Physiol Mol Biol Plants 23(4): 933-943

PMC: PMC5671455

PMID: 29158640

A sucrose non-fermenting-1-related protein kinase 1 gene from potato, <em>StSnRK1</em>, regulates carbohydrate metabolism in transgenic tobacco

Abstract

Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) has been shown to play an essential role in regulating saccharide metabolism and starch biosynthesis of plant. The regulatory role of StSnRK1 from potato in regulating carbohydrate metabolism and starch accumulation has not been investigated. In this work, a cDNA encoding the SnRK1 protein, named StSnRK1, was isolated from potato. The open reading frame contained 1545 nucleotides encoding 514 amino acids. Subcellular localization analysis in onion epidermal cells indicated that StSnRK1 protein was localized to the nucleus. The coding region of StSnRK1 was cloned into a binary vector under the control of 35S promoter and then transformed into tobacco to obtain transgenic plants. Transgenic tobacco plants expressing StSnRK1 were shown to have a significant increased accumulation of starch content, as well as sucrose, glucose and fructose content. Real-time quantitative PCR analysis indicated that overexpression of StSnRK1 up-regulated the expression of sucrose synthase (NtSUS), ADP-glucose pyrophosphorylase (NtAGPase) and soluble starch synthase (NtSSS III) genes involved in starch biosynthesis in the transgenic plants. In contrast, the expression of sucrose phosphate synthase (NtSPS) gene was decreased in the transgenic plants. Meanwhile, enzymatic analyses indicated that the activities of major enzymes (SUS, AGPase and SSS) involved in the starch biosynthesis were enhanced, whereas SPS activity was decreased in the transgenic plants compared to the wild-type. These results suggest that the manipulation of StSnRK1 expression might be used for improving quality of plants in the future.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-017-0473-4) contains supplementary material, which is available to authorized users.

Keywords: Potato, Soluble sugar, Starch content, StSnRK1, Tobacco

Introduction

Biofuel significantly reduces the extraction and use of fossil fuels and therefore decrease environmental damage. There is a global awareness on breeding of non-food energy crops and research on biofuels is under progress (Sanz-Barrio et al. 2013; Wang et al. 2016a). Starch has been used as a main raw material for making first-generation biofuels due to the relatively easy conversion of starch into fermentable sugars (Smith 2008; Sanz-Barrio et al. 2013). Therefore, it is very important to understand how carbohydrates are metabolized in plants, which could greatly help to promote starch synthesis and improve the production efficiency of bio-fuels (Sanz-Barrio et al. 2013; Wang et al. 2016c).

There are three subfamilies of sucrose non-fermenting-1 (SNF1)-related protein kinases [SnRKs] (Halford and Hardie 1998; Celenza and Carlson 1986; Jossier et al. 2009) in higher plants viz. SnRK1, SnRK2 and SnRK3 (Hrabak et al. 2003). Carbohydrate metabolism and starch biosynthesis are regulated by SnRK1 in plants (Polge and Thomas 2007; Halford and Hey 2009; Wang et al. 2012).

Conversion of sucrose to starch is mediated by two key enzymes namely sucrose synthase (SUS) and ADP-glucose pyrophosphorylase (AGPase, Purcell et al. 1998; Mckibbin et al. 2006; Jiang et al. 2013). The redox modulation of AGPase activity is necessary for the reaction of sucrose (Tiessen et al. 2003) and regulates the expression of these two genes associated with biosynthetic pathway.

