Increased energy sequestration in Nicotiana tabacum overexpressing UGPase and SPP genes in mesophyll.

doi:10.21203/rs.3.rs-2214016/v1

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Increased energy sequestration in Nicotiana tabacum overexpressing UGPase and SPP genes in mesophyll.

https://doi.org/10.21203/rs.3.rs-2214016/v1

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Transgenic Nicotiana tabacum with cDNA coding for uridine diphosphate glucose and sucrose phosphate phosphatase from Hordeum vulgare expressed from chrysanthemum rubisco small subunit promoter shows high expression of these transgenes in young leaves and low in roots. Although the activity of this promoter decreased during leaves development, even in fully developed leaves it was over 10 times higher than the expression of the native genes of these enzymes. The plants overexpressing the transgenes grew faster and started the generative phase earlier than the control plants, without any disturbances in leaves, flower and seed development. The dry weight of the transgenic plants at the end of the generative phase was slightly higher than in the control group and showed a greater proportion of carbohydrates. The content of lignin, cellulose and hemicellulose was higher in the transgenic plants than in the control plants, and similar differences showed the energy value of these plants. A comparison of the energy value sequestered in the aerial part of these plant showed that the transgenic plants stored up to 18% more energy than the control plants.

Mesophyll-specific overexpression of the transgenes showed beneficial effects - faster plants growth and higher accumulation of energy in the transgenic plants than in the controls. This effect was achieved in N. tabacum, a plant without specific storage organs or tissues. The use of a promoter directing expression of transgenes into mesophyll cells allowed to increase the efficiency of the selected metabolic pathway - photosynthesis-dependent sucrose synthesis.

Sucrose synthesis

mesophyll-specific promoter

UGPase

SPP

energy value

Nicotiana tabacum

Energy produced from plant feedstock has a significant share of the energy market, and forecasts show that the demand for this type of technology will increase [1–3]. The interest in renewable energy sources, especially green energy, is caused by the ongoing population growth worldwide, which will reach approximately 9 billion people by 2050, and the related increase in energy demand [4, 5]. Moreover, important reason for the development of green technologies is the pressure to decarbonize energy production caused by global warming. Another advantage offered by the bioenergy industry is the economic independence of the country and the development of the local economy. In many regions, including the EU, biomass resources have become the main source of renewable energy [6, 7]. As the decisive factor in the development of the bioenergy industry is the availability of plant feedstock, there is a need to develop specific crops for this industry and plants dedicated to energy production [8–10]. In this context, the photosynthetic sucrose synthesis pathway is an attractive target for modifying plant metabolism. The pathway, which is localized in the cytosol of photosynthetically active cells, is the major route used to incorporate the newly fixed carbon into plant metabolism (Fig. 1).

In this pathway, fructose-6-phosphate produced from triose phosphate exported from chloroplasts is converted to glucose 6-phosphate and, subsequently, to glucose 1-phosphate. The next step is catalyzed by uridine diphosphate-glucose pyrophosphorylase (UGPase, E.C 2.7.7.9) synthesizing uridine-di-phosphate glucose (UDP-glucose) [11]. The last two steps of the pathway depend on sucrose phosphate synthase (SPS, E.C. 2.4.1.14) and sucrose phosphate phosphatase (SPP, E.C. 3.1.3.24), which catalyze the synthesis of sucrose 6-phosphate and its hydrolysis to sucrose and phosphate, respectively [12–14]. The pathway is tightly coordinated with carbon dioxide fixation, triose phosphate transport to the cytoplasm, and the concentration of sucrose itself is a part of other regulatory circuits, such as the diurnal cycle [15, 16]. Sucrose, the final product of this pathway, is the main compound used for the long-distance transport of newly fixed carbon in the majority of plants, and genetic studies have shown associations between the enzymes in this pathway and plant productivity and growth [17–21 ]. The last three steps in this pathway are catalyzed by UGPase, SPS and SPP and are suitable targets for modifying plant metabolism. The reversible reaction of UDP-glucose synthesis catalyzed by UGPase is coupled with sucrose6phosphate synthesis, which ensures UGPase activity toward synthesis. The activity of plant UGPases is regulated by oligomerization, with monomers being the active form of the enzyme [22–24]. Apart from its role in sucrose synthesis, UGPase participates in the synthesis of cellulose, hemicellulose, glycoproteins, glycolipids, and glycosylated secondary metabolites [25, 26]. In sink tissues, UGPase is involved in sucrose breakdown, where UGPase reverse activity allows utilization of UDP-glucose for ADP-glucose synthesis [27, 28]. The subsequent reaction in the sucrose synthesis pathway carried out by SPS is recognized as a major rate-limiting and regulatory step. The activity of the SPS enzyme is allosterically regulated by glucose-6-phosphate and inorganic phosphate as well as by SPS serine phosphorylation dependent on light and osmotic stress [11, 29–32]. SPS genes are also transcriptionally regulated by environmental factors such as light or cold stress and during developmental processes such as leaf maturation [16, 31, 33]. The efficiency of the whole pathway also depends on SPS interactions with SPP, the final enzyme in the pathway [34]. These two proteins form a heterocomplex that facilitates the channeling of the SPS product to SPP [35, 36]. The SPP enzyme plays a key role in the synthesis of sucrose, as the reaction catalyzed by this enzyme is the only irreversible reaction that shifts the pathway balance toward sucrose synthesis [20, 37]. Similar to UGPase, SPS and SPP enzymatic activity is not limited to sucrose synthesis. The role of these enzymes in heterotrophic cells of sink organs may be associated with the cycling of the synthesis and breakdown of sucrose, which allow an increase in the supply of hexoses in these organs while maintaining the sucrose concentration appropriate for its transport and signaling functions [37–39]. Due to the dual role of UGPase and SPS and SPP participating in opposite processes, such as in the synthesis of sucrose from newly fixed carbon and the breakdown of sucrose during its sequestration and sugar metabolism in accumulating organs, the results of the overexpression of these genes from a strong, ectopic promoter do not allow us to answer the question of whether the efficiency of the sucrose synthesis pathway can be enhanced based on accurate results. The use of such promoters to overexpress the transgenes for UGPase, SPS or SPP most often results in plants with accelerated growth and development and increased biomass and/or carbohydrate content but does not allow us to dissect the enhanced yield of the photosynthetic sucrose synthesis pathway from the increased sucrose unloading in sinks [17, 18, 40–42]. Moreover, the use of ectopic promoters makes it impossible to target sucrose sequestration to a selected organ or process of an energy plant.

