Gene expression profiling reveals light regulation on adventitious root formation in lotus (Nelumbo nucifera Gaertn.)

doi:10.21203/rs.3.rs-17592/v1

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Research article

Gene expression profiling reveals light regulation on adventitious root formation in lotus (Nelumbo nucifera Gaertn.)

https://doi.org/10.21203/rs.3.rs-17592/v1

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published 12 Oct, 2020

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Background: Lotus is an aquatic horticultural crop, and widely cultivated in most regions of China. Lotus is often used as a kind of an important off-season vegetable with various nutrients. Principle root of lotus is degenerated, and the role of adventitious roots (ARs) is Irreplaceable for plant growth. We found that no ARs could be formed under darkness condition, and high light significantly promote the development of root primordium. Therefore, four libraries with three light intensities were constructed to monitor metabolism changes, especially in IAA and sugar metabolism.

Results: We found that ARs formation was significantly affected by light, high light intensity accelerated ARs development. The change of metabolism during ARs formation under different light intensity was evaluated according to gene expression profiling by high-throughput tag-sequencing. It was shown that more than 2.2× 10 4 genes was obtained in each library, and the expression level of most genes were distributed between 1e-01 and 1e+03 (FPKF value). Among these identified genes, 1739, 1683 and 1462 genes were up-regulated, and 1533, 995 and 834 genes were down-regulated in D/CK, E/CK and F/CK libraries respectively. In addition, we also found that 1454 and 478 genes changed expression in D/CK and F/CK libraries when compared D/CK with F/CK libraries. In F/D libraries, the transcriptional level of most differentially expressed genes was between -5~5 fold, and twenty differentially expressed genes were involved in signal transduction pathway. Twenty-eight genes related with IAA response and thirty-five genes involved in sugar metabolism were found to change expression. It was elucidated that IAA content was enhanced after seed germinated, even in darkness condition, which was responsible for ARs formation.

Conclusion: The process of ARs formation was very complex, and regulated by multiple factors. The ARs formation was regulated by IAA, even in the dark, induction and developmental process could also be completed. In addition, the genes (36 genes) changed expression level in carbohydrate metabolism showed the third number, so sucrose metabolism was involved in the ARs development (expressed stage) according to genes expression and content change characteristic.

Epigenetics & Genomics

Lotus

ARs

light

gene

IAA

sucrose

Lotus is widely cultivated in the South region of the Yellow River Basi, and about 200,000 hectares of lotus, which commonly is classified in three phenotype, such as flower lotus for ornamental, rhizome lotus for vegetable, and seed lotus for food is mainly distributed in Hubei, Jiangsu, Anhui, Guangdong, and Shandong provinces. As a kind of vegetable, the product organ (rhizome) can be continuously supplied local market due to its easy storage in soil from October to April of the next year. Traditional cuisine such as steamed Lotus, boiled lotus and lotus soup is very popular among consumers. As a kind of processing products, some kinds of lotus product including lotus starch, lotus drink and salted lotus has been imported to some countries of Asia, America and Europe [1]. In addition, a large number of secondary metabolites make the lotus into a constituent of traditional Chinese medicine. Recently, with adjustment of industrialization, lotus has been considered the largest area in cultivation among all the aquatic vegetable. Therefore, related studies with theory and practice has attracted more and more attention [2, 3].

Light including photoperiod, light quality and light intensity is an basic condition to be involved in every aspect of plant development and growth such as root formation, photosynthesis, flowering, fruit development and plant morphogenesis [4–6]. It is evidences that many metabolism processes depending on light signal during plant growth are always induced by the hormone signal [7, 8], suggesting that hormone action happens in downstream of light signal transduction pathway [9–11]. It is shown that light regulates the whole process of root formation [12–14]. Some factors such as reactive oxygen species, abscisic acid, sugar and auxin, which is involved in light signal pathway are found to affect root development [15–17]. IAA is synthesized in vigorous organ under light regulation [18–19], and IAA synthesis depended on light play an criticle role on developmental process of adventitious roots including induction, development and expression of root [20, 21]. it is shown that improvement of endogenous IAA content by exogenous application of IAA significantly promotes cell division of root primordium, which directly lead to positive effect on ARs development [22]. Further study shows that any approach changing auxin metabolis or auxin sensitivity in plant is helpful for the formation of adventitious roots [23, 24]. It is reported that cytokinin, which regulates cell division is also involved in the accident of adventitious root formation due to its effect on auxin metabolism [20]. Therefore, IAA is considered as the direct regulator among the complex network in regulating adventitious root formation.