In plants, SnRK1 from the rye was the first such gene, which was cloned and characterized (Alderson et al. 1991). Subsequently, several SnRK1 genes have been identified from different plant species such as Arabidopsis (Kleinow et al. 2000), maize (Lumbreras et al. 2001), apple (Li et al. 2010) and sweet potato (Jiang et al. 2013). There are a few reports about investigating the function of SnRK1 on carbohydrate metabolism and starch biosynthesis in plants. The inhibition of SnRK1 function by antisense approach showed reduced viability and almost complete loss of starch accumulation in developing pollen grains (Zhang et al. 2001). Kanegae et al. (2005) found that SnRK1 was involved in starch accumulation in rice. The study of McKibbin et al. (2006) reported that overexpression of SnRK1 increased SUS and AGPase expression and enhanced their enzyme activities, leading to increased starch levels in the potato tubers. Jain et al. (2008) further found that the expression of a SnRK1 gene occurred simultaneously with the accumulation of starch in sorghum endosperm and maize endosperm. Wang et al. (2012) and Jiang et al. (2013) demonstrated that overexpression of SnRK1 from apple/sweet potato increased soluble sugar and starch content by increasing the expression of SUS and AGPase and enhancing the activities of SUS and AGPase in transgenic tomato/tobacco plants. It is thought that SnRK1 transfers carbon to starch by means of the storage pathway (McKibbin et al. 2006; Halford and Hey 2009). Furthermore, SnRK1 can inactivate sucrose phosphate synthase (SPS) via phosphorylation to regulate carbohydrate metabolism (Sugden et al. 1999; Halford and Hey 2009). SPS activity has been shown to decrease in the transgenic tomato/tobacco plants overexpressing SnRK1 from apple/sweet potato (Wang et al. 2012; Jiang et al. 2013).

There are information available for SnRK1 characterization in different plant species but the regulatory role of StSnRK1 (Genbank accession No. {"type":"entrez-protein","attrs":{"text":"NP_001274953","term_id":"568214265","term_text":"NP_001274953"}}NP_001274953) from potato in regulating carbohydrate metabolism and starch accumulation has not been reported. In this work, we isolated StSnRK1 from potato and elucidated its roles in transgenic tobacco. StSnRK1-overexpressing tobacco and wild type (WT) tobacco were used to investigate the function of StSnRK1 on carbohydrate metabolism. Overexpression of StSnRK1 was found to significantly increase sucrose, glucose, fructose and starch content in the leaves of transgenic tobacco plants, suggesting a potential use of StSnRK1 in improving quality levels of plants in the future.

Materials and methods

Plant materials

Potato cultivar Zhongshu No. 5 was employed for StSnRK1 gene cloning in this study. Tobacco (Nicotiana tabacum L.) cultivar Wisconsin 38 [wild type (WT)], a model plant, was used to study the functions of StSnRK1.

Cloning and sequence analysis of the potato StSnRK1 gene

Total RNA was extracted from the leaves of Zhongshu No. 5 with the RNA prep Pure Kit (Tiangen Biotech, Beijing, China). RNA samples were reverse-transcribed according to the instructions of Quant script Reverse Transcriptase Kit (Tiangen Biotech, Beijing, China). Based on the sequence of StSnRK1 (Genbank accession No. {"type":"entrez-protein","attrs":{"text":"NP_001274953","term_id":"568214265","term_text":"NP_001274953"}}NP_001274953), we designed one gene-specific primers (GC-F/R) of reverse transcription PCR (RT-PCR) (Table S1) to obtain its full-length cDNA sequence. PCR was performed with an initial denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min and a final extension at 72 °C for 10 min. PCR products were separated on a 1.0% (w/v) agarose gel. Target DNA band was recovered by gel extraction, then cloned into PMD19-T (TaKaRa, Beijing, China), and finally transformed into competent cells of Escherichia coli strain DH5α. White colonies were checked by PCR and the positive colonies were sequenced (Invitrogen, Beijing, China).

The open reading frame (ORF) of the cloned StSnRK1 gene was predicted with ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/). The theoretical molecular weight and isoelectric point (pI) of the StSnRK1 protein were calculated using http://web.expasy.org/protparam/. The nuclear localization signal of StSnRK1 protein was predicted using cNLS Mapper program (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi). The conserved domain of StSnRK1 protein was scanned by the InterProScan program (http://www.ebi.ac.uk/Tools/pfa/iprscan/). The homology of StSnRK1 protein was identified using protein BLAST in the National Center for Biotechnology Information (NCBI) database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A multiple sequence alignment of StSnRK1 with other SnRK1 proteins from different plant species retrieved from NCBI was conducted using the DNAMAN software (Lynnon Biosoft, Quebec, Canada). Phylogenetic analysis was conducted using the MEGA4 software (http://www.megasoftware.net/).