Therefore, we decided to investigate how increasing the expression of UGPase and SPP genes in mesophyll cells affects sucrose metabolism and thus plant physiology, especially the processes that determine its energy value and productivity. We used N. tabacum, which is a convenient model energy plant with secondary cell walls built of cellulose and lignin, as the primary carbon sink [43] and specifically prepared a binary vector addressing the expression of the transgene in mesophyll cells in which the photosynthetic sucrose synthesis pathway is active.

2.1. Isolation and cloning of H. vulgare cDNA for UGPase and SPP

H. vulgare var. Apex was grown in a greenhouse in 5 liter pots under 15 h light/9 h dark at 22°C. The top two leaves of the plants were harvested approximately 30 days after emergence, at the stem elongation stage. The leaves were immediately frozen in liquid nitrogen, ground in a mortar and used for total RNA isolation with an RNeasy Plant Kit (Qiagen). A total of 5.0 µg of total RNA was used as a template for the reverse transcription (RT) reaction using the Maxima H Minus First Strand cDNA Synthesis Kit. The cDNA for HvUGPase and HvSPP was PCR amplified using 2.0 µl of unpurified RT reaction per 50.0 µl of PCR reaction with primers s-33 and s-34 (HvUGPase) and s-36 and s-38 (HvSPP) (Supplementary Table S1). The PCR products were gel purified, cloned into the auxiliary plasmid pGEMT-Easy (Promega) or pJET1.2 (Thermo Scientific), and sequenced. Phusion DNA polymerase (Thermo Scientific) or PrimeSTAR GXL DNA polymerase (Takara) was used for PCR amplification, and E. coli Stellar competent cells (Clontech) or JM109 competent cells (Promega) were used for cloning.

2.2. pB-Ch binary vector construction and construct assembly

The cassette based on the sequence of the Chrysanthemium x morifolium Ramat. ribulose-1,5-bisphosphate carboxylase small subunit gene (Chm rbcS1, AY163904) was prepared using the synthetic DNA fragments Chrbc-P and Chrbc-T (Eurofins), sequences in Supplementary Materials. The Chrbc-P and Chrbc-T DNA fragments used in this cassette correspond to the AY163904 regions 1-1013 bp (promoter and 5’ UTR) and 1724–2658 bp (terminator with 3’ UTR), respectively (Fig. 2) [44].

Briefly, the synthetic Chrbc-P DNA in pEX-A2 was digested with the enzymes AvrII (restriction site in the synthetic fragment) and EcoRI (restriction site in the polylinker) and ligated with a short adapter fragment containing an internal SpeI site and flanked by AvrII and EcoRI restriction sites, which were digested with the same restriction enzymes. The obtained pEX-aux-A auxiliary construct was digested with SpeI and EcoRI restriction enzymes and ligated with the synthetic Chrbc-T DNA digested with the same enzymes to give the pEX-aux-B auxiliary construct. Finally, the pB-Chrbc binary vector was obtained by transferring the cassette containing the Chrbc-P promoter and Chrbc-T terminator from the pEX-aux-B auxiliary construct to pBI121 by digestion with HindIII and EcoRI restriction enzymes followed by ligation. Standard protocols recommended by the manufacturers were used for restriction enzyme digestion, DNA purification, ligation and E. coli transformation. The integration of the HindIII – EcoRI Chm rbcS1 cassette with the vector was checked by restriction enzyme digestion and sequencing.

