Photosynthesis of Sago Palm ( Metroxylon sagu Rottb . ) 2 Seedling at Different Air Temperatures

Photosynthetic activities of the sago palm (Metroxylon sagu Rottb.) were studied to find out its 13 sensitivity to changes in ambient air temperature. The minimum ambient air temperature designed for the 14 experiment was 25–290C, while the higher end was 29–330C. Several photosynthetic parameters were studied to 15 support our analysis in sago photosynthetic activity, including diurnal leaf gas exchange, assimilation rate vs. 16 CO2 concentration, leaf greenness, leaf chlorophyll content, and photosynthetic rate vs. irradiance. We found 17 that sago palm photosynthetic activity tends to be more sensitive to minimum than to maximum ambient air 18 temperature. The plants exposed to higher air temperatures had dark green leaf color associated with higher rates 19 of diurnal photosynthesis, chlorophyll content, and rubisco limited photosynthetic activity. They also exhibited 20 higher trend in optimum irradiance absorption level. Consequently, maximum light energy dissipation occurred 21 at higher temperatures. 22


Introduction
Ambient air temperature generally has a significant impact the physiological performance of plants.Many studies reveal that inhibition of photosynthetic perfomance occurs at severely high (>35⁰C) and low temperatures (<20⁰C) [1].However, in the tropical zone in which the sago palm typically grows, variation in air temperature within a year is less than in temperate zones, so this study focused on a moderate air temperature range from 25-33⁰C to ascertain the sago palm's photosynthetic performance in the ambient air environment of its typical habitat.
The sago palm is a perennial monocot crop well known for its potential to accumulate high amounts of starch in its trunk.It can store approximately 300 kg (dry weight) of starch per tree [2].The importance of sago palm as a staple food is well recognized in some areas of Southeast Asia and South Pacific.The carbohydrate contained in the trunk can be further processed into various basic raw materials for food, animal feed and industrial uses [3].Coming from the arecaceae or palmae family, sago is able to grow in marginal terrain such as submerged and tidal areas where most agronomy crops cannot survive without drainage or soil improvement.As one of the most important crops for sustainable agriculture and for rural development in swampy areas of Indonesia, sago palm has become an important part of peatland restoration projects.
The optimum air temperature range for sago palm is very narrow, reported as from 25⁰C to 29⁰C [4].
Elsewhere it has been reported that the best growing conditions required a minimum of at least 26⁰C [5].The minimum temperature has been cited as an important factor limiting sago palm performance [6], which is likely due to the tendency of the photosynthesis enzyme Ribulose-1,5-bisposphate caboxylase/oxygenasi (Rubisco) to be very sensitive to air temperature.Minimum temperature reduces rubisco activity, which consequently reduces the utilization of RuBP by rubisco.Rubisco activase content is suppressed by up to 80% at 25⁰C and below [7].
A study on hibiscus plants reported that the effective quantum yield of PSII (Ф PSII ) is suppressed below 10⁰C [8].
Regarding maximum temperature, morphological observation of sago palm seedlings reported that at 35⁰C leaves expanded but become less green [9].Higher air temperature also plays an important role in sago seed germination.At 30⁰C, seed germination was about 20% higher than at 25⁰C [10].In physiological studies, moderate heat stress affects light energy harvesting as more light is dissipated for non-photochemical quenching rather than photochemical quenching.Therefore, the CO 2 fixation process is depressed [11].Another study confirmed that carboxylation efficiency decreased at 39⁰C followed by a reduction in photosynthetic efficiency [12].
While it has been considered that variations of ambient air temperature in the sago palm habitat might affect its physiological performance, especially its photosynthetic activity, this has not been definitively proven.This study aimed to provide useful data on sago palm physiology at different air temperatures beyond the little that has so far been published.Understanding the regulation of photosynthesis and chlorophyll fluorescence is very important as a tool in characterising plant reactions under abiotic stress, such as high and low temperature stress [13].Thereby, the area for sago palm cultivation can be effectively selected to meet the need for appropriate air temperature, allowing optimum plant growth to be achieved and producing optimal yields.Due to the abovementioned high degree of photosynthetic sensivity of the plant, especially at lower temperatures, it was hypothesised that even moderate changes in air temperature will inhibit the photosynthetic performance of sago palm.

