Material’s preparation
In this study, “Taikong 36”, which was bred by research base of Guangchang space lotus (this species of lotus has been deposited in a publicly available herbarium) belonging to seed lotus was selected for all the experimental analysis due to enough material resource. “Taikong 36” was harvested from Guangxi province, and grown in the experimental field of aquatic vegetables research group of Yangzhou University, Southeast China with conventional management in spring. We have acquired a permission to acquired an permission to collect the plant samples. In the grown season, the temperature was 30±5°C/day and 25±5°C/night, and the average water depth of field (the plant must be maintained in the water) was 30±10 cm. The seeds were harvested after 30 d of flowering, and kept in the warehouse under normal temperature condition. High activity of lotus seeds could be maintained within ten years.
Paraffin sections experiment
The seed coat of “Taikong 36”lotus were broken and soaked in the water for germinated. The seedlings of lotus were put into various light intensities including darkness, 5000 lux and 20000 lux with the temperature of 30°C/day and 20°C/night. Lotus hypocotyls these treatment were chosen after day of 0, 1, 2, 3, 4, and 5 post-treatment. The hypocotyls sections of 2 mm × 2 mm × 2 mm (length × width × height) were prepared, and placed into a small container with about 10 ml free fatty acid fixing fluid ( the valum of free fatty acid fixing fluid was twenty times than that of each sample). The vacuum condition of above small container was made with a syringe. The samples of these treatments were transferred into vacuum environment for 10 sec, and then exchanged gas for 10 min. Above process was repeated for three time with normal temperature overnight. Various concentrations of ethanol (50%, 60%, 70%, 80%, 90% and 100%) were prepared and used to treat samples for 20 min sequentially. These dehydrated samples were put into a mixed slution (pure xylene: absolute ethanol; 1:1), and then transfer into pure xylene for about 20-30 min respectively. The above samples wrapped in paraffin were placed into a container for at least 12 h at normal temperature, and then transferred into the thawed paraffin wax for 18-24 h for prepared paraffin blocks. An incisive slicer was used to prepare a 10 microns thickness wax tape, which was then placed on the glass slide. The samples on the glass slide were treated in turn by xylene, mixed solution (pure xylene: absolute ethanol; 1:1) and absolute ethanol for about 10 min. At last, these slides were dried in air under normal temperature condition, and the tissues on the slide were identified using an optical microscope.
RNA-sequencing analysis
For this experiment, the lotus seed coat was broken for available uptake of water, and germinated at 28-30 °C, and then transferred into a container with 5 cm of water depth for growth. The one-leaf seedlings were put into three light intensity including darkness, 5000 lux and 20000 lux for investigation of ARs analysis. The materials of these treatments were chosen at day 0 (CK0), and day 3 (CK1: the sample for CK1 library construction under darkness condition, E: The sample for E library construction with 5000 lux treatment, F: the sample for F library construction with 20000 lux treatment) of treatments. The total RNA of hypocotyls was prepared, and digested with DNaseI for purification. About 3 ug RNA of each samples was applied for library construction. Sample preparation kits of Illumina gene expression was used for CK0, CK1, E and F libraries construction. All the processes of libraries was referred by Cheng et al. (2018) [33]. The detail work was completed by Beijing Institute of Genomics (BIG) with a special construct.
Screening of differentially expressed genes (DEGs)
The DEGs of differential libraries were screened according to the method described previously [72-73]. Further analysis of reads derived from all libraries was carried out by NOISeq technique, which was reported by Cheng et al. (2018) [33]. log2 (fold-change) M was used as the relative expression of genes in each library, and the noise distribution model was built according to the absolute value of difference (D). To identiy whether gene A was DEG, the average expression in control group (control_avg) and treatment group (treat_avg) was firstly computed, and then the value of different expressing change and “D” were obtained according to the data of fold change (MA = log2 ((treat_avg)/(control_avg))) and the absolute value of difference (DA = |control_avg - treat_avg|). At last, the gene A was considered as DEG if If MA and DA measured by probability value significantly diverged from the noise distribution model. Totally, the fold change of expressed gene A was more than two and the divergence probability was more than 0.8 was believed as DEGs.