The cDNA of interest was prepared by PCR amplification using primers equipped with proper restriction sites, such as primers MZ38 and Mz39 for HvUGPase and primers Mz42 and Mz43 for HvSPP (Supplementary Table S1), purified, digested with AvrII and StuI or SpeI restriction enzymes, ligated into the pB-Chrbc vector and used for E. coli transformation. The positive clones were verified with restriction enzyme digestion and sequencing. Finally, all the genetic constructs were electroporated into Agrobacterium tumefaciens LBA4404 using A. tumefaciens Electro-Cells (Takara), verified by sequencing and used for plant transformation.

Taq DNA polymerase (Thermo Scientific), Expand High Fidelity PCR System (Roche), Phusion DNA polymerase (Thermo Scientific) or PrimeSTAR GXL DNA polymerase (Takara), and E. coli Stellar competent cells (Clontech) or JM109 competent cells (L2001, Promega) were applied in all the above operations. DNA, after PCR amplification or enzymatic reaction, was purified with agarose/TAE electrophoresis and a QIAquick Gel Extraction Kit (Qiagen).

2.3. Stable transformation and generation of transgenic Nicotiana tabacum

N. tabacum cv. W38 plants were transformed with the constructs assembled in the pB-Chrbc vector using the leaf disc inoculation method [45]. The standard protocol steps are as follows.

The A. tumefaciens LBA4404 clones with binary plasmids were grown for 2 days at 28°C in liquid YEB medium [46] until an OD₆₀₀ of approximately 0.7 was reached, spun down and resuspended in Murashige and Skoog (MS) medium. N. tabacum leaf pieces (approximately 1 cm x 1 cm) were immersed in an A. tumefaciens suspension for 6–10 min, aseptically dried and plated on MS medium containing 1 mg/L BAP and 0.1 mg/L NAA. The explants were maintained by transferring apical regions forming shootlets on MS medium containing carbenicillin (500 mg/L), kanamycin (50 mg/L) and 2% sucrose and cultured in 16/8 h light/dark at 25°C until resistant adventitious buds were formed. The resistant buds were cut down and transferred to the same medium to select rooted, resistant plants. Regenerated plantlets with roots grown in vitro for 5–6 weeks were screened for the presence of transgene mRNA by RT–PCR amplification using specific primers (Supplementary Table S1). Subsequently, plants that expressed the transgene were moved to soil (1:1 perlite:sand) and grown in a greenhouse.

2.4. Transgenic plant culture, growth analysis and sampling

N. tabacum F1 seeds were germinated on MS medium supplemented with kanamycin and carbenicillin to produce F1 transgenic plants, which were grown in the greenhouse. Leaves or roots used for gene expression analysis were flash frozen in liquid nitrogen within a few minutes after collection. Phenotypic characteristics of the transgenic plants were registered at 30, 44, 51, 54, 58, 62, 65 and 69 days after potting (DAP) and included plant height measured from the base (root collar) to the apex, the length of the three longest leaves and the presence of flower buds/flowers. The entire aerial part of the plant at 69 DAP was harvested and used for the analysis of total dry mass, lignin, cellulose and soluble carbohydrate content, and energetic value. The plant material was dried at 70°C for 48 h until no further weight loss could be detected before being used for the analyses.

2.5. Transgenic plant validation and analysis with PCR and RT-PCR

Total RNA was isolated from N. tabacum leaves or roots ground into powder in liquid nitrogen (-196°C) either with an RNeasy Plant Kit (Qiagen) or using extraction with 0.1 M NaCl, 2% SDS, 50 mM Tris/HCl pH 8.0, and 10 mM EDTA buffer in the presence of phenol–chloroform–isoamyl alcohol followed by two precipitations with 2 M LiCl and a final precipitation with ethanol. RNA was resuspended in RNase-free water, and RNA concentration and quality were evaluated using UV absorbance at 260 nm followed by electrophoresis on an agarose gel under denaturing conditions [46] or an RNA 6000 Nano Chip (Agilent).

Prior to reverse transcription, RNA was digested with RNase-free DNase I (Thermo Sci.) according to the manufacturer’s protocol with 0.1 µg/µl RNA, 0.05 U/µl enzyme and 1 U/µl RNase inhibitor and incubation at 37°C for 30 min. The enzyme was inactivated with a 10 min incubation at 65°C in the presence of 1.0 mM EDTA, and RNA was recovered with ethanol precipitation. Finally, RNA was resuspended in water, and the concentration was measured with a NanoDrop (UV absorbance at 260 nm). Reverse transcription (RT) was performed with a Maxima First Strand cDNA Synthesis Kit for RT–qPCR (Thermo Sci.) according to the manufacturer’s protocol with an RNA concentration of 0.2 µg/µl. Aliquots of the RT reaction were used directly as a template for PCR amplification with 0.025 U/µl Taq DNA polymerase (Thermo Sci.), 1.75 mM MgCl₂, 0.2 mM dNTP, and 0.4 µM forward and reverse primers in a 15.0 µl final volume.

