Division, Council of Scientific and Industrial Research e National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India
a r t i c l e i n f o
Article history:Received 15 September 2011Accepted 19 November 2011Available online 28 November 2011
Keywords:Bark photosynthesisChlorophyll a fluorescenceJatropha curcasFruit photosynthesisSeed respirationVapour pressure deficit (VPD)
* Corresponding author. Tel.: þ91 522 2297928; faxE-mail address: [email protected] (P.A. Shirke).
0981-9428/$ e see front matter � 2011 Elsevier Masdoi:10.1016/j.plaphy.2011.11.008
a b s t r a c t
Jatropha curcas (L.) trees under north Indian conditions (Lucknow) produce fruits in two major flushes,once during autumnewinter (OctobereDecember). The leaves at this time are at the senescence stagesand already shedding. The second flush of fruit setting occurs during the summer (AprileJune) afterthe leaves have formed during spring (MarcheApril). Photosynthetic performance of detached jatro-pha fruits was studied at three developmental stages, immature, mature and ripe fruits. Studies weremade in both winter and summer fruits in response to light, temperature and vapour pressure deficit(VPD) under controlled conditions to assess the influence of these environmental factors on thephotosynthetic performance of jatropha fruits. Immature fruits showed high light saturating point ofaround 2000 mmol m�2 s�1. High VPD did not show an adverse effect on the fruit A. Stomatalconductance (gs) showed an inverse behaviour to increasing VPD, however, transpiration (E) was notrestricted by the increasing VPD in both seasons. During winter in absence of leaves on the jatrophatree the fruits along with the bark contributes maximum towards photoassimilation. Dark respirationrates (Rd) monitored in fruit coat and seeds independently, showed maximum Rd in seeds of maturefruit and these were about five times more than its fruit coat, reflecting the higher energy requirementof the developing fruit during maximum oil synthesis stage. Photosynthesis and fluorescenceparameters studied indicate that young jatropha fruits are photosynthetically as efficient as its leavesand play a paramount role in scavenging the high concentration of CO2 generated by the fruit duringrespiration.
� 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
Jatropha curcas (Supplementary Fig. 1A), a member of theEuphorbiaceae family, originated in Central America, but nowthrives in many parts of tropics and subtropics of Asia and Africa.The seed oil of J. curcas has long been used around the world asa source of lamp oil. Recently, it has attracted global attention, sinceJ. curcas is considered to be one of the most prominent species forbiofuel production the world over. It has been claimed that thatJ. curcas has few pests and diseases and grows under a wide rangeof environmental conditions [21,23].
In addition to the green leaves, commonly considered as theprimary sources of photosynthate production, higher plants canpotentially use almost all vegetative and reproductive structures toperform photosynthetic CO2 assimilation. Fruit photosynthesis,either manifested as net photosynthesis or internal CO2 refixationis regarded as an important strategy of additional carbon-acquisition. Chlorophyllous reproductive structures could derive
: þ91 522 2205847.
son SAS. All rights reserved.
up to 60% of their total carbon requirement from own CO2 fixation[1]. In non-foliar plant parts such as fruits and stem, A is believed toassist in the assimilation of respiratory CO2, thus compensating forcarbon loss [3,7,22]. Another plausible function is related to the factthat, A may additionally help to avoid acidification because ofextremely high CO2 concentration, and alleviate the adverse effectsof hypoxia due to a very low partial pressure of O2 because themovement of oxygen through the fruit tissues cannot keep pacewith the rate of oxygen consumption [5,10,15,22]. Rolletschek et al.[25] have also shown in developing soybean seeds that illuminationof seeds causes photosynthetic release of significant amounts of O2.Under internal hypoxia occurring due to high respiration rates ofthe seeds, this O2 is instantly used for respiration which, in turn,elevates the energy supply. Finally, this affects metabolic fluxes andincreases assimilate partitioning towards lipids. Thus, A in fruitsoccurs under a specific microenvironment never encountered byleaves. Moreover, the metabolic status of fruits is quite different toleaves, as fruits are sinks for both carbon and chemical energy [15].Lytovchenko et al. [17] suggest that fruit photosynthesis is notnecessary for fruit energy metabolism or development, but isessential for properly timed seed development and may thereforeconfer an advantage under conditions of stress.
Table 1Characteristics of various parameters in J. curcas fruits: Themean fruit weight and surface area in J. curcas fruits produced inwinter and summer. The dark respiration rates (Rd)measured in the fruit coat and the seeds at 25 �C. The chlorophyll (Chl) content, intrinsic photosynthetic efficiency of PS II, (Fv/Fm) in dark adapted fruits and the stomatacharacteristics of J. curcas fruits produced in summer during different developmental stages. Data represent the means � SD of 8e10 samples. T-tests were performed for pairsof corresponding values between (meanwinter fruit weight andmean summer fruit weight, meanwinter fruit area andmean summer fruit area) and for rest of the parametersbetween (immature fruit and mature fruit, ripe fruit); significantly different values are indicated with asterisks (P � 0.001) and not significant as n.s (P>0.001).
Parameters Immature fruit (mean � SD) Mature fruit (mean � SD) Ripe fruit (mean � SD)
Mean winter fruit weight (g) 3.88 � 0.59 16.38 � 2.14 12.48 � 1.53Mean winter fruit area (cm2) 13.22 � 1.44 31.85 � 4.43 26.23 � 2.43Mean summer fruit weight (g) 2.69 � 0.35* 7.63 � 0.71* 5.3 � 1.26*
Mean summer fruit area (cm2) 11.63 � 0.61n.s. 20.40 � 0.86n.s. 16.49 � 1.91*
Fruit Coat Rd (nmol s�1 g�1 FW) 3.0 � 0.48 2.10 � 0.20n.s. 1.26 � 0.30n.s.