Functional analysis of DEGs
All the DEGs in each library involved in molecular function, cellular component, and biological process were annotated by the Gene Ontology (GO) tool. The biological function of DEGs was obtained according to the comparing result with the National Center for Biotechnology Information (NCBI) database. The number of DEGs involved in above three ontologies were computed after compared with the GO database (http://www.geneontology.org/), and then the DEGs were collected together as GO terms by hypergeometric test. In addition, KEGG tool was used for pathway analysis, and so theses DEGs were enriched into various metabolic processes.
Relative expression analysis
Relative expression of some important genes was documented to identify the changes of metabolism in various light intensity treatment. The lotus seeds treatment and cultivated conditions were the same as mentioned above. The samples were collected at 0 (the germinated seeds) and day 3 (germinated seeds which were cultivated for three days at darkness, 5000 lux and 20000 lux condition respectively). Quantitative polymerase chain reaction (qPCR) was applied to monitor the change of gene expression, and the detailed method was referred as described previously (Li et al. 2019; Zhang et al. 2019). The RNA extraction mini kit (QIAGEN, Germany) was used to obtain the total RNA of lotus hypocotyls, and DNA contamination were removed by DNaseI. First Strand cDNA Synthesis Kit (Fermentas, USA) [74](Jiao et al. 2019) was applied to synthesized cDNA (about 3-5 µg of total RNA). The transcriptional level was investigated by SYBR Green Master Mix (Tiangen, China) on the Mx 3000P machine (STRATAGENE, http:// www.stratagene.com) [62] with three repeated experiments. The primers of selected genes were derived from NCBI database, and β-Actin was considered as referred genes to measure genes relative expression. The primers of β-Actin was: upstream, 5'-AACCTCCTCCTCATCGTACT-3', and downstream, 5'-GACAGCATCAGCCATGTTCA-3', and the primers of chosen genes was listed in additional file 1: Table S1. The total volume of PCR reaction system was 25 μL including Premix Ex Taq II (TliRNaseH Plus) (2X) of 12.5 μL SYBR, forward and reverse primers of 10 μM each, cDNA solution of 2 μL, and distilled water of 8.5 μL. The program of PCR was concluded 94 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 55°C~60 °C for 60 s. The data analysis was performed by 2-△△Ct method described as Cheng et al (2019a) [49].
Analysis of IAA content
The seeds of lotus were broken and then put into a container with 5cm water in depth for germination at 26 °C for about 4-5 d. The germinated seeds were transferred into four light intensities for continue growth (dark, 5 000 lx, 15 000 lx and 30 000 lx), and lotus hypocotyls of seedlings were selected at 0 d 0 d, 2 d, 4 d, 6 d, 8 d and 10 d of each treatment for IAA identification according to the following procedure. Firstly, the hypocotyls of each treatment were put into liquid nitriogen to grind into powder, and then 50 mg of sample powder was transferred into a 2 ml centrifugal tube, which was pre-cooled in advance. Secondly, about 500 μl of extraction reagent (V/V/V: isopropyl alcohol: water: concentrated hydrochloric acid = 2:1: 0.002) were put into the tuber. The tuber were oscillated at 4℃ for 30 min at 100 rpm, and 1 ml dichloromethane was added. After shaking at 4℃ for 30 minutes, the mixed solution were centrifuged with 12,000-13,000 rpm at 4℃ for 5 minutes. 900 ml of lower layer supernatant was extracted, and dried with nitrogen. 100 ml of methanol was added to resolute the dry powder, and then filtered and injected into the bottle. 50 ml of sample was injected into the C18 column of liquid chromatography for IAA identification.
Complementary effect of sucrose on IAA treatment to lotus ARs
The lotus seed (Taikong 36) was broken for available uptaking water, and then was group into four groups. The seeds of first group were placed into water for germination, and the second group were treated with 150 uM IAA for two days and then transferred into water for germination. The seeds of third group were put into 20 g/L sucrouse for two days and then transferred into water, and the seeds of fourth group were placed into the solution with 20 g/L and 150 uM IAA for two days. The conditions of germination including temperature, water deep and light intensity were the same descripted as above. After germination of seeds, the number of ARs was counted with an interval of 1 day within six days. The experiment was repeated for three time.