The results of RT–PCR amplification were analyzed with electrophoresis on a 1.0% agarose/TAE gel stained with Midori Green Stain (Nippon Genetics) using a UV transilluminator and Quantum ST4 and Bio Profil software (Vilber Lourmat).

Genomic DNA was extracted from N. tabacum leaves ground into powder in liquid nitrogen (-196°C) with 0.1 M NaCl, 2% SDS, 50 mM Tris/HCl pH 8.0, and 10 mM EDTA buffer in the presence of phenol–chloroform–isoamyl alcohol, separated from RNA with RNA precipitation in 2 M LiCl, and finally precipitated with ethanol. Finally, DNA was dissolved in water (ddH2O), and the concentration and quality were evaluated by measuring UV absorbance at 260 nm and visually via electrophoresis on an agarose-TAE gel. Aliquots of DNA were used as a template for PCR amplification with 0.025 U/µl Taq DNA polymerase (Thermo Sci.), 1.75 mM MgCl₂, 0.2 mM dNTP and 0.4 µM forward and reverse primers in a 15.0 µl final volume. The results of PCR amplification were analyzed with electrophoresis on an agarose/TAE gel.

2.6. Digital droplet PCR (ddPCR)

The single ddPCR mixture contained 1.0 µL of RT diluted 1:10–1:250 with water, 200 nM primers (Supplementary Table S1), 1 x QX200 ddPCR EvaGreen Supermix (Bio-Rad) and Milli-Q water added to a total reaction volume of 20.0 µL. The reactions were partitioned into droplets by the QX200 Droplet Generator (Bio-Rad), and DNA was amplified using PCR conditions according to the manufacturer’s instructions: initial denaturation at 95°C for 5 min, 40 cycles of 95°C for 30 s, 59.5°C for 30 s and 72°C for 45 s, followed by a final extension at 72°C for 2 min and signal stabilization first at 4°C for 5 min and then at 90°C for 5 min, with a ramping time of 2°C/s. The fluorescence amplitude of the generated droplets was measured by a QX100 droplet reader (BioRad), and the data were analyzed with QuantaSoft (BioRad) software. The N. tabacum gene for the ribosomal L25 protein was used to normalize the expression of genes and transgenes [47]. Primers Mz116 and Mz117 used for amplification of the reference gene were projected based on the L18908 sequence (Supplementary Table S1).

2.7. Cell wall analysis – determination of mono- and disaccharides

N. tabacum stem segments and leaves were dried at 72°C and ground with IKA MF10 and Floss Cyclotec 1093 into grains below 0.25 mm. Determination of the content of soluble carbohydrates in plant materials was carried out using a spectrophotometric method after acid hydrolysis performed with 3,5-dinitrosalicylic acid (Sigma D0550). A Helios Gamma spectrophotometer was used in the analyses, measurements were made at a wavelength of 540 nm, and anhydrous glucose (Sigma G5767) was used to prepare the standard curve. All determinations were performed in triplicate.

2.8. Cell wall analysis – determination of cellulose, hemicellulose and lignin

N. tabacum stem segments and leaves were dried at 72°C, ground with an IKA MF10 grinder into grains below 1.0 mm, and sieved to obtain a fraction with a grain size of 1.0-0.5 mm. Finally, a Binder FD53 drying oven was used to determine the humidity according to the ISO 18134 standard. The hot water soluble substances neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were then determined with an Ankom A200 and Foss Fibertec 2010 Auto Fiber Analysis System (FOSS Analytical AB, Sweden) according to the manufacturer’s protocols: Foss 3434 (compliant with ISO 16472 standard) and Foss 3429 (compliant with ISO 13906 standard). Based on the differences in the determined parameters, the content of lignin, cellulose and hemicellulose in the plant material was calculated based on the formulas: a - hemicellulose = NDF (hemicellulose, cellulose and lignin) – ADF (cellulose and lignin); b - cellulose = ADF – ADL (lignin); and c - lignin = ADL, in accordance with the Foss AN304 protocol. All determinations were performed in triplicate.

2.9. Determination of the energy value of N. tabacum plants

N. tabacum stem segments and leaves were dried at 72°C and ground with an IKA MF10 grinder into grains below 1.0 mm. Finally, a Binder FD53 drying oven was used to determine the humidity according to the ISO 18134 standard. The IKA C2000 combustion calorimeter was used to determine the gross calorific value (GCV) of plant material at a constant volume and at the reference temperature of 25°C in a bomb calorimeter calibrated by the combustion of certified benzoic acid according to the ISO 18125 standard. All determinations were performed in triplicate.