Analysis of gene expression or regulation at the whole genome is the best way to understand ARs formation. Genes involved in auxin metabolism or response related with ARs developmnet has been detail studied in the past decades. It is evidence that many genes participate on IAA synthesis, transport or response undoubtedly accelerates developmental process of ARs [25]. Until now, two kinds of IAA transport (influx carriers and efflux carriers) are found. It is reported that AUX1/LAX family, which belongs to influx carriers have a great influence on root development by triggering the IAA distribution in plant [26]. AUX1/ LAX gene family contain several members, and different expression profiling is found in various tissue [27]. Ahkami et al (2013)[28[] reports that auxin also affects the content of IAA in plant by regulating GH3 expression in petunia hybrida. Above data is indicated that various functions is existed for the member of AUX1/LAX family, although these genes are involved in ARs development. PIN, as a efflux carriers specially expresses in the root primordia and is required for root formation [29, 30]. An auxin-induced gene, ARL1 is found to participate in cell division relevant to ARs formation [31]. In addition, some auxin reponsive factors such as ARF6, ARF8 and ARF17 are also involved in ARs development [32]. Therefore, the biological process of ARs formation is regulated by multiple genes.

Lotus needs great nutrition to support plant growth, especially during the formation of rhizome. However the principal root cannot be developed in the plant due to long period evolution, so ARs become the main way to uptake water and mineral substance. The ARs of lotus is latent primordial form, and while it still needs induced by IAA [33], and then the developmental process is started. ARs has been found to frequently locate two sites of lotus plant including seedling’s hypocotyl and inernodes of storage organ [34]. In general, the number of ARs in seedling hypocotyl is less than that of internodes of storage organ due to great mount of nutrition is needed for plant growth. Primordial of root is differentiated from normal cell triggered by hormone or other environmental factors, and developed at pericycle in lotus or land vegetable [35, 36]. The biological process of ARs formation includes three periods such as induced stages, initial developmental stages, and breaking out from the epidermis [37, 38]. For the induced stage, the meristematic cells is developed from the normal cells, and so the sink establishment phase is established. For the initial developmental stage, primordium relevant to ARs is formed and developed [39], and at last, ARs protrude from the epidermis [40]. Above three biological processes are affected by light.

In the past years, we find that exogenous application of ethylene, IAA and mechanical damage significantly affecting lotus ARs formation is derived from the change of endogenous IAA content under normal light condition. In darkness, no accidence of ARs is happened, although above substances was applied, suggesting that light is a necessary factor for lotus ARs development. However, there were no direct evidence for the light-dependent IAA regulation on ARs formation, therefore, four libraries were constructed to monitor genes expression from induced stage to expression stage of ARs development. At the same time, the change of IAA content was also documented.

Light promotes ARs development

To investigate the effect of light quality on lotus ARs formation, lotus was exposed to various light intensities, including darkness, 5000 lux and 20000 lux. No ARs was formed in the lotus under the darkness (Table.1), whereas lotus could develop ARs when exposed to lights. The ARs formation seems to be relevant to light intensity. After germination, the ARs could be observed at the second day under 20000 lux and at the fourth day under 5000 lux, indicating that light regulates the ARs development (Fig.1a). Next, we observed the microstructure of hypocotyl where the ARs emerged. When exposed to light, ARs development could be clearly divided into three stages, induced process, developmental process and expressed process (Figure 1b). Under the darkness, the AR primordium were present, but failed to break out of the epidermis (Figure 1b).

Light affects IAA contents

IAA has been characterized as an inducer of ARs. To investigate whether the regulation of light on ARs is dependent on IAA, we monitored the IAA contents of lotus seedlings under various light intensities (darkness, 5000 lux, 1500 lux and 30000 lux). Along with light exposure time, IAA contents gradually increased and reached the maximum within 4 days and subsequently decreased. Interestingly, a significant increase of IAA content was also observed in darkness. Among different light intensities, the increased level of IAA in lotus is the highest under 30000 lux. Above result showed that another factor was existed to coordinately decide the ARs development with IAA, which was regulated by light (Fig.2).