Seed Rd (nmol s�1 g�1 FW) 4.37 � 1.33 10.5 � 2.20n.s. 1.2 � 0.05n.s.
Chl (mg g�1 FW) 84.5 � 8.2 72.4 � 6.2* 45.15 � 3.2*
Chl a/b ratio 2.93 � 0.14 3.24 � 0.18* 3.0 � 0.41n.s.
Fv/Fm 0.79 � 0.019 0.77 � 0.042n.s. 0.63 � 0.06*
Stomata density (mm�2) 72.22 � 10.93 66.79 � 6.39n.s. 65.0 � 5.92n.s.
Guard cell length (mm) 15.03 � 0.76 18.03 � 0.70* 16.08 � 0.41n.s.
Guard cell width (mm) 4.17 � 0.28 4.60 � 0.20n.s. 4.17 � 0.17n.s.
S. Ranjan et al. / Plant Physiology and Biochemistry 52 (2012) 66e76 67
Under north Indian climatic conditions (Lucknow), J. curcasshows major fruit setting during its two flushes in a year. Duringspring in early March new leaves flush followed by flower initiationand subsequently fruit setting during AprileJune, which are thesummer months. The second flush of fruiting sets in October andfruiting occurs until December. During this winter period the leavessenesce and leaf fall occurs in J. curcas trees. The major photosyn-thetic tissues in J. curcas during this period are the fruits and thebark.
In leaves under the natural conditions the major environmentalfactors that influence the gas-exchange parameters are light,temperature and vapour pressure deficit (VPD) [28]. It has beenshown in several plants that there is a reduction in steady-statestomatal conductance with an increase in VPD [20,6]. This isinterpreted as a means by which plants can minimize water loss[28,30]. Hence, we were interested to understand as to do theseenvironmental factors also influence gas exchange in the jatrophafruits? We have studied the response of temperature and VPD onthe gas-exchange characteristics of jatropha fruits in winter andsummer during their developmental stages. The light responsestudies and net photosynthesis rate versus internal CO2 concen-tration (ACi) curves in developing jatropha fruits were carried out inthewinter fruits. The leaf photosynthetic characteristic was studiedin summer and the contribution of bark photosynthesis wasstudied in the winter.
Fig. 1. J. curcas fruits studied at different developmental stages. Immature stage fruits wer17days (B) and ripe stage fruits were collected between 30 and 32 days (C) after anthesis.
2. Results and discussion
2.1. Fruit development, stomata and chlorophyll content in J. curcasfruit
The jatropha fruits (Supplementary Fig. 1C) were studied fortheir developmental size during the two major fruiting periods ofwinter and summer (Table 1). The fully developed mature fruitsformed in winter were more than 1.5 folds in surface area and 2times more inweight than the fruits produced in summer (Table 1).The stomatal density, length and width of guard cells wereobserved in the peels of immature, mature and the ripe fruits(Fig. 1), which were collected on 8e10 days, 15e17 days and 30e32days after anthesis in case of immature, mature and ripe stagesrespectively. The density was higher in the immature fruits at 72stomata mm�2 as compared to the mature and ripe fruits where itwas 67 and 65 stomata mm�2 respectively however, these differ-ences were not statistically significant. The size of stomata wassmaller in immature fruits than in the mature fruits (Table 1). Thenumber of stomata is set at anthesis and remains constant, whilethe stomatal frequency decreases as the fruit surface expands [3].
Chlorophyll content was maximum in the immature fruits and itwas just around 50% in ripe fruits due to the degradation of chlo-rophyll (Table 1). The fruit chlorophyll amounted only about 17% ofthe leaf chlorophyll content (Table 2). However, the chl a/b ratio
e collected between 8 and 10 days (A), mature fruits were collected between 15 and
Table 2Gas exchange characteristics (net photosynthesis, A; transpiration, E and stomatalconductance, gs), Chl fluorescence characteristics (electron transport rates, ETR;effective quantum yield of PSII, FPSII; photochemical quenching, qP; non-photochemical quenching, qN and intrinsic photosynthetic efficiency of PS II, Fv/Fm) and chlorophyll (Chl) content of young mature leaves of J. curcas under ambientconditions of photosynthetic photon flux density (PPFD), leaf temperature (LT) andvapour pressure deficit (VPD) in summer. Data represent the means � SD of fiveindependent experiments.