2.10. Bioinformatics, phylogenetic analysis and programs

The sequences were assembled, edited and analyzed with the programs BioEdit (https://bioedit.software.informer.com/7.2/), BLAST, The Sequence Manipulation Suite [48] and Mega7 [49].

The phylogenetic trees were constructed using protein sequences aligned with ClustalW, the maximum likelihood method based on the JTT matrix-based model [50] and 1000 bootstrap replications [51]. All positions containing gaps and missing data were eliminated.

Primers were generated with Primer3 (http://bioinfo.ut.ee/primer3).

Figures and diagrams were prepared with Illustrator for Biological Sequences (IBS) [52] and Gene Structure Display Server (GSDS) [53] or Paint.

Transcription factor binding sites and polyadenylation signals in promoter and terminator of Chrysanthemum rbcs S1 gene (AY163904) (Supplementary Tables S2 and S3) were identified with a database of plant cis-acting regulatory elements (PLACE), coordinates according to AY163904 [54].

3.1. Constructs for HvUGPase and HvSPP expression in mesophyll cells

The pB-Chrbc vector was prepared by replacing the 35S CaMV promoter and nopaline synthase terminator in the pBI121 binary plasmid with the promoter and terminator derived from the Chrysanthemum morifolium ribulose-1,5-bisphosphate carboxylase small-subunit gene, GenBank acc. nr. AY163904 [44, 55]. The promoter and terminator corresponding to the AY163904 regions 1-1013 bp and 1719–2662 bp, respectively, were used in the pB-Chrbc vector. The regions used in the genetic constructs also contained UTRs and the first three codons in the case of promoter (Fig. 2a). The proximal and terminal parts of the original promoter and terminator sequences were modified by adding restriction sites allowing the insertion of a gene of interest and named Chrbc-P and Chrbc-T, respectively (Fig. 2b, c) (the sequences of Chrbc-P and Chrbc-T are provided in the Supplementary Materials). The numerous highly conserved regions from rubisco small subunit promoters and cis-acting regulatory elements responsible for efficient expression, enhancement of transcriptional activity and response to light are present in the Chrbc-P promoter, which suggests that the region is functional (Fig. 2b and Supplementary Table S2). The 72–98% conservation between the proximal part of the Chrbc-P promoter and rubisco small subunit promoters from the Solanaceae family indicates that this promoter may be active in N. tabacum (Fig. 2b and Supplementary Fig. S1). In the case of the Chrbc-T terminator, its functional activity is supported by the presence of eight polyadenylation signals organized into two clusters (Fig. 2c and Supplementary Table S3). The synthetic DNA fragments harboring the Chrbc-P and Chrbc-T sequences were digested with restriction enzymes and used to prepare a cassette that was subsequently inserted between the EcoRI and HindIII sites of pBI121, yielding a binary plasmid named pB-Chrbc (Fig. 3a).

The cDNAs encoding UGPase and SPP, which were used herein as transgenes, were cloned from H. vulgare, and their sequences were deposited in the Genbank repository under accession numbers MN584934 and MN584936, respectively. The HvUGPase MN584934 cDNA encodes a protein of 473 amino acids, with a calculated molecular mass of 51.6 kDa and pI of 5.41. The HvUGPase MN584934 protein differs from HvUGPase CAA62689 by one amino acid, Gly-160, which is substituted as Glu-160 and lies outside of the following regions of functional importance: N-glycosylation motif, nucleotide-binding loop, 14-3-3-protein binding site, and motifs involved in phosphorylation [56]. The conserved amino acids involved in maintaining UGPase catalytic activity and substrate binding, such as Trp-191, Trp-302, Lys-260, Lys-326, and Lys-364, are preserved in the MN584934 protein [28, 57–60]. Phylogenetic analysis revealed that HvUGPase MN584934 is found in a cluster of monocotyledonous UGPases along with H. vulgare UGPase CAA62689 and Saccharum hybrid UGPase AIB06871, and the structure and enzymatic activity of these genes have been well characterized (Fig. 4) [24].

The 16-1486 bp region of HvUGPase MN584934 was used as the transgene in the pB-Chrbc-UGPase construct for HvUGPase overexpression in N. tabacum (Fig. 3b). The complete sequence of the construct is submitted in the Supplementary Materials as pB-HvUGPase-construct.txt.

The H. vulgare SPP cDNA MN584936 used in this study encodes a protein of 422 amino acids with a calculated molecular mass of 47.22 kDa and pI of 5.7. The MN584936-encoded protein shares 100% identity with the H. vulgare SPP protein encoded by AK250551 cDNA, 99% identity with another H. vulgare SPP protein, AAO33159, and 82% identity with Zea mays SPP AF283564, which has experimentally confirmed enzymatic activity [13, 14] (Fig. 5).