Plant material and culture conditions
The experiment was conducted in two phytotrons (glass house) with air temperatures ranging from 25-29⁰C and 29-33⁰C respectively at Nagoya University, Japan, from January to March 2017.These air temperatures were considered as the range of ambient temperature in sago palm habitats associated with tropical rainforest climate.Air relative humidity ranged from 30-50% and irradiance flux density from 600-800 µmol m -2 s -1 .Six one-year-old sago palm seedlings with six fully-developed leaves, grown individually in 1/10000a Wagner pots (diameter 115 mm and height 184 mm), were tested.The plant materials were obtained in seed form from Sentani District, Jayapura, Indonesia.Vermiculite was applied as the growing media for each plant.
The nutrients were supplied through the application of Kimura B culture solution.The second youngest leaves were selected for all measurements.At the beginning, all plants were placed in the same phytotron with air temperature set at 25-29⁰C.After that six plants were moved to a phytotron with air temperature at 29-33⁰C.
After one month of acclimation, measurement was conducted.

Diurnal leaf gas exchange
Diurnal leaf gas exchange was measured hourly from 7:00 AM to 05:00 PM.The diurnal leaf gas exchange measurement was end at 03:00 PM to the plants grown at 25-29⁰C as the photosynthetic value was zero after 03:00 PM.The portable photosynthesis system, Li-6400XT (LiCor Inc., USA) with 6 cm 2 leaf chamber was utilized during the measurement.The CO 2 concentration was controlled at 400 µmol and photosynthetic photon flux density (PPFD) was set at 750 µmol m -2 s -1 .The CO 2 mixer was adjusted at 500 µmol and relative humidity in the leaf chamber was controlled at 40% in the phytotron.During measurement, leaf temperature was set at 25⁰C.Net photosynthetic rate (P N ), stomatal conductance (g s ), transpiration rate (T r ), and intercellular CO 2 concentratio (C i ) parameters were obtained from this measurement.

Assimilation rate vs. CO 2 concentration (A/Ci curve)
A carbon response curve was constructed following the Farquhar photosynthesis model [14,15] to conceive the photosynthesis interference at different air temperatures.Changes in rates of assimilation in response to carbon dioxide variation was studied by setting several levels of CO 2 concentration with constant PPFD intensity set at 750 µmol.At the beginning, CO 2 concentration was set at 400 µmol and gradually reduced to the lowest concentration at 50 µmol.After reaching the lowest level, CO 2 concentration was gradually increased to a maximum level of 2000 µmol.Finally, CO 2 concentration was returned to 400 µmol with irradiance in the "off" mode.There were three replicates for each treatment.
The A/Ci curves data were obtained by A/Cc curve fitting utility version 1.1 developed by Sharkey [15].
The derived variables obtained from the fitting curve are maximum carboxilation capacity (V cmax ), and electron transport rate (J) [16].The Rubisco limited photosynthesis (V cmax ) was calculated using the following equation: V cmax represents maximum Rubisco rate in CO 2 reduction, C c is partial CO 2 pressure at rubisco, K c is the Michaelis constant of Rubisco for CO 2 , O is the partial pressure of O 2 at rubisco, K o is the inhibition constant of Rubisco for O 2 , Γ * is the compensation point of photorespiration, and R D is dark respiration in which CO 2 is released by the non-photorespiration process.
The following equation was used to calculate the RuBP limited photosynthesis: 3 of 9 J represents the RuBP limited photosynthesis for NADPH formation which is utilized in RuBP regeneration which takes four electrons per carboxylation and oxygenation [15].