Parameters Leaf, in summer (mean � SD)
PPFD (mmol m�2 s�1) 1075 � 85LT (�C) 34.9 � 0.68VPD (KPa) 2.05 � 0.2Maximal A (mmol m�2 s�1) 21.62 � 1.52E (mmol m�2 s�1) 5.5 � 0.48gs (mmol m�2 s�1) 270 � 28Maximal ETR (mmol m�2 s�1) 165 � 3.5FPSII 0.361 � 0.054qP 0.641 � 0.055qN 0.71 � 0.029Fv/Fm (dark adapted) 0.828 � 0.022Chlorophyll (mg g�1 FW) 488 � 71Chlorophyll a/b ratio 3.35 � 0.14
A (µ
mo
l m
-2
s
-1
)
-2
0
2
4
6
8
10
E (m
mo
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s
-1
)
0.0
0.3
0.6
0.9
1.2
1.5
1.8
1
)80
100
A
C
B
S. Ranjan et al. / Plant Physiology and Biochemistry 52 (2012) 66e7668
was around 3.0 in fruits of all three stages and was comparable toleaf chl a/b ratio of 3.35 (Tables 1 and 2). Similar chlorophyll chl a/b ratio has been observed in other fruits [2,17].
gs (m
mo
l m
-2
s
-
0
20
40
60
PPFD (µmol m-2
s-1
)
0 400 800 1200 1600 2000
Ci (µ
mo
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0
200
400
600
800D
Fig. 2. Photosynthetic light response curve in fruits of jatropha, (A). The concurrenttranspiration rates, E (B), stomatal conductance, gs (C) and internal CO2 concentration,Ci (D), in immature (C), mature (-) and ripe (:) fruits. Measurements were madebetween 8:00 and 11:00 h at a fruit surface temperature of 28e29 �C and VPD wasmaintained below 2.0 kPa. Data represent the means � SD of five independentexperiments. The linear and quadratic terms for the polynomial regressions for A underincreasing photosynthetic photon flux density (PPFD) was significant at P < 0. 0001and r2 values were 0.99 for immature and mature and 0.98 for ripe fruits.
2.2. Gas-exchange and fluorescence in response to light
Detached jatropha fruits during the different developmentalstages (Fig.1) showed a vast variation in their light response (Fig. 2).The immature stage fruit showed a light saturation point (ALSP) of2155 (�340) mmol m�2 s�1, while the mature and ripe showed 1108(�185) and 144 (�30) mmol m�2 s�1 respectively. The lightcompensation point (ALCP) was 4.0 (�0.65) mmol mmol m�2 s�1 inimmature and 64 (�10.7) mmol m�2 s�1 in mature fruits and was16 mmol m�2 s�1 in ripe fruit. Maximum assimilation rate (Amax)was 9.8 (�1.55), 5.0 (�0.84) and 0.64 (�0.13) mmol m�2 s�1 inimmature, mature and ripe fruits respectively. The apparentquantum use efficiency (AQE) was 0.007 mol CO2 mol�1 incidentphotosynthetic photon flux density (PPFD) in all three stages offruits (Fig. 2A), fruits in general have a very low AQE as compared toleaf [2]. The developing immature fruit showed a light compensa-tion point of 4.0 mmol m�2 s�1 indicating a capacity to utilize evenextremely low light for its A. Immature fruits also showed a veryhigh light saturation point (ALSP) of 2155 mmol m�2 s�1 and the ALSPin mature fruit was above 1100 mmol m�2 s�1, these were very highin comparison to ALSP in leaves of most of the C3 plants [16] or infruits of Cinnamomum camphora, which showed ALCP of around100e400 mmol m�2 s�1 [14]. The high ALSP is due to the high CO2availability for photosynthesis produced due to the high respirationin jatropha fruits. A similar high photosynthesis at high lightintensity is also observed in tomato fruits [17].
The rate of transpiration (E) showed an increasing trend withincrease in PPFD in immature fruits even at 2000 mmol m�2 s�1
PPFD (Fig.1B). E and the gs weremore or less constant at all the lightlevels in the mature and ripe stages of fruits studied (Fig. 2B, C). Theinternal CO2 concentration (Ci) showed a decreasing trend withincrease in PPFD and A in immature and mature fruits (Fig. 2D). Inripe fruits where the A was very low at around 0.5 mmol m�2 s�1
(Fig. 2A) the Ci did not show much variation and was around600 mmol mol�1 (Fig. 2D).
The effective quantum yield of PSII (FPSII) indicates the energyutilisation by the photosynthetic tissue, as it measures theproportion of the photos absorbed by chlorophyll associated with
PSII that is used in the photochemistry and thus indicates overallphotosynthesis [11]. The FPSII values observed at zero PPFD wasabove 0.8 in immature and mature fruits, while it was 0.66 in ripefruit (Fig. 3A). FPSII decreased with increase in PPFD in all threestages of fruits however, the decrease was drastic in ripe fruitsfollowed by mature and immature fruits respectively (Fig. 3A). Theelectron transport rates (ETR) in immature fruits (Fig. 3B) werecomparative to the rates of maximal ETR in leaves of jatropha
A B
DC
Fig. 3. Fluorescence parameters in response to light in fruits of jatropha. The effective quantum yield of PSII (FPSII) (A), electron transport rates, ETR (B), photochemical quenching, qP(C) and non-photochemical quenching, qN (D), in immature (C), mature (-) and ripe (:) fruits. Data represent the means � SD of five separate measurements. The linear andquadratic terms for the polynomial regressions for (FPSII, ETR, qP and qN) under increasing photosynthetic photon flux density (PPFD) were significant at P < 0. 0001 and notsignificant value is mentioned as n.s. and r2 values are mentioned in figures.
S. Ranjan et al. / Plant Physiology and Biochemistry 52 (2012) 66e76 69
(Table 2). However, the maximal rates of ETR decreased with theage of fruit.