All the amino acids differentiating the proteins HvSPP MN584936 (used in this study) and ZmSPP AF283564 are located outside of conserved motifs I – III representing active sites in the N-terminal HAD phosphatase domain [37]. The 52-1455 bp region of HvSPP MN584936 cDNA was used to prepare the pB-Chrbc-SPP construct for HvSPP overexpression in N. tabacum (Fig. 3c). The complete sequence of the construct is provided in the Supplementary Materials as pB-HvSPP-construct.txt.

In addition, a reference construct, pB-Ref, containing a cassette with an unexpressed GUS gene was prepared. The pB-Ref construct is based on the pBI121 vector in which the 35S CaMV promoter was replaced with a 586 bp fragment of the Sorghum bicolor intergenic region (see pB-Ref-construct in the Supplementary Materials). The plants transformed with this construct did not show GUS expression after GUS staining and RT–PCR.

3.2. Transgenic N. tabacum plants expressing HvUGPase and HvSPP

Transgenic N. tabacum plants with pB-Chrbc-UGPase and pB-Chrbc-SPP expression constructs as well as with the reference construct pB-Ref were generated by Agrobacterium-mediated transformation of leaf explants. When the plants grown in vitro on selective media had 4–6 leaves, the activity of the expression constructs was analyzed using standard RT–PCR with the transgene-specific primers and RNA from the leaf as the template. Next, the HvUGPase and HvSPP F0 plants that had transgene expression were rooted and transferred to the soil where they were grown for seeds without selection. At 69 days after transferring the plants to pots, the presence of the transgene transcripts was confirmed using standard RT–PCR, and RNA from leaves that formed after potting the plant was used as a template.

In the case of the F0 reference plants, RT–PCR showed no transgene expression despite the detection of the GUS gene in genomic DNA by PCR. The reference F0 plants grown in soil were tested using PCR with genomic DNA from leaves that formed after potting as a template.

The seeds of self-pollinated F0 transgenic plants were used for growing F1 plants, which were subsequently analyzed with PCR and RT–PCR for the presence of the cassette with the transgene in genomic DNA and its expression activity. The plant material, young leaves and whole roots, was collected 7 weeks after sowing when the plants had 4–6 leaves and the leaves were 6–10 cm in length and still under development.

The analyses revealed that the F1 progeny of plant #24, transformed with the pB-Chrbc-UGPase construct, showed the presence of the transgene in its genomic DNA and transgene expression (Figs. 6 and 7). The same procedure allowed to identify two lineages of plants transformed with the pBChrbc-SPP construct whose F1 progeny possessed and expressed the HvSPP transgene: #22 and #23 (Figs. 6 and 7). Additionally, two lineages of the reference plants, #34 and #35, were identified and applied in further experiments. The F1 progeny of reference plants (in the figures marked as Ref plants) #34 and #35 possess the transgene for GUS integrated into their genome, but its expression was not observed (Figs. 6 and 7).

Two independent sets of F1 plants were analyzed: 1 - young plants grown for 7 weeks after sowing from which young, still developing leaves and whole roots were collected to analyze the promoter specificity, including plants 22 − 11, 22 − 12, 23 − 11, 23 − 12, and 24 − 11, 24 − 12; and 2 - mature plants grown up to 69 days after potting used for analysis of the energy value and cell wall composition, including plants: 22 − 1, 22 − 2, 23 − 1, 23 − 2, and 24 − 1, 24 − 2.

3.3 F1 transgenic plants of the same lineage show the same or a similar number of transgene copies in the genome

The number of transgene copies integrated into the genome of F1 transgenic plants was analyzed with digital droplet PCR (ddPCR). To make this analysis independent of the measurement of absorbance or fluorescence of DNA, the NtL25 gene fragment flanked by the primers Mz116 and Mz117 was used as the standard for genomic DNA. The results showed that F1 transgenic plants from the same lineage had the same or a very similar number of transgene copies in the genome (Table 1 and Supplementary Fig. S2). However, plants from different lineages showed large differences, up to one order of magnitude, in the number of copies of the transgene per genome (Table 1 and Supplementary Fig. S2).

Table 1

Number of HvUGPase and HvSPP transgene copies in the genomes of transgenic plants, average values from two ddPCR experiments. F1 plants of line 22 show a great variability in the number of transgene copies per genome, presumable due to the multiple copies of the transgene integrated into the genome of the F0 plant. The ddPCR with genomic DNA from reference plants as template and primers targeting HvUGPase or HvSPP did not produce any product (positive droplets).
Lineage	Plant	Transgene/1 copy of NtL25 gene	Copies of transgene per genome assuming 3 or 4 copies of NtL25 gene	Plant	Transgene/1 copy of NtL25 gene	Copies of transgene per genome assuming 3 or 4 copies of NtL25 gene
a	b	c	d	e	f	g
	Promoter specificity analysis / young plants			Energy value analysis / mature plants
Transgene - HvSPP				Transgene - HvSPP
22	22 − 11	5.3	15.9–21.20	22 − 1	3.9	11.7–15.6
22	22 − 12	4.2	12.6–16.80	22 − 2	2.24	6.72–8.96
23	23 − 11	0.41	1.23–1.64	23 − 1	0.73	2.19–2.92
23	23 − 12	0.83	2.49–3.32	23 − 2	0.34	1.02–1.36
Transgene - HvUGPase				Transgene - HvUGPase
24	24 − 11	0.36	1.08–1.44	24 − 1	0.32	0.96–1.28
24	24 − 12	0.39	1.17–1.56	24 − 2	0.34	1.02–1.36