Subcellular localization of StSnRK1

Subcellular localization of StSnRK1 in onion (Allium cepa) epidermal cells was analyzed as described by Wang et al. (2016b). The ORF of StSnRK1 was cloned and then inserted into the pMDC83 expressing vector containing the green fluorescent protein gene (GFP) at SpeI and AscI restriction sites under the control of the CaMV35S promoter and NOS (nopaline synthase) terminator (Table S1). Both the fusion construct (StSnRK1-GFP) and the control vector (GFP) were transformed into living onion epidermal cells by particle bombardment with a GeneGun (Biorad HeliosTM) according to the instruction manual (helium pressure 260 psi). After incubation on MS medium (pH 5.8) solidified with 3% agar at 28 °C for 24–36 h, the onion cells were observed with bright field and fluorescence using confocal microscopy (Nikon Inc., Melville, NY).

Transformation of tobacco with StSnRK1

The binary vector pCAMBIA1301-StSnRK1 used in this study contained the StSnRK1gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) terminator and β-glucuronidase (gusA) and hygromycin resistance (hptII) genes driven by a CaMV 35S promoter, respectively. The primers used to amplify StSnRK1 were designed with the Primer5 (Table S1). The vector CAMBIA1301-StSnRK1 was transformed into the Agrobacterium tumefaciens strain EHA105 cells by the electroporation method for tobacco transformation (Lou et al. 2007). Transformation and plant regeneration were performed according to the method of Jiang et al. (2013).

Molecular confirmation of transgenic plants

The putatively transgenic tobacco plants were identified using the histochemical GUS assay according to Jefferson et al. (1987). The blue staining of tissues indicated a positive reaction. Genomic DNA was extracted from the leaves of the GUS-positive plants, and PCR amplifications were performed using specific primers (Table S1) to amplify fragments of the hptII coding sequence.

Analyses of sucrose, glucose, fructose and starch content

The content of sucrose, glucose and fructose were measured using the anthrone method according to the method of Zhang (1977). Starch extraction and quantification were performed based on the means of Smith and Zeeman (2006). The transgenic plants and WT were grown in pots containing a mixture of soil, vermiculite and humus (1:1:1, v/v/v) under normal conditions for 4 weeks at 27 °C under standard long day conditions (14 h light and 10 h dark). Leaves of plants were harvested to determine sucrose, glucose, fructose and starch content in light at 10–11 a.m. All treatments were performed in triplicate.

Southern blot analysis

Genomic DNA was extracted from the leaves of transgenic and WT plants by cetyltrimethylammonium bromide (CTAB) method (Rogers and Bendich 1985). Southern blot analysis was conducted as described by Wang et al. (2016c). Coding sequence of the 501bp StSnRK1 was used as probe (Table S1). The labeling of probe, prehybridization, hybridization and detection were performed using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics GmbH, Germany).

Expression analysis of the related genes

The expression of StSnRK1 and starch biosynthesis related genes was analyzed by real-time quantitative PCR (qRT-PCR) as described by Wang et al. (2016c). The cDNA solution was used as templates for PCR amplification with gene specific primers (Table S1). Tobacco Nt actin gene was used as an internal control (Jiang et al. 2013) (Table S1). Quantification of gene expression was done with comparative C_T method (Schmittgen and Livak 2008). All experiments were repeated three times and each data represents the average of three experiments.

SnRK1, SUS, SPS, AGPase and SSS activity assays

The activities of SnRK1, SUS, SPS, AGPase and SSS involved in starch biosynthesis were measured using the methods described by Nakamura et al. (1989), Zhang et al. (2009), McKibbin et al. (2006) and Wang et al. (2012).

Statistical analysis

All experiments were repeated three times and the data presented as the mean ± standard error (SE). Where applicable, data were analyzed by Student’s t test in a two-tailed analysis. Values of P < 0.05 or < 0.01 was considered to be statistically significant difference.