The actual photosynthetic efficiency is determined by the rela-tive distribution of absorbed photon energy to photochemical andnon-photochemical pathways [26]. Relative allocation of energy tothese pathways is indicated by the quenching coefficients qP and qN.A qP of 1.0 indicates there is maximal availability of the acceptor, forPS II, that is, oxidized. The immature fruits at PPFD of1200 mmol m�2 s�1 showed a qP value of around 0.46, while it waslower in mature and the ripe fruits (Fig. 3C). It indicates that theimmature fruit is able to regulate to maintain the PS II acceptor inan oxidized form by about 50% at 1200 mmol m�2 s�1, PPFD. Thesevalues are close to that of jatropha leaf at a PPFD of about1100 mmol m�2 s�1 (Table 2). The allocation of photon energy tonon-photochemical pathway increases with increase in PPFD(Fig. 3D). The initial increase in qN up to 0.4e0.5 reflects theincreased thermal dissipation at the pigment level, the furtherincrease in qN above about 0.5 at PPFD level of above1000 mmol m�2 s�1 in immature and mature fruits (Fig. 3D) reflectsadditionally the contribution of an energy loss process in whichenergy of photons absorbed by chloroplast pigment is transferredto zeaxanthin to be dissipated as heat [13].
The intrinsic photosynthetic efficiency of PS II, measured as Fv/Fm in dark adapted fruits was determined in the three stages of fruitdevelopment. These values were around 0.8 in immature andmature fruits, while Fv/Fm value was low at 0.63 in ripe fruits(Table 1). These results indicate that the immature and maturefruits had a high efficiency of light utilization in PS II [28,2].
As in the ripe fruits which are turning yellow, there is degra-dation of chlorophyll (Table 1), this results in the use of less radi-ation energy due to low chlorophyll content and lower
photosynthetic capacity as is evident from low assimilation rates(Fig. 2A) and low ETR (Fig. 3B), leading to low FPSII, qP and higher qN(Fig. 3A, C, D) [27].
2.3. Response to temperature of gas-exchange
The response to temperature of gas-exchange parameters wasstudied in the immature, mature and ripe fruit stages during bothseasons of winter and summer. In winter fruit surface temperaturewas maintained from around 20 �C to 35 �C and in summer the fruitsurface temperature was maintained from 22 �C to 38 �C. Duringwinter the immature fruits showed a steady A with increase intemperature upto 30 �C, later on A decreased with increase intemperature (Fig. 4A). The mature and ripe fruits showed steady Afrom 20 to 35 �C (Fig. 4A) and were thus insensitive to the increasein temperature. The E increased linearly with increase in temper-ature, while gs decreased with temperature in all the three stagesstudied (Fig. 4B, C). During summer in immature fruits A showed anincrease by two folds from 22 �C to 38 �C (Fig. 4D). However, inmature fruit there was no much variation in A with increase intemperature, while A was very poor in the ripe fruits duringsummer (Fig. 4D). Jatropha fruits showed that the temperatureoptima for maximumphotosynthesis were different during the twoseasons, with 25 �C in winter and above 35 �C in summer fruits.Thus the fruits too showed an adaptation like leaves to the changein temperature [27]. However, temperature had no pronouncedeffect on A in cucumber fruits [18]. The E and gs trend was similar inthe summer fruits as in winter fruits. However, the rates of E and gswere lower during summer (Fig. 4E, F) as compared to that inwinter. In winter the immature fruits were more sensitive toincrease in temperature as compared to mature and ripe stage
A D
EB
C F
Fig. 4. Effect of temperature on the gas-exchange parameters of jatropha fruits in winter (left panel) and summer (right panel). Photosynthesis, A (A, D), rate of transpiration, E (B, E)and stomatal conductance, gs (C, F), in immature (C), mature (-) and ripe (:) fruits. The photosynthetic photon flux density (PPFD) was 1000 mmol m�2 s�1 and vapour pressuredeficit (VPD) was maintained <2.5 kPa upto 30 �C fruit surface temperature and <4.0 kPa above 30 �C fruit surface temperature. Data represent the means � SD of five independentexperiments. The linear and quadratic terms for the polynomial regressions in winter and summer under increasing temperature for A, E and gs were performed, significantlydifferent values (P < 0.05) are indicated with asterisks and not significant as n.s. and r2 values are mentioned in figures.
S. Ranjan et al. / Plant Physiology and Biochemistry 52 (2012) 66e7670
fruits. The decrease in A was steep in the immature fruits, while Eincreased by three folds from 20 �C to 35 �C (Fig. 4B). It wasobserved that the summer fruits were better adapted to regulate itsE than the winter fruits.
Rd was monitored at different temperatures in the three stagesof fruit development in both winter and summer season, and isexpressed on a fresh per mass basis. The fruits of all three stagesshowed an increase in Rd with increase in temperature in both theseasons of winter (Fig. 5A) and summer (Fig. 5B) however, the rateswere higher during summer as compared to that inwinter (Fig. 5B).
Our studies on Rd of the fruit coat and the seeds in the summerfruits showed that in the immature stage fruits the seeds showedabout one and a half times more respiration than the fruit coat. Rdin the seeds of mature fruits increased by five folds in comparisonto its fruit coat. While in the ripe fruits there was no much differ-ence in Rd between the fruit coat and the seeds (Table 1). Thus withthe development of fruit there was an increase in the respiration,reflecting the higher energy requirement of the developing fruit [4].