Photosynthetic rate vs. Irradiance
A light response curve was constructed using a photosynthesis yield analyzer (MINI-PAM, Walz-Germany) after dark adaptation for 20 minutes.Nine irradiance levels were given from zero to 1300 µmol m -2 s -1 and each erradiance had an interval of 10 seconds to reach to steady state level.Three sago seedlings were choosen as replicates.Three leaflets from the second younger leaf of each plant were measured to obtained the mean value of each replicate.
The fluorescence data including quantum yield photosystem II (Φ PSII ), electron transport rate (ETR), nonphotochemical quenching (NPQ), and coefficient of non-photochemical quenching (qN), were computed with WinControl software (Walz -Germany).The fraction of energy photo-chemically converted in photosystem II is represented by Ф PSII which is calculated as: Fm′ is the maximum fluorescence yield in light adapted sample where all PSII is the open stage.F is yield fluorescence measured briefly before saturation pulse application, and is the increase of fluorescence induced by a saturation pulse [17].The equation used to fit Φ PSII is simple exponential decay function of the form Ф PSII = e -x after appropriate scalling, Φ PSII is quantum yield, Φ PSII max is maximum quantum yield at theoritical zero irradiance, k y is a scaling constant, and PPFD is photon flux density (µmol (CO 2 ) m -2 s -1 ).
Electron transport rate (ETR) is calculated by estimating gross photosynthesis using the following equation: Ф PSII is the effective quantum yield, PPFD is the irradiance, allocation factor (0.5) is the partitioning energy between PS II and PS I, and 0.84 is the leaf absorbance factor (α leaf ) [18].Following Ritchie and Bunthawin [19], ETR data was fit using non-linear least squares methods calculated as: The excel routine for fitting Waiting-in-Line curves was utilized to fit the ETR vs. several levels of irradiance [20].The excel routine was obtained personally from Ritchie.The non-photochemical quenching (NPQ) parameter corresponds to the loss of potential energy which is dissipated as heat also referred to as thermodynamic loss.NPQ is calculated as: qN is calculated as: F is yield fluorescence measured briefly before saturation pulse application, F m is the maximum fluorescense of dark adapted leaf, F o is the minimum fluorescence, and F′ is the maximum fluorescence measured at saturation pulse.The equation used to fit qN and NPQ vs. irradiance curves is simple exponential saturation functions: while NPQ was calculated as: PPFD is photon flux density, K qN and K NPQ are exponential constants, qN max is the asymptotic maxima for qN, and NPQ max is the asymptotic maxima for NPQ.

Chlorophyll content
A portable chlorophyll meter, SPAD-502Plus (Konica Minolta, Japan) was utilized to measure leaf greenness of the same leaves chosen for photosynthesis measurement.The same leaves were harvested for chlorophyll content analysis.

Statistical analysis
This experiment employed completely randomized design with two ranges of air temperature, 25-29⁰C and 29-33⁰C, with three replicates.The second upper-most leaf from each replicate was used for measurement.
Each datum is presented as mean ± SE.Statistical differences were tested by student t-test.

Diurnal leaf gas exchange
Data for sago palm data show considerable differences in net photosynthetic rate (P N ), stomatal conductance (g s ), and leaf transpiration rate (T r ) values between the two air temperature ranges (Fig. 1A).The open circles represent the P N of sago palm seedlings growing at 25-29⁰C room temperature while the closed circles represent the P N of seedlings grown at 29-33⁰C room temperature.In the first daily measurement at 7:00 h sago seedlings growing at the higher room temperature started with a higher P N than the seedlings growing at lower temperature.The plants grown at 25-29⁰C maintained the optimum P N for a short period as the downward trend began around 12:00 h.
Stomatal conductance (g s ) and leaf transpiration rate (T r ) showed the same trend with P N .At 25-29⁰C, g s showed a slight upward trend during the observation.Low stomatal aperture at 25-29⁰C caused a reduction in T r (Fig. 1B,D).At 29-33⁰C, the higher P N trend was followed by higher g s and T r .However, intercellular CO 2 concentration (C i ) at higher temperature showed a lower rate only in the first two hours of measurement (7:00 to 8:00 h) and the last two hours of measurement.They exhibited almost the same trend in C i rate between 9:00 and 13:00 h (Figure 1C).