Plant materials

Cloning and sequence analysis of the potato StSnRK1 gene

Subcellular localization of StSnRK1

Transformation of tobacco with StSnRK1

Molecular confirmation of transgenic plants

Analyses of sucrose, glucose, fructose and starch content

Southern blot analysis

Expression analysis of the related genes

SnRK1, SUS, SPS, AGPase and SSS activity assays

Statistical analysis

Results

Cloning and sequence analysis of StSnRK1

The StSnRK1 gene contained a 1545 bp ORF encoding a 514 amino acids polypeptide with a molecular weight of 58.74 kDa and a pI of 8.03. The InterProScan domain analyses of StSnRK1 amino acid sequence showed the presence of Serine/Threonine-protein kinase catalytic domain (S-TKc) and the Serine/Threonine-protein kinase active site at 138–150 position, which are the characteristic features of plant SnRK1 protein (Fig. 1a). The cNLS mapper program detected a putative nuclear localization signal (NLS) at 222–233 position in the sequence (Fig. 1a).

Fig. 1

Fig. 2

Nuclear localization of StSnRK1

The subcellular localization of StSnRK1 protein was investigated in onion epidermal cells. During transient expression experiment, we observed the fluorescence of StSnRK1-GFP exclusively localized in the nuclei of the cells (Fig. 3). The control experimental set showed that GFP fluorescence was present throughout cells (Fig. 3). These results suggests that StSnRK1 is a nuclear-localized protein and therefore may be involved in the regulation of downstream gene tragets.

Fig. 3

Subcellular localization of the StSnRK1 protein in onion epidermal cells. The StSnRK1-GFP fusion protein was localized to the nucleus. Scale bar = 100 μm

Regeneration and identification of transgenic tobacco with StSnRK1 gene

The binary vector containing StSnRK1 ORF (pCAMBIA1301-StSnRK1) was used to ectopically express the gene in tobacco (Fig. 4a). A total of 250 leaf discs of tobacco cultivar Wisconsin 38 co-cultivated with the A. tumefaciens produced 24 putatively transgenic plants from 24 leaf discs. GUS assay showed that 6 of them had visible GUS activity in leaf, stem and root tissues, indicating stable integration of gusA gene in the genome of the plants (Fig. 4b, c, d). We could not detect the GUS expression in the remaining 18 transgenic and WT plants (Fig. 4b, c, d). Six independent transgenic lines overexpressing StSnRK1 were obtained by Hyg resistance selection, numbered #1-#6, respectively. PCR analysis further confirmed that these 6 plants were transgenic (Fig. 4e).

Fig. 4

Molecular confirmation of transgenic plants. a Schematic diagram of the T-DNA region of binary plasmid pCAMBIA1301-StSnRK1. LB, left border; RB, right border; hptII, hygromycin phosphotransferase II gene; StSnRK1, potato sucrose non-fermenting-1 related protein kinase 1 gene; gusA, β-glucuronidase gene; 35S, cauliflower mosaic virus (CaMV) 35S promoter; 35S T, CaMV 35S terminator; NOS T, nopaline synthase terminator. b, c andd GUS expression in leaf, stem and root of a transgenic plant and no GUS expression in the wild-type (WT). ePCR analysis of StSnRK1-overexpressing tobacco plants. Lane M: DL2000 DNA marker; Lane W: water as negative control; Lane P: plasmid pCAMBIA1301-StSnRK1 as positive control; Lane WT: wild type; Lanes #1-#6: different transgenic lines

Assay of sucrose, glucose, fructose and starch content in transgenic tobacco

Quantitative transcript analyses by qPCR showed the significantly increased expression level of StSnRK1 in the transgenic lines, especially #2, #3 and #4, while no transgene expression was observed in WT (Fig. 5). Furthermore, we found that SnRK1 activity was significantly enhanced in transgenic lines (#2, #3 and #4) (Fig. 6). Therefore, transgenic lines #2, #3 and #4 were selected for further analysis.