2.4. Influence of VPD and temperature on gas-exchange
The influence of VPD on the photosynthetic characteristics wasobserved at three different temperatures of 25, 30 and 35 �C in
immature and mature jatropha fruits during winter and summer.During winter in both immature andmature fruits the A showed anincreasing trend with increase in VPD at all the three temperatureregimes (Fig. 6A, D). During winter in immature fruits at 30 and35 �C regimes E showed a constant rate beyond 4.0 kPa VPD(Fig. 6B). While in mature fruits E showed an increase even beyond4.0 kPa VPD (Fig. 6E). The gs declined with increase in VPD in bothimmature and mature fruits at all the three temperature regimesstudied (Fig. 6C, F).
During summer the A in the immature fruits at 25 �C and 35 �Cwere more or less constant at all VPD levels, while at 30 �C Aincreased with VPD and became constant after 3.0 kPa (Fig. 7A). Themature fruits showed an increase in Awith increase in VPD levels at25 and 30 �C (Fig. 7D). At 35 �C the Awas lower and steady than at25 �C at all VPD levels studied (Fig. 7D). E in both immature andmature fruits showed an increasing trend with increase in VPD(Fig. 7B, E). gs like in winter in summer also showed a negativetrend with rise in VPD levels, in both immature and mature fruits(Fig. 7C, F).
With increasing VPD a decrease in the gs was evident, accom-panied by linear increase in E, in immature and mature fruits, at alltemperatures studies (Figs. 6 and 7), in both winter and summerfruits The study shows that the stomata of the fruit responded to
Fruit Surface Temperature (o
C)
20 25 30 35 40
Rd (n
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g
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F
W)
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Fruit Surface Temperature (o
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Rd (n
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W)
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1
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3
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5
6
A (Winter) B (Summer)
Fig. 5. Effect of temperature on the dark respiration (Rd) of jatropha fruits in winter (A) and summer (B), in immature (C), mature (-) and ripe (:) fruits. Data represents themeans � SD of five independent experiments. The linear and quadratic terms for the polynomial regressions in winter and summer for Rd under increasing temperature wassignificant at P < 0.01 and r2 values were above 0.98 for immature and ripe and above 0.84 for mature fruits in winter, while for summer fruits it was above 0.99 for immature,mature and ripe fruits.
S. Ranjan et al. / Plant Physiology and Biochemistry 52 (2012) 66e76 71
increase in VPD by decreasing their conductance to decreasedwatervapour. It has been shown in leaves of several plants that there isa reduction in steady-state gs with increase in VPD [20,6]. This is
A
B
C
Fig. 6. Effect of VPD at different fruit surface temperature regimes in winter on the photosynNet photosynthesis, A (A, D), transpiration, E (B, E) and stomatal conductance, gs (C, F). Ptemperatures were 25 �C (C), 30 �C (-) and 35 �C (:). Data represents the means � SD ofterms for the polynomial regressions under increasing VPD for A, E and gs at 25, 30 and 35 �
and not significant as n.s. and r2 values are mentioned in figures.
interpreted as a means by which plants can minimize water loss[28,30]. However, in case of jatropha fruits even after a decrease ings, the water loss via E did not restrict itself.
D
E
F
thetic characteristics of jatropha immature (left panel) and mature (right panel) fruits.hotosynthetic photon flux density (PPFD) was 1000 mmol m�2 s�1 and fruit surfacefive independent experiments. In immature and mature fruits the linear and quadraticC were performed, significantly different values (P < 0.05) are indicated with asterisks
A D
EB
C F
Fig. 7. Effect of VPD at different fruit temperature regimes in summer on the photosynthetic characteristics of jatropha immature (left panel) and mature (right panel) fruits. Netphotosynthesis, A (A, D), transpiration, E (B, E) and stomatal conductance, gs (C, F). Photosynthetic photon flux density (PPFD) was 1000 mmol m�2 s�1 and fruit surface temperatureswere 25 �C (C), 30 �C (-) and 35 �C (:). Data represents the means � SD of five independent experiments. In immature and mature fruits the linear and quadratic terms for thepolynomial regressions under increasing VPD for A, E and gs at 25, 30 and 35 �C were performed, significantly different values (P < 0.05) are indicated with asterisks and notsignificant as n.s. and r2 values are mentioned in figures.
S. Ranjan et al. / Plant Physiology and Biochemistry 52 (2012) 66e7672
2.5. CO2 concentration in fruit
Net photosynthesis rate versus internal CO2 concentration (ACi)curves were monitored in the three developmental stages ofjatropha fruits during winter. In all the stages the internal CO2concentration (Ci) was higher than the CO2 injected inside thechamber or the chamber CO2 concentration (Ca) (Fig. 8D). Thephotosynthesis rates did not show much variation with increase inCi in all three stages of jatropha fruit (Fig. 8A). Similarly E and gs toowere steady over the range of Ci in all three fruit stages (Fig. 8B, C).
We observed a very high Ci as compared to Ca in all our gas-exchange measurements, Lytovchenko et al. [17] in tomatao fruitshave also observed that the rates of carbon assimilation rarely exceedthose of CO2 release. During the light response study theminimum Ciwas around 500 ppm in the immature andmature fruits, while itwasaround 600 ppm in the ripe fruit (Fig. 2D). The temperature responsestudy too showed similar high Ci in all the three stages (Data notshown).During theACi response theminimumCi possiblewere about130, 200 and 250 mmol mol�1 in immature, mature and ripe staged
fruits, respectively at around 50 mmol mol�1 chamber CO2 concen-tration even after 15 min (Fig. 8A, D). Goffman et al. [12] have shownthat CO2 concentrations in oil seeds peaked during the stage ofmaximum oil synthesis and declined as seeds matured. As the fruitpericarp display a low stomatal density, accordingly, diffusion ofgases becomes restricted and the internal atmosphere becomesextremely enriched in CO2 [3,12]. Rolletscheck et al. [24] showed thatoxygen concentration decreases sharply to about 1% within the seedcoat of Vicia faba and Pisium sativum creating a hypoxic environment.However, oxygen concentration increased upon illumination, indi-cating that photosynthesis significantly contributes to internaloxygen levels in these green seeds. Thus fruit photosynthesis plays animportant role in maintaining the oxygen levels inside the fruit.