The genome of N. tabacum has not yet been sequenced, and therefore, there are no data on the copy number of the reference sequence. Thus, the number of copies of the reference sequence in the genomes of closely related plants, such as Solanum tuberosum and Solanum lycopersicum, was estimated using the blastn program, and the L18908 cDNA fragment from N. tabacum flanked by primers Mz116 and Mz117 as a query [61]. The genome of S. lycopersicum contains four regions with high similarity (77–89% identity) to this sequence along its entire length, while the S. tuberosum genome has three such regions with 84–90% identity and a fourth with 85% identity to part of this DNA fragment. Thus, it is further assumed that the N. tabacum genome contains 3 to 4 copies of the reference sequence (hereafter referred to as the NtL25 gene), similar to the genomes of the closely related plants S. tuberosum and S. lycopersicum. Based on this assumption, it was estimated that HvUGPase transgenic plants 24 − 1 and 24 − 2 or 2411 and 24 − 12 possess one copy of the transgene per genome. HvSPP F1 plants from lineage #23 possess from one to three copies of the transgene per genome and the F1 HvSPP plants in lineage #22 have from 6 to 21 copies of the transgene per genome. This variability is most likely due to the presence of multiple copies of the transgene in the ancestor of lineage #22, (Table 1).

3.4. Chrbc-P promoter specificity and the expression level of transgenes

The specificity of the Chrbc-P promoter and its efficiency were assessed by comparing the expression levels of the transgenes in leaves and roots. The preliminary results obtained from standard RT–PCR showed a much higher promoter efficiency in leaves than in roots (Fig. 6), so the ddPCR technique was used for accurate, quantitative analysis. Relative transgene expression in leaves and roots was used in these analyses to make the results independent of RNA concentration estimation by measuring absorbance or fluorescence. The NtL25 gene was used as a reference gene together with the primers Mz116 and Mz117, the same as in the genomic DNA analysis [47]. The plant material used for Chrbc-P promoter analysis was collected from young F1 plants grown for 7 weeks after sowing and included young, still developing leaves (6–10 cm in length) and whole roots.

The expression level of the transgenes expressed from the Chrbc-P promoter in leaves was 7 to 48 times higher than that of the reference gene NtL25 (Table 2, Supplementary Figs. S3 and S4). Conversely, in the roots, the activity of the Chrbc-P promoter was 10 to 50 times lower than that of the reference gene. The comparison of the Chrbc-P promoter performance in leaves and roots revealed that the frequency of the transcripts generated from this promoter in leaf cells was 67 to 2489 times higher than that in root cells (Table 2). The leaf-to-root ratio of relative expression of the transgene was similar in transgenic plants of the same lineage but varied between different lineages by as much as 30-fold.

Table 2

Specificity of the ChmrbcS promoter – relative expression (RE) of the transgenes in young leaves and roots, average of the two ddPCR experiments.
Plant ID	Aver. RE in young leaves (+/- standard dev.)	Aver. root RE (+/- standard dev.)	Aver young leaves RE / Aver. root RE
HvSPP transgene; HvSPP plants, lineage #22
HvSPP 22 − 11	48.8 (+/- 16.0)	0.020 (+/- 0.003)	2440.0
HvSPP 22 − 12	35.8 (+/- 9.2)	0.016 (+/- 0.004)	2237.5
HvSPP transgene; HvSPP plants, lineage #23
HvSPP 23 − 11	19.3 (+/- 2.6)	0.036 (+/- 0.004)	536.1
HvSPP 23 − 12	15.5 (+/- 4.1 )	0.040 (+/- 0.007)	387.5
HvUGPase transgene; HvUGPase plants, lineage #24
HvUGP 24 − 11	7.3 (+/- 1.5)	0.109 (+/- 0.019)	67.0
HvUGP 24 − 12	7.6 (+/- 0.3)	0.084 (+/- 0.016)	90.5

3.5. Expression of the transgenes and native genes for UGPase and SPP in mature leaves

The level of transgene expression in mature HvUGPase- and HvSPP-overexpressing F1 plants was analyzed by RT ddPCR. The plant material, two leaves at least 20 cm long, from the third and fourth lower internodes, was collected from mature, glasshouse-grown F1 plants at 69 DAP.

The results showed a higher average relative expression level of HvUGPase compared to HvSPP in transgenic lines, with values of 2.93 and 0.91–1.21, respectively (Table 3). The difference in the expression level of HvSPP varied between transgenic plants by as much as 2.7-fold. Due to the small representation of plants overexpressing HvUGPase, less variation was observed in the relative expression level of HvUGPase between individuals.