The effects of light in transcriptome profiling

To dissect the underlying mechanism of light regulates ARs development, we comparatively analyzed the transcriptome profiling of lotus before and after the exposure of various light intensities (darkness, 5000 lux and 20000 lux) by constructing four different libraries, which are CK0 (before treatment), D (3-d exposure under darkness), E (3-d exposure under 5000 lux) and F (3-d exposure under 20000 lux). Analysis of quality control (QC) showed that the reads derived from RNA-seq libraries covered the whole lotus genome, evidenced by the flat curve of obtained reads (Additional file 1: Fig S1). Approximate 1.2× 10⁹reads was obtained, and more than 97% were clean reads. Around 83% reads were successfully mapped into lotus genome and 73% reads were unique (Additional file 1: Table S1). PCA analysis showed a high correlation among three biological replicates (Fig 3a). In total, 25766 genes were obtained and over 86% of genes were present in each library (Fig 3b). The FPKM value of most gene ranges from 1e^-01~1e⁺⁰³(Fig 3c,d). Analysis of differentially expressed genes (DEGs) showed that, compared with before treatment (CK0), 1739, 1683, and 1462 genes were upregulated and 1533, 995, and 834 genes were downregulated when lotus were exposed 3 days under darkness (D), 5000 lux (E) and 20000 lux (F), respectively (Fig.4a,b, Additional file 1: file S1 ). Further DEGs analysis between 3-d exposure of darkness (D) and 20000 lux (F), only 240 genes satisfied the threshold of DEGs (Fig.4c,d).

Light influence carbohydrate metabolism and hormone signal transduction

In term of dramatic difference on ARs development between 3-d exposure of darkness (D) and 20000 lux (F), we analyzed their DEGs with by the KEGG tool. These DEGs could be classified into five groups, including cellular processing, environmental information processing, genetic information processing, metabolism and organismal system. Further analysis showed that 20 DEGs were involved in signal transduction in the group of environmental information processing and 36 DEGs were related with carbohydrate metabolism in the group of the metabolism processing (Fig. 5a), indicating they might be the major regulatory pathways during light-dependent ARs development. In support, the expressions of genes involved in plant hormone signal transduction and the metabolism of starch and sucrose were changed (Fig. 5b).

Furthermore, we employed qPCR to confirm the results of RNA-seq. Ten genes such as pectinesterase, peroxisomal adenine nucleotide carrier 1-like, indole-3-acetic acid-amido synthetase, ethylene-responsive transcription factor ERF118, peroxisomal(S)-2-hydroxy-acid oxidase GLO1-like, pyruvate decarboxylase 1, respiratory burst oxidase homolog protein B-like, sucrose synthase, light-regulated protein, photosynthetic NDH subunit of liumenal location 1, which were involved in various process such as sugar metabolism, IAA signal transduction, energy metabolism, photosynthesis, ethylene signal transduction and respiratory metabolism were chosen to identify their expression under three light intensities (darkness, 5000 lux and 30000 lux) by qPCR technique. Generally, the tendency of these genes expression was similar to that derived from the dataset of RNA-seq (Fig.6).

Role of sucrose in lotus AR formation

In order to analyze role of sucrose in AR formation, complementary experiment between IAA and sucrose was carried out under normal light condition. Based on the data, we found that 60 mg/L sucrose and 150 μmol IAA significantly inhibited AR development, while 20 mg/L sucrose and 10 μmol IAA dramatically promoted the formation of lotus ARs. The inhibition by 60 mg/ sucrose could be compensated by application of exogenous 10 μmol IAA, although no obvious difference was found with control seedlings. Furthermore, exogenous application of 20 mg/L sucrose dramatically increased AR development in seedlings treated with 150 μmol IAA (Fig. 7). According to the change in IAA under various light intensities, IAA was an absolutely inducer of ARs in lotus. We observed that ARs could be developed at the induced and developed stage under 150 mg/L IAA treatment (although they could not break out of the epidermis), and remove the negative effect of sucrose. Based on these observations, we believe that sucrose might be involved in the expressed state of lotus AR formation (Fig. 8).

