Ethephon

Two wild type Saccharum species, Molokai6081 (S. robustum, SR 2n = 8× = 80), and SES208 (S. spontaneum, SS, 2n = 8× = 64), one cultivated species, LA Purple (S. officinarum, SO, 2n = 8× = 80), and one hybrid cultivar ROC-22 (SH 2n = 8× = 100–130) were used in this study [43 (link)]. Plants were grown in the field on the campus of Fujian Agricultural and Forestry University (Fuzhou, China) in the February of 2015. Tissue samples from leaf roll, leaf, top internode (i.e., internode number 3), maturing internode (i.e., internode number 9 for ‘LA Purple and Roc-22, internode number 8 for Molokai6081, and internode number 6 for SES208) and mature internode (i.e., internode number 15 for ‘LA Purple’ and Roc-22), internode number 13 for Molokai6081, internode number 9 for SES208) were collected from premature 7-month-old sugarcane plants and 11-month-old mature sugarcane plants from different branches of the same individuals (as replicates). Internodes were numbered from the top, as previously described [44 (link)], and the corresponding internode number for the different Saccharum species, due to the variation in number of stems, was also established according to the previously described approach [45 (link)].
The plants for PEG and hormone treatment were grown in a growth chamber at 30 °C, 70% RH, and a 14 h:10 h L:D photoperiod. Seedlings were treated with PEG6000 (30%) for 48 h, and the leaf tissue was collected for RNA isolation. Seedlings were treated with gibberellin (GA,200 μM), abscisic acid(ABA, 200 μM), indole-3-acetic acid(IAA, 200 μM), or ethephon (Et, 200 μM) for 24, 48, and 96 h. Stem and leaf tissues from the seedlings of the four sugarcane species were collected from 35-day-old plants. Harvested tissue was immediately frozen in liquid nitrogen and stored at −80 °C prior to RNA isolation.

The deduced amino acids of HbAQPs available in the NCBI GenBank were used as queries to search the available RRIM600 genome and our in-house RY7-33-97 genome for rubber tree homologs. Sequences with an E-value of less than 1e⁻⁵ in the tBlastn search [61 (link)] were selected for further analyses. The gene structures were firstly predicted using GeneMark.hmm [62 (link)], and the gene models were further validated with ESTs and raw RNA sequencing reads available at GenBank. The exon-intron structures of AQP genes detected in the laticifer transcriptome were also confirmed by aligning the cloned cDNAs to the corresponding gene sequences. Gene structures were displayed using GSDS [63 (link)]. Homology search for nucleotides or Sanger ESTs was performed using Blastn, and sequences with an identity of more than 98 % were taken into account. RNA sequencing reads were mapped using Bowtie 2 [64 (link)] with default parameters, and mapped read number of more than one was counted as expressed. Unless specific statements, the tools used in this study were performed with default parameters.