Fig. 5

Expression analysis of the StSnRK1 gene in the transgenic tobacco plants by real-time quantitative PCR. The transgenic plants and WT were grown in pots under normal conditions for 4 weeks. The tobacco Ntactin gene was used as an internal control. Data are presented as mean ± SE (n = 3). * and ** indicate a significant difference from that of WT at P < 0.05 and < 0.01, respectively, by Student’s t-test

Fig. 6

SnRK1 activity assay in the leaves of WT and transgenic plants. The transgenic plants and WT were grown in pots under normal conditions for 4 weeks. Data are presented as mean ± SE (n = 3). * and ** indicate a significant difference from that of WT at P < 0.05 and < 0.01, respectively, by Student’s t-test

The transgenic plants (lines #2, #3 and #4) and WT, grown in pots under normal condition for 4 weeks, had no significant differences in growth (Fig. S1). However, the sucrose, glucose, fructose and starch content in the leaves of these plants were different. The results showed that the StSnRK1-over expressing tobacco plants had significantly higher sucrose, glucose, fructose, soluble sugar and starch content, which were increased by 34–92%, 62–234%, 19–31%, 42–54% and 14–34%, respectively, compared to WT (Figs. 7, ,88).

Fig. 7

Sucrose, glucose, fructose and soluble sugar content analyses in the leaves of WT and transgenic plants. The transgenic plants and WT were grown in pots under normal conditions for 4 weeks. Data are presented as mean ± SE (n = 3). * and ** indicate a significant difference from that of WT at P < 0.05 and < 0.01, respectively, by Student’s t-test

Fig. 8

Starch content analysis in the leaves of WT and transgenic plants. The transgenic plants and WT were grown in pots under normal conditions for 4 weeks. Data are presented as mean ± SE (n = 3). * and ** indicate a significant difference from that of WT at P < 0.05 and < 0.01, respectively, by Student’s t-test

Southern blot analysis of transgenic tobacco plants

Southern blot analysis indicated that 3 transgenic plants (lines #2, #3 and #4) with higher starch content displayed different integration patterns. The copy number of integrated StSnRK1 gene varied from 1 to 2. We could not detect any evident relationship between expression levels of related genes and copy number of integrated StSnRK1 gene (Fig. 9). Also, no clear relationship between the starch accumulation and the copy number was ascertained (Fig. 9).

Fig. 9

Southern blot analysis of the transgenic plants to detect the copy number of integrated StSnRK1 gene. WT, wild type; #2, #3 and #4, transgenic plants with higher starch content

Expression analysis of starch biosynthetic genes in transgenic tobacco plants

qRT-PCR analysis showed that sucrose synthase (NtSUS), AGPase (NtAGPase) and soluble starch synthase (NtSSS III) genes related to starch biosynthesis pathway were all up-regulated (Fig. 10), whereas the expression level of NtSPS was decreased in transgenic plants (Fig. 10). These results demonstrated that StSnRK1 gene might be involved in the regulation of starch biosynthetic processes.

Fig. 10

Transcript levels of starch biosynthesis genes in the leaves of WT and transgenic plants. The transgenic plants and WT were grown in pots under normal conditions for 4 weeks. The tobacco Ntactin gene was used as an internal control. Results are expressed as relative values with respect to WT, which was set to 1.0. Data are presented as mean ± SE (n = 3). * and ** indicate a significant difference from that of WT at P < 0.05 and < 0.01, respectively, by Student’s t-test

Enzyme activity assays in transgenic tobacco plants

The enzymatic analyses indicated that the activities of SUS, AGPase and SSS were significantly enhanced, whereas the activity of SPS was decreased in transgenic plants compared to that in the WT (Fig. 11). The changes in enzyme activity of transgenic plants showed correlation with the transcription levels of their corresponding genes. All these findings indicate that StSnRK1 has significant effects on the activities of SUS, SPS, AGPase and SSS in transgenic tobacco plants.