Light (light quality, photoperiod and light intensity) is essential environmental factor to affect most of plant metabolism processes. It is an evidence that light intensity and light quality affect adventitious root development and the photosynthate quantity of Hypericum perforatum [41]. Chen et al. (2019) report that root generation and development can be dramatically promoted under available light intensity condition in Haworthia [42]. At the same time, light quality is shown to influence ARs in number [43]. We found that different light intensity showed various roles on ARs formation in lotus. The seedling under darkness condition could not form ARs, and high light intensity promoted the developmental process of ARs (Fig.1, Table.1). Interaction of light with other factors is found to be cooperatively involved in root development [44]. Light regulating ARs development often derives from sucrose content of photosynthate, and this effect mainly reflected on root number [45,46]. Sorin et al. (2005) find that IAA role on regulating ARs formation of Arabidopsis thaliana is depended on light condition. IAA synthesis and accumulation in plants can induce the formation of founder cells of ARs [21]. In our study, we found that IAA content was increased with or without light treatment, and while plant under high light intensity had more IAA content than low light intensity or darkness (Fig.2), suggesting that IAA synthesis was affected by light intensity in lotus. Therefore, we believed that ARs formation affected by light was directly regulated by IAA.

Lotus is an important aquatic ornamental plant, and at the same time it is also a kind of vegetable in China. Adventitious roots is a necessary secondary organs for mainly water and nutrition uptake because no principle roots was happened in plant [33,47]. Similar with reported previously, three obvious developmental periods such as induced period of root primordium, developmental period and expressed period (stages of breaking out epidermis) was found for lotus ARs [33. We found that many factors including plant hormone [33,48], mechanical damage [34] and sucrose [data is not shown] are all involved in ARs development. In addition, some important genes or regulator (miRNA) are shown to play critical role for ARs formation [49]. In order to monitor metabolism mechanism regulated by light, genes expression was identified under various light intensities. We found that a total of genes enhanced expression and decreased expression in the three libraries (D/CK0, E/CK0, and F/CK0) respectively. Based on these dataset, some important genes which obviously changed expression were also implored in F/D libraries (Additional file 1: file S1). Therefore, we concluded that the biological process of ARs formation regulated by light was very complex.

The role of IAA or sucrose on ARs developmentAuxin has been believed as a criticle hormone to participate in various biologicalprocesses such as organogenesis, fruit development, flowering and adaptation tostresses [40, 50- 53 ]. It is shown that auxin metabolism including auxin synthesis,transport and homeostasis are involved in the regulation of plant development such asroot formation, shoot development and reproduction [54]. At the same time, auxin isbelieved as an necessary regulator to switch the process from xylogenesis formation(this process is adverse to ARs formation) to root development [55]. In the pastdecades, many important genes relevant to IAA metabolism or response involved inroot formation have been identified [56-57]. In this study, four libraries treated withdifferent light intensities were constructed with solexa technology (Fig 3), which hasbeen proved as an efficient way to analysis genes expression under certain condition(Mach 2011, Wang et al 2012). We found that a total of 4884 genes and 2040 genesenhanced expression and decreased expression in the three libraries (D/CK0, E/CK0,and F/CK0) respectively (Fig.4). A total of twenty-eight genes including auxinsynthesis, responding (inducing), auxin transporter, and auxin repressed protein wereidentified in F/D libraries. However, it was showed that GH3 which was responsivefor IAA synthesis could not change expression (Table. 2), suggesting that another wayfor IAA synthesis was existed according to the change of IAA content. As IAAtransporter, auxin is consider as an important factor to involved in ARs developmentat induction stages of root primordium development [26,58-59]. Interestingly, weobserved that the expression of auxin transport relevant genes such as auxin effluxcarrier component 1c and Auxin transporter like protein 2 were not severely affectedby light intensity, which indicated that the difference of ARs formation was not resultfrom the IAA transport in the darkness and 20000 lux light intensity (Table. 2). Plant growth is depended on cell division, differentiation and elongation. It is evidence that sugar (especially sucrose) signal is involved in above biological process by inducing many important genes [60-63]. In the past years, sucrose is found to regulate root growth by increasing root length [64]. Le et al. (2019) report that application of available concentration of sucrose in culture medium can significantly promote ARs growth [65]. At the same time, sucrose not only affects ARs growth, but has influence on substance metabolism of ARs in Echinacea pallida [66]. Takahashi et al. (2003) show that the role of sucrose on ARs formation is similar with hormone, and induction stage of ARs development is strictly controlled by sucrose [67]. In this study, we found that some important genes related with sucrose synthesis were enhance expression in F/D libraries, and the content of sucrose was more increased than that of darkness. Sucrose synthase and sucrose-phosphatase were the criticle enzymes in sucrose synthesis (Fig.5, Table.3). It is shown that improvement of these genes expression can lead to increase content of sucrose [68] Change of sucrose content by modification of the activity of sucrose synthase and sucrose-phosphatas is benefit to maintain fruit storage quality [69-70]. We observed that genes encoding sucrose synthase and sucrose-phosphatase were enhanced 3.91 and 1.29 fold in expression in F/D libraries (Table.3). High correlation was found between genes expression and sucrose content, suggesting that sucrose metabolism, suggesting that sucrose was necessary for ARs development in lotus. In addition, we observed that complementary phenomenon was existed between IAA and sucrose during ARs formation, which could explain IAA and sucrose cooperatively regulated lotus ARs formation according the change of IAA and sucrose. Until now, a crosstalk is existed between ethylene and auxin in controlling root development, exogenous application of auxin can increase ethylene content and plant treated with ethephon also improve the content of endogenous IAA content [22,24,71]. Therefore, we believed that a new model of interaction between sucrose and IAA was involved in lotus root development.

