Dependance of isoprene emission flux on leaf mass per area of Phyllostachys pubescens (moso bamboo)

It is challenging to estimate isoprene emissions from plants and determine the basal isoprene emission rate ( i.e., isoprene emission capacity under a specific light and leaf temperature ) of plant species. Previous studies have investigated the effect of physiological factors on isoprene emission capacity; however, the effect of leaf morphology on isoprene emission capacity has seldom been mentioned. This study aims to clarify the relationship between the basal isoprene emission rate and leaf mass per area ( LMA ) of a woody bamboo ( Phyllostachys pubescens ) . Since there was no observation of isoprene emission from low-LMA leaves of P. pubescens , we conducted measurements on culms exhibiting lower LMA ( 27.5 - 47.9 g m - 2 ) . By observing leaf-scale isoprene emission flux under a specific incident light ( 1000 μ mol m - 2 s - 1 ) and temperature ( 30 ° C ) to represent basal isoprene emission fluxes, we found a series of varied area-based isoprene emission rate among leaves ( 1.4 - 32.2 nmol m - 2 s - 1 ) and a strong correlation between area-based isoprene emission rate and LMA without any distinction between culms. A further comparison with other studies demonstrated that even for the culms that exhibited larger LMA and isoprene emission flux, a generally consistent pattern in the relation of area-isoprene emission flux and LMA could be found across these sites. This result suggests the importance of detecting LMA in the determination of the basal isoprene emission rate, which can improve the current emission estimation method.


Introduction
Largescale emissions of isoprene from vegetation have been revealed by several global estimation studies, which show that plant-based emissions can be 4 -7 times the global anthropogenic emissions of non-methane volatile organic compound emissions Guenther et al., 2006;Guenther et al., 2012;Kansal, 2009;Saunois et al., 2020;Sindelarova et al., 2014 . The emission of isoprene can affect atmospheric chemistry and cause negative effects such as the formation of air pollution e.g., ozone, oxidants, and secondary organic aerosols with nitrogen oxides Claeys et al., 2004;Kanakidou et al., 2005;Paulson and Seinfeld, 1992;Teng et al., 2017 ; moreover, isoprene emissions can alter the global warming potential by changing the lifetime of methane and as a carbon emission itself Archibald et al., 2011;Fehsenfeld et al., 1992;Pike and Young 2009 . The assessment of global isoprene emissions is necessary for the mitigation of isoprene-induced negative effects.
The current global estimation of isoprene emissions, such as the Model of Emissions of Gases and Aerosols from Nature MEGAN, Guenther et al., 2006 combines meteorological data, land use maps, emission inventories i.e. basal isoprene emission, the isoprene emission capacity under a specific light and leaf temperature , activity factors including responses to light, temperature, leaf age, soil moisture, leaf area index, and CO 2 inhibition. It has been shown that estimation results are highly sensitive to emission inventory Henrot et al., 2017 . Since isoprene emission capacity could change among different species and have intraspecific variation, determination of emission inventory should be carefully obtained from field observations or scientific estimations.
Recent reports have indicated that, Phyllostachys pubescens moso bamboo , a woody bamboo that exhibits invasion and expansion Akutsu et al., 2012;Bai et al., 2013;Kudo et al., 2011;Takada et al., 2012 , showed a potential of high isoprene emission, however, with discrepancies in basal isoprene emission flux detected among sites, or even among leaves. Chang et al., 2019;Okumura et al., 2018 . Physiology-linked factors e.g., temperature, leaf nitrogen concentration, photosynthetic limitations of isoprene emission capacity of the plant leaves have been well shown by previous studies e.g., Beckett et al., 2012;Litvak et al., 1996;Niinemets et al., 1999;Oku et al., 2014;Rosenstiel et al., 2004 . Currently, however, knowledge of the relationship between morphologic effect and isoprene emission is limited, only Harley et al. 1997 reported that sunlit leaves with higher leaf mass per area LMA showed higher area-based isoprene emission rate than shaded leaves for deciduous oak species. Because leaf morphology could determine the abundance of chloroplast in unit area, the area-based isoprene emission flux is expected to respond to the change in leaf morphology since the production of isoprene is dependent to chloroplast. Therefore, by clarifying the relationship between leaf morphology and isoprene emission, a better determination of the emission factor for isoprene could be achieved.
According to previous research, LMA could be an indicator of number of chloroplasts per area Hanba et al., 1999;Ivanova et al., 2018;Liakoura et al., 2009 , therefore, this study hypothesizes a linkage between area-based isoprene emission flux and LMA to explain the variation in isoprene emission capacity for moso bamboo and aims to determine the relationship between isoprene emission capacity and LMA. To test this hypothesis, we conducted isoprene emission measurements in constant environmental conditions on a hillslope that demonstrates a high morphological diversity for moso bamboo culms. It has been showed that the LMA of moso bamboo leaves could vary largely, from 25 to 70 g m -2 Lin et al., 2020 , but only isoprene emission from leaves with higher LMA > 55 g m -2 has been observed e.g., Chang et al., 2019;Chang et al., under submission . Due to the lack of isoprene emission observations of moso bamboo leaves with LMA of < 55 g m -2 , this study conducted measurements on leaves of overtopped moso bamboo culms to fill the gap in isoprene emission traits with lower LMA. Because the photosynthetic rate and nitrogen concentration could also influence isoprene emission capacity Harley et al., 1994;Litvak et al., 1996;Niinemets et al., 1999;Rasulov et al., 2009;Rosenstiel et al., 2004 , we also recorded these factors to help in determining the attribution of LMA.

