DOI: 10.5061/dryad.brv15dvf9
title: Contents and isotope carbon compositions (δ13C) of main biochemical fractions in organs at different growth stages in Phyllostachys edulis

description: Phyllostachys edulis is a spectacularly fast-growing species that
 completes its height growth within two months after the shoot emerges
 without producing leaves (fast-growing period, FGP). This phase was
 considered heterotrophic, the carbon necessary for the growth being
 transferred from the mature culms via the rhizomes, although previous
 studies observed key enzymes and anatomical features related to C4-carbon
 fixation in developing culms. We tested whether C4-photosynthesis or
 dark-CO2 fixation through anaplerotic reactions significantly contributes
 to the FGP, resulting in differences in the natural abundance of δ13C in
 bulk organic matter and organic compounds. Further, pulse-13CO2-labelling
 was performed on developing culms, either from the surface or from the
 internal hollow, to ascertain whether significant CO2 fixation occurs in
 developing culms. δ13C of young shoots and developing culms were higher
 (-26.3–-26.9‰) compared to all organs of mature bamboos (-28.4–-30.1‰).
 Developing culms contained chlorophylls, most observed in the skin
 tissues. After pulse-13CO2-labelling, the polar fraction extracted from
 the skin tissues was slightly enriched in 13C, and only a weak 13C
 enrichment was observed in inner tissues. Main carbon source sustaining
 the FGP was not assimilated by the developing culm, while a limited
 anaplerotic fixation of respired CO2 cannot be excluded and is more likely
 than C4-photosynthetic carbon fixation.
description: <strong>Fraction of
 carbon compounds in organs </strong>
 All samples were freeze-dried
 for at least 72 hours using a vacuum freeze dryer (FDU-1200, EYELA, Tokyo,
 Japan), and ground to a fine powder in a ball mill (MM-400, Retsch,
 Düsseldorf, Germany). Weight 50 mg per sample generally (30 mg per sample
 for bamboo shoots in this study). Soluble and insoluble compounds were
 separated based on their polar properties in a mixture of
 methanol–chloroform–water (MCW, 12/5/3, v/v/v). After centrifugation, the
 supernatant was mixed with 0.5 ml of methanol–chloroform (MC, 1/1, v/v) to
 separate pigments and lipids in the lower phase from the polar compounds
 in the upper phase. The pellet was digested with pronase in Tris buffer.
 Proteins were solubilised while starch remains in the insoluble fraction.
 The insoluble fraction was washed with ethanol and starch was gelatinised
 by hydrochloric acid. The starch fraction, recovered in the supernatant
 after centrifugation, was precipitated with absolute methanol. The
 structural fraction was recovered in the pellet washed with water at least
 3 times. All fractions were oven-dried at 65°C and weighed.
 <strong>Carbon isotope
 composition (δ<sup>13</sup>C
 values)</strong> For the determination of the carbon isotope
 composition, 1 mg of dried samples or dried fractions were weighed in tin
 capsules and combusted in elemental analysers coupled to isotope ratio
 mass spectrometers (Thermo FLASH2000, DeltaV advantage with ConfloIV
 system, Thermo Fisher
 Scientific, Massachusetts, USA; for unlabelled samples, and Thermo NC2500,
 MAT252 with ConfloIII system, Thermo Fisher Scientific, Massachusetts,
 USA; for labelled
 samples). Several international standards [IAEA-CH-3 (-24.724‰), USGS40
 (-26.39‰), and USGS41a (36.55‰)] were used for calibrations. USGS 41a was
 further used running standard for every 11 samples. The analysis of the 11
 samples would have been rerun if the running standard had been an outlier
 of all standards, which did not happen. Results
 (δ<sup>13</sup>C) were expressed in ‰ as the relative
 deviation of the isotope ratio of the sample
 (<sup>13</sup>C/<sup>12</sup>C,
 R<sub>sample</sub>) compared to that of the international VPDB
 standard (Vienna PeeDee Belemnite)
 <strong>Chlorophyll in developing
 culms</strong> Two-mm wide
 vertical sections of the skin, middle, and inner parts of the
 7<sup>th</sup> internode in developing culms were cut for
 chlorophyll extraction. Chlorophyll was extracted from 500 mg of fresh
 samples using 4 mL of N, N-dimethylformamide (DMF) as the extraction
 solvent. Extraction was conducted in darkness at 65°C for 2 h followed by
 1 h at room temperature. At the end of the extraction, the samples were
 colourless. The absorbance of the extract was measured at 470, 663.6, and
 646.6 nm using a spectrophotometer (ASV-S3, As One Corporation, Osaka,
 Japan). The amounts of chlorophyll <em>a </em>and
 <em>b</em> were calculated using the extinction
 coefficients.
 <strong>Excess</strong><strong><sup>13</sup>C in after a pulse<sup>13</sup>CO<sub>2</sub> labelling</strong> Six immature, leafless but vigorous culms of similar size were selected (mean DBH = 11.7 cm, mean height = 6 m). Three labelling chambers were installed on three developing culms around the 7<sup>th</sup> internode from the ground without sheaths (chamber size, Φ=20 cm, height =15 cm). The chambers were made of a transparent thin polycarbonate sheet, wrapped around the culm on polypropylene (PP) half-circular plates at both the bottom and the top of the chamber, with semi-circular holes at their centre to accommodate the culm. They were affixed to the surface of the culm with neutral seal putty and a silicon sealant. At 13:20, 75 mL of 99% <sup>13</sup>CO<sub>2 </sub>was injected into each chamber from three directions (three times 25 mL with a needle inserted through the polycarbonate sheet). The concentration of <sup>13</sup>CO<sub>2</sub> in the chambers was 2.4%, based on the volume of the chamber and the amount of <sup>13</sup>CO<sub>2</sub> injected. In the other three developing culms, 50 mL of 99% <sup>13</sup>CO<sub>2</sub> was injected with a needle inserted into the hollow of the 7<sup>th</sup> internode without sheaths. The concentration of <sup>13</sup>CO<sub>2</sub> in the hollows was 3.7%, based on the volume of the hollow and the amount of<sup> 13</sup>CO<sub>2</sub> injected. The small pinhole was sealed by tape. Two hours after the injection of <sup>13</sup>CO<sub>2</sub> in either the chamber or the culm hollow, the 5<sup>th</sup>, 7<sup>th</sup>,and 9<sup>th</sup> internodes of the six labelled and two unlabelled developing culms were sampled. Disks of 100-200 g were collected using a hand saw shortly after labelling to limit the proportion of labelled products potentially transported away from the assimilation site. Samples were frozen in liquid nitrogen and first stored at -15 °C in a portable freezer in the field before being transferred to -20 °C in the laboratory. The method of purifying the polar fraction is the same as the "<strong>Fraction of carbon compounds in organs</strong>" and the method of measuring the carbon isotope composition is the same as the method of <strong>"Carbon isotope composition (δ<sup>13</sup>C values)"</strong>.
description: Excel is required to open these data files.