In this study, we observed that lotus ARs was regulated by light intensities. Therefore, genome-wide gene expression was analyzed by RNA-seq technique. Approximate 1.2 × 109 reads was sequenced, and more than 97% was clean reads after remove of low quality reads. Among which, 1739, 1683, and 1462 genes expression were increased, and 1533, 995, and 834 genes decreased expression in the D/CK0, E/CK0, and F/CK0 libraries respectively. We observed that 20 DEGs were involved in signal transduction, and 36 DEGs were related with carbohydrate metabolism. In F/D libraries, twenty-eight genes relevant to auxin synthesis, transport, response or binding were found to participate in ARs development, and twenty-four genes related with sucrose metabolism, glucose metabolism and fructose metabolism changed their expression. According to changes of IAA and sucrose content, we believed that IAA and sucrose cooperatively regulated lotus ARs formation.

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.

ARs

adventitious roots; IAA:indole-3-acetic acid; DEGs:differentially expressed genes; QC:analysis of quality control; PCR:polymerase chain reaction; qPCR:real-time Quantitative PCR.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The materials of all the experiment was supported by aquatic vegetable Lab of Yangzhou University. The detail data has been deposited in NCBI database (Biosample number, CK0_1~CK0_3: SAMN13000907~SAMN13000909, CK0_1~ CK0_3: SAMN13000910~SAMN13000912; E_1~ E_3: SAMN13000913~SAMN13000915; E_1~ E_3: SAMN13000916~SAMN13000918; Bioproject number: 576670).

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by Jiangsu Agriculture Science, technology Innovation Fund (CX (18) 3066), and the modern agriculture of Yangzhou (YZ2017044), and special project of north Jiangsu (SZ-YC2019008). The costs of experiment was supported by CX (18) 3066 and YZ2017044, and the publication costs was supported by SZ-YC2019008. All the funder list in this manuscript has no role in design and performance of all the experiments, analysis of data and in the writing of the text file.

Authors’ contribution

The manuscript was revised and approved by all the authors. CL, HZ and LS designed the experiment; HY and SZ performed research; ZM and HY analyzed data. XX and CL participated in the sequence analysis. CL and LS wrote the paper, and HZ modified the manuscript.

Acknowledgements

We extend our thanks to some members of BIG for their cooperation in obtaining the data during ARs formation of the lotus by RNA-seq technique. The authors also thank Editage Ltd for their editorial assistance.

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Due to technical limitations, tables are only available as a download in the supplemental files section

Table.1. The role of various light intensities (darkness, 5000 lux and 30000 lux) on

number and rates of ARs formation within 6 d.

Table.2. Identification of genes expression involved in IAA metabolism or response

in D/CK, F/CK and F/D libraries.

Table.3. Identification of genes expression involved in sucrose, glucose and fructose

metabolism or response in D/CK, F/CK and F/D libraries.

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Gene expression profiling reveals light regulation on adventitious root formation in lotus (Nelumbo nucifera Gaertn.)

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