Site description
The field work was conducted in an unmanaged pure moso bamboo stand on a hillslope in Fukuoka Prefecture, Japan 33 38' N, 130 33' E with a slope angle of 42.8 . This site has a subtropical monsoon climate with an average annual temperature of 15.9 C and annual precipitation of 1833 mm.
Average culm density, diameter at breast height DBH , and height of 8000 480 culms per hectare, 9.5 0.7 cm, and 11.1 0.7 m were recorded, respectively. Previous investigations of vegetation and soil indicated large spatial variations in culm density, culm height, DBH, biomass distribution, soil nitrogen content, and soil moisture at this site Ichihashi et al., 2015;Shimono et al., 2021 . Eight moso bamboo culms Culm A to Culm H were chosen for measurement at the site, each of which demonstrated different DBH and culm height; the age of these culms were less than two years during measurement. Note that the chosen culms demonstrated lower culm height 4.2 -7.9 m and DBH 2.0 -5.2 cm to their neighboring culms and had weaker light exposure than the top of the canopy Table 1 . For each culm, four leaves near the top of the crown were measured for isoprene emission flux, photosynthetic rate, nitrogen concentration, and LMA.

Measurements
In this study, the culm height and DBH for each of the selected culms were measured, as were the isoprene emission flux, photosynthetic rate, LMA, and nitrogen concentration for each of the chosen leaves.
The measurement period of isoprene emission rate and photosynthetic rate was August 14 -17, 2019. A portable photosynthesis measuring system LI-6400, Li-Cor Inc., Lincoln, NE, USA equipped with an LED cuvette LI-6400-02B, Li-Cor Inc. was used to conduct the measurements. To capture isoprene, a T-junction made of Teflon to avoid adsorption of VOCs was added to replace the original tube between the leaf cuvette of LI-6400 and its embedded infrared gas analyzer IRGA , adding another channel that can be plugged to an adsorbent tube; a granular filter filled with activated charcoal was connected to the air inlet of the LI-6400 system to supply VOC-free air. The adsorbent tube used for isoprene collection is made of glass and filled with 250 mg Tenax-TA 60/80 mesh GL Science Inc., Tokyo, Japan , based on the method tested and verified by Chang 2009 . During sampling, the light and leaf temperatures in the cuvette were set at a photosynthetic photon density flux PPFD of 1000 μmol m -2 s -1 and a temperature of 30 C. Leaves were clamped by the leaf cuvette for approximately 5 min to stabilize gas exchange under the controlled light and leaf temperatures. During this period, the photosynthetic rate was recorded once it were determined as follows: is the measured value of the photosynthetic rate with the default leaf area R Origin set at 6 cm 2 , R Cuvette cm 2 is the actual in-cuvette leaf area, M CO 2 is the molecular mass of CO 2 44.01 g mol -1 , R Leaf cm 2 and M Leaf g are the whole leaf area and dry mass of the measured leaf, respectively. The LMA g m -2 of the leaves was determined using R Leaf and M Leaf . To obtain R Leaf , the leaves were scanned rapidly after excision with a scanner GT-S650, Seiko Epson Corporation, Nagano, Japan before deformation due to dehydration, and measured using an image processing software ImageJ, National Institutes of Health, Bethesda, MD, USA . The scanned leaves were then dried in an oven at 60 C for 72 h for M Leaf measurement with a microbalance accuracy: 0.1 mg . The quantification of isoprene emissions was determined by gas chromatography-mass spectrometry. The isoprene content in the adsorbent tube was first desorbed and re-trapped with a preconcentrator Model 7100A, Entech Instruments Inc., CA, USA , and then introduced into a gas chromatography system with a mass spectrometer HP6890, Agilent Technologies Inc., CA, USA for identification and quantification. A calibration line R 2 > 0.995 was obtained by testing standard samples at different isoprene concentrations 5, 10, 20, 50, and 100 ppb with the same air flow as the actual field measurements. The obtained isoprene concentration C Isoprene was then used to calculate the area-based isoprene emission flux I Area , nmol m -2 s -1 and mass-based isoprene emission flux I Mass , μg g -1 hr -1 using the following equation: where F μmol s -1 is the flow rate of the LI-6400 air inflow, and M isoprene is the molecular mass of isoprene 68.12 g mol -1 .
The whole leaf nitrogen content N Content , mg was determined using an element analyzer JM1000 system, J-SCIENCE LAB, Co., Ltd., Japan based on the Pregl-Dumas method. A calibration line R 2 > 0.999 was established by testing the standard material hippuric acid, C 9 H 9 NO 3 in different masses 3, 6, 9, 20, 30, and 50 mg . Area-based nitrogen concentration N Area , g m -2 and mass-based nitrogen concentration N Mass , are defined as follows:

Results
The moso bamboo culms selected for measurement exhibited various morphologies, with differing DBH and culm height measured; observations including I Area , I Mass , LMA, A Area , A Mass , N Area , and N Mass also demonstrated variations among culms Table 1 . Even under the same irradiance PPFD = 1000 μmol m -2 s -1 and leaf temperature ~30 C , large variations in isoprene emission fluxes were recorded  I Area and I Mass were more likely to be associated with varying leaf morphology instead with DBH or culm height though the culms exhibited a large variety in these culm morphologies Table 1 . As shown in Fig. 1, I Area significantly increased with LMA. Basing on Pearson' r value R and t-test P value P , a strong correlation between I Area and LMA P < 0.001; R = 0.666 were observed, and LMA was determined to be the most significant factor influencing I Area ; the effect of LMA on I Mass was less, but still significant Table 2 .
Both A Area and N Area exhibited significant positive correlations with I Area Table 2 . No relationship was detected, however, between N Mass and I Mass or between A Mass and I Mass . Note that all three observations in the area-based units demonstrated strong correlations with LMA, which explained most of the variation in them Table 2 .
By comparing our results with other studies on isoprene emission from moso bamboo Chang et al., 2019;Chang et al., under submission , we can see that I Area in this study was remarkably lower than those in other studies Fig. 2a  Although the height of the measured culm was relatively low 6.5 m , the leaves were well exposed to sunlight due to a far distribution between each culm at this site and demonstrated a moderate LMA among the sites 52.7 2.3 g m -2 . The data collected from this site were recorded in August 2019 with a leaf temperature of 31.4 1.0 C and a PPFD of 1000 μmol m -2 s -1 . .235 ---*p-value < .05; **p-value < .01; ***p-value < .001

Fig. 2.
Relationship between a leaf mass per area LMA and area-based isoprene emission flux I Area , and b LMA and I Area adjusted by G93 algorithm Guenther et al., 1993 in different sites This study, Chang et al., 2019, and Chang et al., under submission . Open circles represent the observation averaged by each culm in this study N = 4 per culm, leaf temperature = 29.9 0.0 C ; solid square represents the averaged observation from a newly abandoned moso bamboo stand in Taiwan conducted by Chang et al., 2019 N = 4, leaf temperature = 25.7 1.6 C ; solid triangle represents the averaged observation from a moso bamboo plot in a specimen garden in Kyoto conducted by Chang et al.,under submission N = 3; leaf temperature = 31.4 1.0 C .
With I Area adjusted by the G93 model Guenther et al., 1993 to simulate a temperature of 30 C and PPFD of 1000 μmol m -2 s -1 , a pattern between the adjusted I Area and LMA that was generally consistent across the different sites is demonstrated Fig. 2 .