Fig. 11

SUS, SPS, AGPase and SSS enzyme activity assays in the leaves of WT and transgenic plants. The transgenic plants and WT were grown in pots under normal conditions for 4 weeks. Data are presented as mean ± SE (n = 3). * and ** indicate a significant difference from that of WT at P < 0.05 and < 0.01, respectively, by Student’s t-test

Cloning and sequence analysis of StSnRK1

Fig. 1

Fig. 2

Nuclear localization of StSnRK1

Fig. 3

Subcellular localization of the StSnRK1 protein in onion epidermal cells. The StSnRK1-GFP fusion protein was localized to the nucleus. Scale bar = 100 μm

Southern blot analysis of the transgenic plants to detect the copy number of integrated StSnRK1 gene. WT, wild type; #2, #3 and #4, transgenic plants with higher starch content

Expression analysis of starch biosynthetic genes in transgenic tobacco plants

Fig. 10

Enzyme activity assays in transgenic tobacco plants

Fig. 11

Discussion

There are a few findings that SnRK1 is involved in carbohydrate metabolism and starch biosynthesis of plants. Kanegae et al. (2005) reported that SnRK1 had a role in starch accumulation in rice. The inhibition of SnRK1 led to the almost complete loss of starch accumulation and viability in developing pollen grains (Zhang et al. 2001). Overexpression of SnRK1 from apple/sweet potato increased soluble sugar and starch content in transgenic tomato/tobacco plants (Wang et al. 2012; Jiang et al. 2013). In this work, we isolated a StSnRK1 gene from potato. Bioinformatic analyses of StSnRK1 showed the presence of characteristic domain present in plant SnRK1 proteins (Fig. 1). Transient expression of StSnRK1 in onion epidermal cells suggest StSnRK1 protein is localized to the nucleus (Fig. 3), similar to the results of Vincent et al. (2001) and Jiang et al. (2013). Our results also revealed that overexpression of StSnRK1 was found to increase soluble sugar and starch content in transgenic tobacco plants (Figs. 7, ,88).

The reversible conversion of sucrose and UDP to UDP-glucose (UDPG) and fructose is catalyzed by enzyme SUS (Lee et al. 2009, 2010; Ovono et al. 2009; Jiang et al. 2013). The SUS activity was closely related to the strength of the sink and the accumulation of starch (Zrenner et al. 1995; Jiang et al. 2013; Wang et al. 2016a). SPS is involved in the conversion of UDPG and fructose into sucrose (Sugden et al. 1999; Halford and Hey 2009). AGPase converts G-1-P to ADP-glucose (ADPG) (Tiessen et al. 2003; Geigenberger 2011). It is reported that AGPase is regulated by transcriptional redox, which provides a mechanism for regulating the starch synthesis rate in potato tubers to sucrose supply (Tiessen et al. 2003; Geigenberger et al. 2005).

The SnRK1 was involved in the control of SUS gene in potato and it has been shown that SnRK1 regulate SUS transcription and thus affects various aspects of carbon metabolism in plants (Purcell et al. 1998). SnRK1 can regulate carbohydrate metabolism by phosphorylating and inactivating SPS (Sugden et al. 1999 and Halford and Hey 2009). The study found that the expression of SUS and AGPase was up-regulated and their enzyme activities were also increased in the SnRK1-overexpressing potato plants, in which starch levels in the tubers were increased by up to 30%, while the expression of gene encoding SPS showed no change from the wild-type (McKibbin et al. 2006). Wang et al. (2012) and Jiang et al. (2013) demonstrated that overexpression of SnRK1 from apple/sweet potato up-regulated the expression of SUS and AGPase, and increased the activities of SUS and AGPase and decreased the activity of SPS, leading to increased soluble sugar and starch content in transgenic tomato/tobacco plants. In our work, the expression of NtSUS and NtAGPase was up-regulated, while the expression of NtSPS was slightly inhibited in transgenic tobacco plants (Fig. 10). Consistently, enzymatic activity of SUS and AGPase were also significantly increased, and SPS activity was also decreased in the StSnRK1-overexpressing tobacco plants (Fig. 11).