Photosynthetic activity and chlorophyll content
Photosynthetic activity and chlorophyll contetn of sago palm seedlings showed lower rates and concentrations respectively at 25-29⁰C air temperature.The low P N value was followed by lower values in biochemical limiting photosynthetic activities such as electron transport rate (J) and maximum carboxilation capacity (V cmax ).The other parameters to explained the low P N at minimum air temperature was pigmentation.
Chlorophyll content seems to be lower at minimum than at higher air temperatures tested.Although only Chl b showed significant difference in pigment content, all parameters were considerably higher in sago seedlings grown at 29-33⁰C air temperature (Table 1).The leave color of sago palm grown at 29-33⁰C was obviously darker than at the lower air temperature tested and was confirmed by significantly higher SPAD values.In addition, we noticed that most plants showed leaf emergence at the higher air temperature, although no measurement in leaf emergence rate undertaken.

Photosynthetic rate vs. Irradiances
Photosynthetic rate vs. Irradiances was measured and fit using non-linear least square fit.The graphics of quantum yield of PSII at both air temperatures were obtained (Fig. 3A).The curve of quantum yield of PSII (Φ PSII ) vs. irradiance at both air temperatures provided a maximum effective quantum yield (Φ PSII max ) value at

Discussion
At the beginning of measurement (7:00 h) sago palm seedlings revealed low photosynthetic rates in both treatments.This might be due to the light not reaching sufficient levels for optimum stomatal aperture as blue light induces the aperture of stomata [23].Moreover, air temperature at that time has not reached the optimum level for higher rubisco activity.Consequently, with an increase in light intensity and air temperature from 8:00-11:00 h, P N , g s , and T r trended upwards in both treatments.However, the plants showed midday depression as photosynthetic rate reduced from 12:00.This might be caused by stomatal and other non-stomatal limitations such as photoinhibition, photorespiration and reduction of rubisco activity under high temperature [24].This is consistent with most field work cases, where it is difficult to obtain optimum P N rate in sago palm when the measurement is conducted after 12:00 h.This information was confirmed by our findings from diurnal leaf gas exchange data.In addition, our data suggests that lower air temperature inhibited the seedlings' capacity to maintain a longer P N rate.Higher temperatures (29-33⁰C) appear to induce higher rubisco activity in sago palm seedlings than that achieved at lower temperatures (25-29⁰C) (Table 1).Although producing the same diurnal leaf gas exchange trend across both air temperature ranges, the sago seedlings growing at 29-33⁰C room temperature showed higher photosynthetic rate.
According to the data from figure 1A, a lower net photosynthetic rate at 25-29⁰C was followed by lower stomatal conductance results in lower leaf transpiration rate.Low transpiration rate is considered to be one of the factors causing low P N rate as CO 2 can only enter leaves through gas diffusion [23].However, the intercellular CO 2 concentration (C i ) did not a show higher rate at 25-29⁰C.C i revealed almost the same trend in both air temperature ranges, except in the first two hours of measurement and the last two hours of measurement (Fig. 1C).When plants performed higher assimilation rates, intercellular CO 2 value should show a lower rate as the CO 2 is utilized during photosynthesis activity.Therefore, we assume that at 25-29⁰C, the photosynthetic activity was not only limited by those components but also the other components such as rubisco activity, leaf chlorophyll content and the light harvesting system.
In the lower air temperature range (25-29⁰C), the performances of rubisco activity (V cmax ) tend to be low.
Low rubisco activity at 25⁰C might be due to the reduction in RuBP regeneration [25,7].RuBP regeneration might be affected by the lower RuBP consumption by rubisco.This could suggest that the activation state of rubisco could be different in plants grown at different air temperatures.