Discussion
According to Poorter et al. 2009 , leaf morphology is strongly related to light of growth environment in most vegetation. Furthermore, bamboo leaves grown under shaded condition were reported to be thinner than leaves grown under sunlit condition March and Clark, 2011 . Therefore, a lower LMA would be expected in the overtopped moso bamboo culms in this site. These overtopped leaves demonstrated lower LMA 27.5 -47.9 g m -2 when compared to recorded averages according to Lin et al. 2020 . The large range of LMA recorded in this study could be due to the consequently various canopy gap sizes to the large spatial variation in culm density in this site. Even under the same incident light and temperature, isoprene emission fluxes demonstrated large variance among the leaves in this study. By plotting the isoprene emission fluxes and LMA, we found that LMA explained most of the variation in I Area and part of the variation in I Mass . Considering the measurements in a previous study on moso bamboos i.e., Chang et al., 2019;under submission conducted under similar incident light and season, a consistent pattern was observed between the I Area and LMA across these sites. The linkage between I Area and LMA could be explained by the higher quantity of chloroplasts per unit leaf area for leaves with higher LMA. According to an allometry study of bamboo species, leaf density is proportional to about -3/4 power of leaf thickness Lin et al., 2018 . Since LMA can be decomposed as the product of leaf density and leaf thickness LMA = leaf density leaf thickness , a higher LMA implies thicker mesophylls, which tend to have larger chloroplasts per area Hanba et al., 1999;Ivanova et al., 2018;Liakoura et al., 2009 ; isoprene is produced only by chloroplasts in leaves Sasaki et al., 2005;Wildermuth and Fall, 1996;Wildermuth and Fall, 1998 , meaning a larger quantity of chloroplasts per area could induce larger I Area at the leaf scale. Nevertheless, the amount of chloroplast per area or efficiency of chloroplast could be affected by many factors confound with LMA e.g., water stress, growth temperature, and growth light Gamble and Burke, 1984;Gotoh et al., 2018;Taylor and Craig, 1971 . Thus, further research is needed to identify actual mechanism causing the dependence on LMA of I Area .
The positive correlation between I Mass and LMA Table 2 could be partially explained by an increased proportion of mesophyll in leaves with larger LMA. The linkage between these factors also infers potential effects from factors beyond morphological characteristics, indicating that leaves with higher LMA may exhibit more efficient isoprene production per unit mass. By analyzing the effect from nitrogen concentration and photosynthetic rate on isoprene emission capacity, we detected correlations between I Area , N Area , and A Area Table 2 . However, since I Area , N Area , and A Area were all demonstrated strong correlations with LMA, LMA could be a confounding variable and lead to spurious correlations between N Area and I Area and between A Area and I Area . To exclude this effect, we analyzed the mass-based form and found no correlation between A Mass and I Mass , nor between N Mass and I Mass . Since nitrogen in ammonium form could potentially enhance isoprene production by enlarging the substrate dimethylallyl diphosphate pool of isoprene synthesis Rosenstiel et al., 2004 , no correlation between I Mass and N Mass implies that the substrate for isoprene production was not constrained by nitrogen status of leaf during our measurement. The dependency of isoprene production on photosynthesis mainly comes from the energetic and reductive agents produced in light-dependent reactions. Previous studies have revealed that this dependency is more likely to relate to the electron transport chain rather than the whole photosynthesis process since photosynthetic rate could be limited by other factors such as stomatal conductance Rodrigues et al., 2020 . Although A Mass did not explain I Mass , we could not exclude the effect of electron transport rate, which has been reported to have a significant influence on isoprene emissions Rasulov et al., 2009 .

Conclusion
This study measured isoprene emission flux from low-LMA moso bamboo leaves under a constant light of 1000 μmol m -2 s -1 and leaf temperature of ~30 C. By combining the observations of moso bamboo with higher LMA conducted in previous studies, we verified a consistency between isoprene emission capacity and LMA of moso bamboo leaf under a variety of LMA. Because area-based isoprene emission capacity is a critical factor in current global-scale isoprene emission estimation methods, the detection of LMA can provide a better way to determine the isoprene emission capacity of plant leaves.