Soluble starch synthase (SSS) is responsible for the synthesis of starch in plants (Delvallé et al. 2005; Ren et al. 2007; Geigenberger 2011). Wang et al. (2016a, c, d; 2017a, b) reported that the activated starch biosynthesis in the ZmTrxF/SlAATP/SlTrxF/StAATP/StTrxF-overexpressing Arabidopsis plants was related to the enhanced enzyme activity of SSS and the increased expression of StSSS involved in starch biosynthesis. In our study, the activity of SSS was increased and the expression of NtSSS III was up-regulated in the StSnRK1-overexpressing tobacco plants (Figs. 10, ,11).11). Thus, it is thought that overexpression of StSnRK1 up-regulates the expression of NtSUS and NtAGPase and increases the activities of SUS and AGPase, which further increase the expression of the genes and enhance the activity of the major enzymes related to starch biosynthesis, leading to increased starch accumulation in transgenic plants (Fig. 12).

Fig. 12

A proposed model of the regulatory network of StSnRK1 in starch accumulation. The pathway for carbon destined for conversion to starch is shown with solid arrows and regulatory interactions are shown with broken arrows

The presence of sufficient substrate (sucrose) is a very important factor in the starch biosynthesis pathway. The studies found that the expression of SUS and AGPase was induced by sucrose in potato tubers (Müller-Röer et al. 1990; Fu and Park 1995). In our work, the increased content of sucrose was observed (Fig. 7), and the up-regulation of NtSUS and NtAGPase genes was also found in transgenic tobacco plants (Fig. 10). It is thought that the high level of sucrose can induce the expression of NtSUS and NtAGPase, further modulating starch metabolism in transgenic plants (Fig. 12). All of the findings suggest that StSnRK1 transfers carbon through the storage pathway to starch by regulating the activities of SUS, SPS and AGPase. Our results support the hypothesis proposed by Halford and Hey (2009) and Jiang et al. (2013) that SnRK1 is activated by sucrose, and then SnRK1 increases flux through the starch biosynthesis pathway by up-regulating the expression of NtSUS and NtAGPase and down-regulating the expression of NtSPS (Fig. 12).

In addition, the present results indicate that there was no clear relationship between the starch accumulation and the copy number of integrated StSnRK1 gene, similar to the results reported by Wang et al. (2016b), in which the copy number of integrated SlAATP gene ranged from 1 to 2 in transgenic plants exhibiting higher starch content.

Taken together, the StSnRK1 gene was successfully isolated from potato. Overexpression of StSnRK1 in tobacco showed significant increase in soluble sugar and starch content. Our results suggest that StSnRK1 plays an important role in starch metabolism, and has great impact in the engineering of alternative energy crop plants with improved starch accumulation.

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 538 kb)^{(539K, docx)}

^{School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huai’an, 223003 Jiangsu China}

^{College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095 Jiangsu China}

Feibing Wang, Phone: 86-517-83591100, Email: moc.361@6891gnibiefgnaw.

Contributor Information.

^{Corresponding author.}

Received 2016 Oct 24; Revised 2017 Aug 1; Accepted 2017 Sep 19.

Supplementary material 1 (DOCX 538 kb)^{(539K, docx)}

Acknowledgements

This work was supported by the Natural Science Research Project in Colleges of Jiangsu Province of China (17KJB210001), the Natural Science Foundation of Jiangsu Province of China (BK2013256), the National Spark Plan Project of China (2014GA69002), the Research Project of Talent Introduction of Huaiyin Institute of Technology (Z301B16534) and the Support Project of Jiangsu Provincial Department of Agriculture (BE2012445).

Acknowledgements

Footnotes

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-017-0473-4) contains supplementary material, which is available to authorized users.

Footnotes

Contributor Information

Feibing Wang, Phone: 86-517-83591100, Email: moc.361@6891gnibiefgnaw.

Qing Zhou, Phone: 86-517-83591100, Email: nc.ude.tiyh@zcjkgh.

Contributor Information