The higher performance in net photosynthetic rate of sago palm seedlings at higher temperatures tested also could not be separated from the support of higher photosynthetic apparatus formation, such as leaf chlorophyll.The higher temperatures induced higher formation of leaf pigments such as Chl a, Chl b and carotenoid.The sago seedlings grown at higher air temperatures produced leaves with a dark green color, while sago seedlings grown at lower air temperature produced light green leaves.An appropriate air temperature increases the capacity for thermotolerance which increases chlorophyll a:b ratio [26,27].Air temperature also influences the formation of chlorophyll as temperature regulates the synthesis of chlorophyll precursor [28].In our study, the phytotron at 29-33⁰C provided an appropriate growth environment for sago palm seedlings.Those growing at 31⁰C revealed higher uptake in macronutrients such as N, P, K, and Ca, which contribute to the maximum leaf area [9].Therefore, it appears the higher uptake of nutrients induced the higher formation of leaf chlorophyll leading to greater capacity to harvest light energy.In addition, although no measurement in leaf emergence rate undertaken, we noticed that most plants showed leaf emergence at the higher air temperature, the same occurence had previously been found in the study of sago palm seedlings' response to various ranges of air temperature.It was found that at 35⁰C leaf emergence rate increased although shoot elongation rate, leaf area and root growth rate decreased.However, at 23⁰C leaf emergence rate decreased along with increased root growth rate [9].
In general, sago palm reach light saturation point from 600-750 µmol m -2 s -1 PPFD, although this point may vary depending on the leaf age and shaded conditions [29,6].According to our finding, air temperature is also one of the factors affecting the light saturation point of sago palm seedlings, especially in the lower air temperature range tested.The optimum irradiances (PPFD opt ) of sago palm seedlings was rather low at 25-29⁰C followed by early reduction in electron transport rate as photo inhibition might have occurred due to excess light energy.This can be seen from the down-ward trend which occurred when the light intensity increased above 600 µmol (Figure 3B).The sago palm seedlings grown at 29-33⁰C maintained higher performance in light energy utilization for electron transport than those at 25-29⁰C (Figure 2B).The increase in leaf temperature as long as it does not exceed the upper thermal limit, may enhances photon flux density which consequently affects the adjustment of thermotolerance in PSII and results in optimum photosynthetic rate [30,31,27].In our study, sago palm photosynthetic optimum irradiance was higher when the plants were growing at the higher air temperature.The reduction in electron transport rate due to photo inhibition occured when PPFD increased above 800 µmol (Fig. 3B).However, the higher maximum electron transport rate showed not significant higher between the treatment.Although the sago seedlings grown at higher temperature performed higher optimum irradiance, the utilization of light energy for photosynthetic activity tends to be less efficient.High dissipation of light energy in non-photochemical quenching (NPQ) at 29-33⁰C also supports this analysis.The process is a plant mechanism to protect the photosynthetic apparatus from photo damage due to excess light energy [32].Nonphotochemical quenching dissipates the excess of light energy as heat.

Conclusion
According to the above findings, we conclude that sago palm photosynthetic performance is affected by changes in ambient air temperature especially at the minimum air temperature tested.This refers to the lower P N performance at air temperatures ranging from 25-29⁰C as compared to those at 29-33⁰C.Low P N was brought about by low values in other supporting variables such as stomatal conductance, leaf transpiration rate, maximum rubisco rate in CO 2 reduction, and optimum irradiance.A low level of formation of photosynthetic pigment also becomes a limiting factor in photosynthetic rate at the lower temperatures tested.Consequently, the harvesting and utilization of light energy for photosynthetic activities was affected.From this it could be concluded that the optimum air temperature would be associated with higher sago palm yield.Further investigation of photosynthetic response to different air temperatures using fully grown sago palms is needed to ascertain whether they show the same photosynthetic performance as in the seedling stage.If such study is able to confirm the broader representativeness of the present study, the findings might be used to make important decisions regarding the location of sago palm cultivation areas in the future for optimal production.

25 -
photochemical quenching (qN max ) between the two air temperature ranges.