Durian (Durio zibethinus Murr.) cv. Monthong and Chanee fruits were collected from a commercial orchard located in Trat province in the eastern part of Thailand in early April 2017. Fruit samples of similar size and weight (~3–4 kg each) were harvested at two different stages: immature (at 70 days (for Chanee) and 85 days (for Monthong) after anthesis) and mature (at 90 days (for Chanee) and 105 days (for Monthong) after anthesis). Immature samples were peeled immediately after harvesting, whereas some mature samples were peeled immediately, and some were kept at room temperature (30 °C) until peeling. Five types of samples were used in this study: immature, mature, unripe (harvested at the mature stage and kept at room temperature for one day (both cultivars) and then peeled), midripe (harvested at the mature stage and kept at room temperature for two days (Chanee) or three days (Monthong) and then peeled), and ripe (harvested at the mature stage and kept at room temperature for four days (Chanee) or five days (Monthong) and then peeled). After peeling, two of the central pulps were collected from each fruit sample, following the method described by Pinsorn et al.^{15 (link)}. Briefly, the first pulp was collected along with a seed, and was used to measure fruit firmness with a texture analyser (TA-XT2i; Stable Micro Systems, Godalming, UK) to assure that samples of the two cultivars were compared at the same ripening stages. A puncture test with a 6-mm probe, at a test speed of 2 mm/s and testing distance of 5 mm, was carried out on five random points in each pulp. Midripe and ripe pulps had a mean ± SD firmness of 3.4 ± 0.81 and 1.55 ± 0.45 N, respectively, in both cultivars. After this test, the second pulp was collected without a seed, immediately frozen in liquid nitrogen, and stored at −80 °C for further use.
To further investigate the role of ethylene during post-harvest ripening, three different ripening treatments were applied as follows: natural, ethephon-induced, and 1-methylcyclopropene (1-MCP)-delayed ripening. Mature durian samples were collected and treated with either ethephon (48% 2-chloroethylphosphonic acid; Alpha Agro Tech Co., Ltd., Thailand) or 1-MCP (0.19% 1-MCP tablet; BioLene Co., Ltd., China) for ethephon-induced and 1-MCP-delayed ripening, respectively. For ethephon treatment, the ethephon solution was applied to the upper area of each fruit stalk. For 1-MCP treatment, each fruit was placed inside a closed 20-L chamber. Then, one tablet of 1-MCP was placed into a beaker inside the chamber. Water (5 mL) was added to the beaker to generate gaseous 1-MCP and the chamber was immediately closed for 12 h at room temperature (30 °C) while the control samples were kept under similar conditions without 1-MCP. After treatment, control and treated samples were kept at room temperature (30 °C) until the ethephon-treated samples ripened. All samples from the three ripening conditions were then peeled. After that, two central pulps were collected from each sample and processed as mentioned previously. In this study, for each type of sample (immature, mature, unripe, midripe, and ripe, as well as natural ripening, ethephon-induced ripening, and 1-MCP-delayed ripening), three biological replicates were used. Each biological replicate was defined as one durian fruit harvested from a separate tree.
For the agroinfiltration experiment, N. benthamiana seeds were sown in pots containing peat moss and were grown under controlled conditions (temperature 25 °C and 16/8 h light/dark photoperiod; artificial light of 4,500 Lux). Two-week-old plants were transplanted individually into pots and were grown under similar conditions.

Exogenous treatments were performed in cv. Cuiguan fruit at approximately 80 DAFB. At least 40 fruits on the tree were soaked in 300 μL l⁻¹ ethephon or 1.5 μL l⁻¹ 1-methylcyclopropene (1-MCP) solutions for 5 min. To block light and limit gas exchange, the treated fruits were bagged with black airtight papers that were removed after 24 h. The fruits treated with ethephon were collected at the ripening stage, while the fruits treated with 1-MCP were collected on the same day as the control fruits at the ripening stage. At least six fruits were included in each of six biological replicates. The sampling regime was identical to a previous report [32 (link)].

Rabbiteye blueberry ‘Powderblue’ plants grown at the Durham Horticulture Farm in Watkinsville, GA were used for ethephon treatments on June 23rd, 2017. Ethephon (250 mg·L^− 1) and control treatments were applied following the method described in [75 (link)]. Each treatment had four biological replicates. Phenotypic data collected included ethylene evolution from fruit and the rate of ripening. Ethylene evolution from fruit was measured using a closed system. Approximately 25 g of fruit were harvested from each blueberry plant (biological replicates) two days after treatment and incubated in an air-tight 125 mL glass jar with a lid fitted with a rubber septum, for 4 h at room temperature. Headspace samples (1 mL) were analyzed by GC-17 A gas chromatography (GC-17 A, Shimadzu, Japan) equipped with a 2 m micropacked column (Hayesep N, Restek, PA, United States) and a flame ionization detector. The temperature of the injection port and the detector of the GC were set at 200 ^oC. The temperature program was 60 ^oC for 4 min; increased by 20 ^oC.min^− 1 to 150 ^oC; and held at 150 ^oC for 1 min. The peak area from the resulting chromatograph and a standard curve generated using various concentrations of ethylene were used to determine ethylene evolution from the fruit sample and expressed as nL·g^− 1·h^− 1. The rate of ripening was determined by visual color assessment, following the method described in [75 (link)]. In short, 3 branches were tagged on each blueberry plant, included around 100 fruits in total. The color of Green, Pink, and Ripe (blue) fruits on the 3 branches were counted when fruits were still attached to the plant at 0, 1, 2, 3, 5, 7, 10, 14 days after the treatment. Green fruit were classified as mature and appearance of color (~ 25% pink coloration) indicating ripening initiation. Pink fruit displayed predominantly pink with some blue (< 10%) and Ripe as fully blue fruit.