2.6. Photosynthetic contribution of the fruit, bark and leaves
The A observed during summer, in young and fully developedjatropha leaves (Supplementary Fig. 1B) were twice the ratesobserved in immature fruits in winter. The rates were about four
A
B
C
D
Fig. 8. Photosynthetic response of jatropha fruits to internal CO2 concentration, Ci (A). The corresponding stomatal conductance, gs (B), transpiration, E (C) and the chamber CO2
concentration, Ca (D) during the ACi measurement in immature (C), mature (-) and ripe (:) fruits. Measurements were performed at photosynthetic photon flux density (PPFD)1000 mmol m�2 s�1, 28e29 �C fruit surface temperature and VPD was <2.0 kPa. Data represents the means � SD of five independent experiments. The linear and quadratic terms forthe polynomial regressions for A under increasing internal CO2 concentration (Ci) and chamber CO2 concentration (Ca) was significant at P < 0. 0001 and r2 values were above 0.95for immature, mature and ripe fruits. While the level of significance and r2 for the linear and quadratic terms for the polynomial regressions for E and gs under increasing Ci aremarked with asterisks and not significant as n.s. in the figure.
S. Ranjan et al. / Plant Physiology and Biochemistry 52 (2012) 66e76 73
times to that observed during summer in immature fruits (Table 2).gs and E in leaves were usually higher by at least two times to thatobserved in the fruits. The fluorescence parameters observed inleaves were similar in values to those observed in fruits (Table 2,Fig. 3). The chlorophyll content was higher in jatropha leaves byabout six times in comparison to the fruit chlorophyll content.However, the chl a/b ratio was similar at above 3.0 in fruits andleaves (Tables 1 and 2). However, a broad range of Chl a/b ratio wasobserved in different cultivars of tomato [29].
Comparison of the FPSII and the ETR values at about 1100 mmolm�2 s�1 PPFD in the leaf and immature fruit were very close(Fig. 3A, B and Table 2), indicating that the young jatropha fruitswere photosynthetically as efficient as its leaves. The results werein contrast to those observed in tomato [13] and Dalbergia mis-colobium fruits [9] where the ETR were about three times higher inleaves than in their fruits. Immature fruits have more light har-vesting chlorophylls than the mature and ripe fruits and thus moreA, and higher ETR and qN. The lower rate of FPSII, ETR, qP and higherqN in ripe fruit suggest that the photon energy is not been utilizeddue to less number of chloroplast (Fig. 3).
The green tissues of jatropha fruits are photosynthetically quiteactive and potentially contribute significantly to fruit growth on perunit area basis, which decreased with fruit development. During
winter the A of immature fruits were high by about two fold ascompared to those during summer (Figs. 6 and 7). However, the A inleaves during autumn when the leaves were in senescence stage,were about 11.0 mmolm�2 s�1, whichwas almost 50% less than thosein summer leaves (Table 2), while in winter there are no leaves onthe trees. Thus to assess the contribution of the bark tissue towardsphotosynthesis the maximal A were estimated in young-green barktissues (Supplementary Fig. 1C) and mature bark tissue(Supplementary Fig. 1D). The young bark showed A rates compara-tive to the immature and mature fruits, while in mature bark tissuethe A was about half of that observed in young bark (Table 3). Thechlorophyll contentwas also low inmature bark tissue than inyoungbark and fruits (Tables 3 and 1). However, the Fv/Fm was high inyoung and mature bark tissue (Table 3) and comparative to the leafand young and mature fruits (Tables 2 and 1).
Thus during autumn and winter the participation of developingimmature fruits and the bark tissues towards photosynthesisbridge the phases until new leaves develop and are able tocontribute to the carbon and oxygen cycles. During summer thecontribution of immature fruits towards A was around 25% ascompared to its leaves on per unit area basis. It has been observedin tomato [13] that the fruit contributes about 15%, while leaves by71% and rest by stem and other tissues towards its photosynthetic
Table 3Net photosynthesis (A), intrinsic photosynthetic efficiency of PS II (Fv/Fm) andchlorophyll (Chl) content in bark of J. curcas under mentioned conditions ofphotosynthetic photon flux density (PPFD), leaf temperature (LT) and vapour pres-sure deficit (VPD) in winter. Data represent the means � SD of five independentexperiments. T-tests were performed for pairs of corresponding values betweenyoung bark and mature bark and significantly different values are indicated withasterisks (P � 0.001) and not significant as n.s (P>0.001).
Parameters Young bark (about3 months old)(mean � SD)
Mature bark(about 1.5 yearsold) (mean � SD)
PPFD (mmol m�2 s�1) 1099 � 107 1100 � 173LT (�C) 29.22 � 0.32 29.34 � 0.61VPD (KPa) 0.9 � 0.23 1.2 � 1.2Maximal A (mmol m�2 s�1) 8.7 � 0.63 4.78 � 0.32*
Fv/Fm (dark adapted) 0.822 � 0.022 0.827 � 0.045n.s.
