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.

Fraction of carbon compounds in organs

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.

Carbon isotope composition (δ¹³C values)

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 (δ¹³C) were expressed in ‰ as the relative deviation of the isotope ratio of the sample (¹³C/¹²C, R_sample) compared to that of the international VPDB standard (Vienna PeeDee Belemnite)

Chlorophyll in developing culms

Two-mm wide vertical sections of the skin, middle, and inner parts of the 7^th 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 a and b were calculated using the extinction coefficients.

Excess¹³C in after a pulse¹³CO₂ labelling

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^th 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% ¹³CO₂ was injected into each chamber from three directions (three times 25 mL with a needle inserted through the polycarbonate sheet). The concentration of ¹³CO₂ in the chambers was 2.4%, based on the volume of the chamber and the amount of ¹³CO₂ injected. In the other three developing culms, 50 mL of 99% ¹³CO₂ was injected with a needle inserted into the hollow of the 7^th internode without sheaths. The concentration of ¹³CO₂ in the hollows was 3.7%, based on the volume of the hollow and the amount of ¹³CO₂ injected. The small pinhole was sealed by tape. Two hours after the injection of ¹³CO₂ in either the chamber or the culm hollow, the 5^th, 7^th,and 9^th 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 "Fraction of carbon compounds in organs" and the method of measuring the carbon isotope composition is the same as the method of "Carbon isotope composition (δ¹³C values)".

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