Chlorophyll A

Growth was measured in terms of fresh weight. Seedlings were selected randomly from control and treated samples and then their fresh weight was determined. For the estimation of photosynthetic pigments (total chlorophyll, chlorophyll a + chlorophyll b), the method of Lichtenthaler (1987) (link) was adopted. For the assessment of photosynthetic performance, chlorophyll a fluorescence measurements were taken in the dark adapted leaves of control and treated seedlings using hand held leaf fluorometer (FluorPen FP 100, Photos System Instrument, Czech Republic). The estimation of NO was performed according to the method of Zhou et al. (2005) (link) as described in Singh et al. (2015) (link).

To analyze the chlorophylls and total carotenoids content in the transgenic lines, 200 mg of leaves homogenized in liquid nitrogen was extracted twice with 2 ml of 100% methanol. Extraction was carried out at room temperature for 1 h in the dark with constant shaking. Methanol fraction from both extracts was pooled and diluted 5 folds before their absorbance values at wavelengths 666 nm, 653 nm and 470 nm were determined using an Infinite M2000 microplate reader (Tecan, Switzerland). The relative amount of chlorophyll a, chlorophyll b and total carotenoids were calculated from their absorbance values using previously reported formula [46 (link)].

Samples were collected during the European leg of The Sorcerer II Global Ocean Sampling expedition in July 2009 with parallel environmental characterization using an integrated CTD-oxygen, chlorophyll a, and pH sensor package. For each country, a Marine Science Research Permit was submitted under the U.N. Conference on Law of the Sea to the U.S. State Department which contacted the Ministry of Foreign Affairs in the respective country. Submissions were then distributed to the appropriate agency in each country. For Sweden, permission was granted by the Swedish Coast Guard. For Denmark, permission was granted by the Danish Ministry of Foreign Affairs. For Germany, permission was granted by the German Federal Maritime and Hydrographic Agency (BSH). In addition, the Access and Benefit Sharing Focal Point was contacted under the U.N. Convention on Biological Diversity to ascertain whether additional permissions were required to access genetic resources. Separate applications were not required in the visited countries.
A total of 21 microbial communities were sampled at 11 locations within the brackish Baltic Sea area (Fig. 1A, Table S1). Eight locations were sampled at two depths (including seven surface/chl a maximum pairs and one chl a maximum/suboxic zone pair), one location was sampled at three depths (surface/chl a maximum/72 m) and at two locations only surface samples were taken. Environmental metadata and coordinates for each sampling location are given in Table S1. Sample preservation, extraction, and sequencing was conducted as described previously [3] (link). Reads were annotated using the JCVI metagenomic annotation pipeline [18] (link), along with APIS [3] (link), and fragment recruitment [2] (link). A global assembly of all sequence data was performed using the Newbler Assembler [19] (link), with a final assembly of 490 million basepairs, a N50 of 1.5 Kbp, and a largest contig of 106 Kbp. Approximately 15,000 16S rRNA subunit amplicons were also sequenced for each location and size fraction and phylogenetically annotated as described previously [16] (link). All sequence and annotation data is available at CAMERA [20] (link) under accession number CAM_P_0001109 and Metarep [21] (link) (metarep.jcvi.org). Detailed descriptions of community composition determinations and regularized canonical correlation analysis (RCCA) are available in the Supplementary information.

Seeds of ETH3 and control were collected at the fruit mature stage of ‘Huashuo’. The soluble sugar content was determined using anthrone colorimetry (Liu et al., 2015 (link)). The contents of sucrose and reducing sugars were evaluated using the 3,5-dinitrosalicylic acid method (Yang et al., 2017 (link)). Endogenous ethylene content was evaluated by the ACC content (Hu et al., 2021 (link)). The grinded samples of 0.5 g were homogenized in phosphate-buffered saline, and then centrifuged at for 20 min (4°C, 12000 rpm). These supernatants were used to measure the ACC contents. The ACC contents of the seed and shell were measured according to the Plant 1-aminocyclopropane carboxylic acid ACC kit (Shanghai Jingkang Bioengineering, Co., Ltd., Shanghai, China) instructions (Hu et al., 2021 (link)). The OD450 value was determined using a microplate reader (BioTek, Winooski, Vermont, USA).
Ten leaves from one tree were randomly selected to measure the chlorophyll content for each biological replicate. Leaves of ethephon treatment and control were cut into filaments. The filaments of 0.2 g were immersed in an acetone–ethanol mixture (2:1, v/v) for 24 h (4°C, darkness). The samples were shaken several times during the experiment. The absorbance indexes at 663 and 645 nm of the solution were assessed by a spectrophotometer (UV-1100, Mapada, China). The chlorophyll a and chlorophyll b contents were calculated, referring to the method of Zhang et al. (Zhang et al., 2021 (link)).

Four weeks after the beginning of the water deficit treatment, plants were harvested. Before harvesting, we measured chlorophyll a fluorescence and collected leaves for physiological parameters on 10 plants per population and water treatment. Plant biomass was collected, and relative water content, cell membrane permeability, pigments and carbohydrates were analyzed on fresh leaves, and antioxidant activity was analyzed on oven-dried leaves.

Photosynthesis was assessed by measuring the chlorophyll a fluorescence using a portable fluorometer (FluorPen FP100 PAM, Photo System Instruments, Czech Republic). After dark adaptation (for at least 30 min), the minimum fluorescence was measured by applying a weak-intensity modulated light and the maximum fluorescence in the dark was measured after using a saturating pulse of light. Then, leaves were adapted to light conditions. The steady-state fluorescence was established, and the maximum fluorescence in light was assessed after a saturating light pulse. The maximum quantum efficiency of photosystem II (F_v/F_m), the effective quantum efficiency of PSII (Φ_PSII), the photochemical quenching (qP) and non-photochemical quenching (NPQ) were calculated according to van Kooten and Snel (1990) (link).
Chlorophyll a (Chl a), chlorophyll b (Chl b), carotenoids and anthocyanins were quantified as described by Sims and Gamon (2002) (link). For the photosynthetic pigments’ extraction, leaf discs were homogenized with an acetone:50 mM Tris (80:20) buffer, and for the anthocyanins’ extraction, leaf discs were homogenized with a methanol/HCL/H₂O (90:1:1) solution. After centrifugation (5 000 g for 5 min at 4°C), the absorbance of the acetone extracts was read at 470, 537, 647 and 663 nm and the methanolic extracts were read at 529 and 650 nm using a Jenway 7305 spectrophotometer. The contents of pigments were calculated according to Sims and Gamon (2002) (link).