Chl (mg g�1 FW) 60.12 � 7.16 27.31 � 5.48*
Chl a/b ratio 2.56 � 0.29 2.55 � 0.17n.s.
S. Ranjan et al. / Plant Physiology and Biochemistry 52 (2012) 66e7674
activity. Due to the higher A in jatropha fruits in winter, the maturefruits were larger and heavier in size as compared to the summerfruits (Table 1). The significant amount of photosynthetic activity bythe jatropha fruits thus play a paramount role in scavenging thehigh concentration of CO2 generated in the fruit.
3. Materials and methods
3.1. Plant material
Jatropha curcas (L.) fruits were collected from two year old trees.The immature stage of fruit were collected from the 8th to 10th day,while mature fruits were collected at 15th to 17th day after anthesison attaining its complete development. The ripe stage was whenthe fruit just started to turn yellow, which was usually 15 days afterthemature stage (Fig.1). The experiments were conducted from the2nd week of October till mid-December. The second major flush offruiting is observed inMayeJune. The two seasons have contrastingtemperatures of 15e33 �C in October - December (winter) and28e45 �C in MayeJune (summer) and maximum VPD levels arearound 3e4 kPa during winter and >7.0 kPa in summer. Therefore,the influence of temperature and VPD on gas-exchange character-istics of jatropha fruits was also carried out in fruits obtained inJune. Care was taken to harvest fruits fully exposed to sunlight,wrapped in moist tissue towels and then put in air-tight plasticenvelopes, brought to the laboratory and experiments were initi-ated typically after 15e20 min after excision.
3.2. Stomata studies
The epidermal layer of the fruits were peeled and studied for itsstomatal density, length and width of the guard cells under theLeica DM 500 microscope (Leica Microsystems Ltd., Heerbrugg,Switzerland). The density was measured in an area of 0.0314 mm2
and fifty such measurements were made. Length and width wasmeasured in at least twenty stomata. These observations weremade in at least 10 fruits of each stage collected from differentplants.
3.3. Chlorophyll estimation
Chlorophyll content in the fruit samples of jatropha was deter-mined in the epidermal layer by grating and collecting the sample,while disc of leaves were punched and epidermal layer wasscrapped from the bark tissue for estimating chlorophyll, accordingto Arnon as, described in Coombs et al. [8].
3.4. Measurements of gas exchange and fluorescence
Gas exchange rates, Fv/Fm and chlorophyll a fluorescence weremeasured with an open infrared gas-exchange analyser system(GFS-3000; Heinz Walz GmbH, Effeltrich, Germany). Parameterswere monitored using the Walz, “Cuvette for Lichen/Mosses 3010-V32”.
During A and Rd measurements a CO2 level of 400 mmol mol�1
was maintained inside the cuvette. All the measurements weremade with the detached whole fruit by placing them verticallyinside the chamber. As the jatropha fruit showed a high rate ofrespiration, even in the presence of light, for all measurements of A,the steady-state respiration rates in dark were monitored at therequisite temperature and VPD prior to subjecting the fruit to light.The rate of respiration obtained in dark was subtracted from therate in presence of light, for the calculation of rate of A. The rate of Awas estimated on the basis of the fruit surface area. As the wholefruit respires, the dark respiration rates are expressed on a fresh permass basis.
3.5. Photosynthetic light response curves
The photosynthetic light response curve data were obtained bymaking measurements on four detached fruits of each stage duringOctobereNovember, between 0800 and 1100 h (to avoid middaydepression in photosynthesis). The light was provided by the inte-grated LED light source, Walz LED-Array/PAM-Fluorometer 3055-FL”module head attached to the cuvette. The PPFD varied from 0, 50,100, 200, 400, 600, 800, 1,000, 1,200, 1600 and 2000 mmol m�2 s�1.The fruit was acclimatized at each intensity for about 15 min. A ateach PPFD was recorded when it was stable (usually 5e8 min). TheCO2 concentration of the air entering the chamberwasmaintained at400 mmol mol�1. During the measurements, the VPD for allmeasurements was maintained below 2.0 kPa. The fruit surfacetemperature was maintained between 28 and 29 �C for all themeasurements. The initial slope of the light response curvewas usedto calculate the apparent quantum use efficiency (AQE), the lightsaturation point (ALSP), light compensation point (ALCP) andmaximum assimilation rate (Amax), using the ACi and AQ ResponseCurve Analysis Software (Version 1.0, Li-COR, 2/2008).
Chlorophyll fluorescence parameters were monitored simulta-neously along with the gas-exchange parameters duringmeasurements of photosynthetic light response curves. Calcula-tions of various chlorophyll fluorescence parameters were done asgiven in Maxwell and Johnson [19]. The maximum photochemicalefficiency of photosystem II (PSII), (Fv/Fm: where Fm is maximumfluorescence of the dark-adapted fruit under a light saturating flashand Fv is maximum variable fluorescence, FmeF0) was measured onfruits after 20 min of dark adaptation. The effective quantum yieldof PSII (FPSII) was calculated as (Fm’-Fs)/Fm’ and apparent electrontransport rates through PSII (ETR) were calculated as 0.5*FPSII*PPFD.Photochemical quenching (qP) was computed as qP ¼ (Fm’�Fs)/(Fm’�F0’) and non-photochemical quenching (qN) was calculated asqN ¼ (Fm�Fm’)/(Fm’�F0’).
3.6. Temperature response
Measurements of gas exchange in response to temperatureweremade in detached fruits during winter and summer. The study wasinitiated at around 20 �C, and fruit surface temperature wassubsequently increased in increments of 2e3 �C to 35 �C. The fruitwas allowed to equilibrate for at least 7e8 min before data wererecorded at each measurement temperature. The vapour pressuredeficit (VPD) was maintained below 2.5 kPa up to 30 �C, and
S. Ranjan et al. / Plant Physiology and Biochemistry 52 (2012) 66e76 75
>4.0 kPa above 30, the PPFDwas 1000 mmol m�2 s�1 during studiesin both the seasons.
Rd was monitored in detached fruits of both seasons (winterand summer) under similar conditions of temperature and VPDlike A. The rates are expressed on fresh per mass basis. Unlike Awhere only the epidermal surface contributes towards A, thewhole fruit respires. Rd was also measured in fruit coat (pericarpand mesocarp) and seeds of immature, mature and ripe fruits insummer.
3.7. VPD studies
The studies were carried out on four independent detachedfruits of immature andmature stages inwinter and summer. All thestudies were made between 8:00 h to 12:00 h to avoid any middaydepression. The PPFD was maintained at 1000 mmol m�2 s�1. Thestudies were carried out at three different fruit surface temperatureregimes of 25, 30 and 35 �C. After the fruit was enclosed inside thechamber, the A was allowed to stabilize at the ambient VPD andcontrolled PPFD and fruit temperature. Later on the humidity wasgradually increased or decreased till the desired VPD was attained.The steady-state Awas allowed to be attained before recording thereadings, which typically took 15e20 min. The maximum VPD thatwas practically achievable in both the seasons at 25, 30 and 35 �Cwas 3.0, 4.0 and 4.8 kPa respectively.
3.8. Measurements of ACi
ACi curves, each consisting of measurements in 4e5 detachedfruits, were performed at 1000 mmolm�2 s�1 PPFD, fruit surfacetemperature was maintained at 28e29 �C and VPD was <2.0 kPainside the cuvette. The measurements were started at ambient CO2,then the CO2 concentration of the air entering the chamber wasdecreased up to 50 mmolmol�1 CO2 so as to have at least 3e4 points<400 mmol mol�1 ambient CO2, then CO2 concentration wasincreased, stopping again at ambient CO2 and then increasing up toabout 1600 mmolmol�1 CO2 of the air passed inside the chamber, soas to have at least 5e6 points above 800 mmol mol�1 CO2. Each setof experiment was completed in about 40e45 min.
3.9. Measurements in leaf and bark
The gas exchange and fluorescence parameters were monitoredin attached young fully mature leaves under natural conditions inthe field in early May. The gas-exchange parameters in the leaveswere monitored using a clear top cuvette, “Standard MeasuringHead 3010-S” and “Leaf Area Adapter 3010-2�4”. The measure-ments were made between 08:00 to 09:00 h, as the A rate wasmaximum during this hour, as determined earlier from our diurnalstudies.
The fluorescence studies were made on separate days at samehour as above, using the Walz, GFS 3000 system with the LED-Array/PAM-Fluorometer 3055-FL” module head attached to thecuvette. Fv/Fm determinations were made after dark adapting theleaf for 20 min. All the gas-exchange and fluorescence determina-tions on attached leaves were made on six independent plants ondifferent days.
Stem samples of young, about 3.0 months old and maturesamples of about 18 months were harvested, wrapped in moisttissue towels, put in air-tight plastic envelopes and brought to thelaboratory from the field. The bark was separated from the stemsample for the photosynthesis and Fv/Fm determinations, usingWalz, GFS 3000 system. Studies were made between 8:00 h to12:00 h to avoid any midday depression.
3.10. Statistical analysis
All values reported are mean of at least four-five independentexperiments. The means � SD are shown in the figure unlessmentioned otherwise. Standard polynomial (quadratic) functionswere fitted to observed responses of A to increasing PPFD, and of A,E and gs to increasing VPD, increasing fruit surface temperature andCi and of A to Ca using SigmaPlot (ver. 9.0.1), these differences wereevaluated at a significance level of 0.0001, 0.01 or 0.05.
The relationship between mean values of winter fruit weightand summer fruit weight; winter fruit area and summer fruit area;immature fruit and mature, ripe fruits and young bark and maturebarkwere tested using T-test (P� 0.001). Significant differences aremarked with asterisks while not significant as n.s.
4. Conclusion
Our results demonstrate that during autumnwhen the jatrophaleaves are in senescence or in winter when there are no leaves thejatropha fruits along with the bark contributes maximum towardsphotoassimilation. We have shown that the jatropha fruits are welladapted to utilize low as well as very high light for its photosyn-thesis. VPD did not have much influence on photosynthesis of thefruit. Although stomatal conductance did show regulation inresponse to VPD, however, transpiration remained unrestrained.Seeds during maturation showed an increased respiration,reflecting the higher energy requirement during maximum oilsynthesis. It would be interesting in future studies to evaluate therole of photoassimilates formed during fruit photosynthesis on theseed formation and the oil synthesis in jatropha.
Acknowledgements
A Senior Research Fellowship provided to Sanjay Ranjan byCouncil of Scientific and Industrial Research (CSIR), New Delhi,India, is gratefully acknowledged.We are thankful to Director, CSIR-NBRI for supporting this work.
Appendix. Supplementary material
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.plaphy.2011.11.008.
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