Table 3

The relative expression levels of the transgenes - HvUGPase and HvSPP and native genes - NtUGPase and NtSPP - in mature leaves of transgenic and reference *N. tabacum* plants, the average values from three individual ddPCR experiments. Expression of transgenes – HvUGPase and HvSPP (column d) is 10–14 times higher than the corresponding native genes (column f).
Lineage	Plant	Aver. RE of given transgene	Aver.. RE of the transg. in given lineage	Aver. RE of given native gene	Aver. RE of nat. gene in given lineage	Aver. RE of a native gene as a % of the aver. RE of a given gene from all reference plants
a	b	c	d	e	f	g
Transgene - HvSPP				Native gene - NtSPP
HvSPP plants, 22	22 − 1	0.717	0.912	0.079	0.090	93.7%*
HvSPP plants, 22	22 − 2	1.107	0.912	0.100	0.090	93.7%*
HvSPP plants, 23	23 − 1	1.953	1.504	0.127	0.106	110.4%*
HvSPP plants, 23	23 − 2	1.055	1.504	0.085	0.106	110.4%*
Ref. plants, 34	34 − 1	-	-	0.094	0.09	93.7%*
Ref. plants, 34	34 − 4	-	-	0.086	0.09	93.7%*
Ref. plants, 35	35 − 1	-	-	0.083	0.101	105.2%*
	35 − 2	-		0.115
	35 − 3	-		0.104
				All Ref. plants	0.096	100%*
Transgene - HvUGPase				Native gene - NtUGPase
HvUGPase plants, 24	24 − 1	3.440	2.927	0.267	0.243	91.7%**
HvUGPase plants, 24	24 − 2	2.413	2.927	0.219	0.243	91.7%**
Ref. plants, 34	34 − 1	-	-	0.246	0.283	106.8%**
Ref. plants, 34	34 − 4	-	-	0.320	0.283	106.8%**
Ref. plants, 35	35 − 1	-	-	0.243	0.254	95.8%**
	35 − 2	-		0.250
	35 − 3	-		0.268
				All Ref. plants	0.265	100%**

The results showed high expression of both transgenes, similar to or 2.9 times higher than that of the native NtL25 gene. However, compared to young leaves, the expression of the transgenes in mature leaves decreased 11–46 times in plants with HvSPS overexpression and 2.5 times in plants with HvUGPase overexpression, Table 2 and Table 3. The expression of the native genes for NtUGPase and NtSPS was not disturbed by the activity of the transgenes and remained in the transgenic plants similar to that of the reference plants. The expression of the transgenes in mature leaves significantly exceeded the expression of the corresponding native genes: 9.0–14 times for SPP and 10.9–12.7 times for UGPase, see Table 3 column d and f.

3.6. The phenotypes of plants overexpressing HvUGPase and HvSPP

The F1 plants overexpressing HvUGPase and HvSPP showed faster growth and earlier flowering than the control reference plants. The F1 HvUGPase and HvSPP transgenic plants showed a greater increase in height that was most evident between 30–69 DAP, and they started the generative phase 11 days earlier than the reference plants (Figs. 8 and 9).

The F1 plants overexpressing HvUGPase were 35.7% taller than the reference plants, and plants overexpressing SPP were 41.5% taller. Additionally, the dry mass (d.m.) of F1 HvUGPase and HvSPP plants, measured at 69 DAP (when the cultivation was completed), was greater than that of the reference plants (Fig. 9b). The differences in d.m. were smaller, amounting to 9% for HvUGPase plants and 6% for HvSPP plants, but still evident, Fig. 9b. No differences in leaf length and width were noted between HvUGPase or HvSPP plants and reference plants, and no disturbances in the flower morphology of the transgenic plants were observed.

3.7. The composition of carbohydrates in plants overexpressing HvUGPase and HvSPP

The carbohydrate composition of the aerial part of mature plants was analyzed to determine the effect of HvUGPase or HvSPP overexpression on plant metabolism and physiology. The aboveground part of mature plants was harvested at 69 DAP, and the plant material, consisting of leaves and stems, was dried and ground. The sum of all carbohydrates, including mono- and disaccharides, hemicellulose, cellulose, and lignin, in HvUGPase and HvSPP plants was higher than that in reference plants, but this difference was subtle, with values of 1.71 [% d.m.] for HvUGPase plants and 3.16 [% d.m.] for HvSPP plants (Fig. 10). Monosaccharides are one of the major constituents of the analyzed plant material, and their content in HvUGPase and HvSPP plants was slightly lower than that in reference plants. Disaccharides, which constitute only approximately 1–2% of the dry mass of the mature N. tabacum plant, have a much smaller impact on the energy value of the analyzed plant material. In the case of HvUGPase plants, their content in d.m. was slightly lower than that for reference plants, and for HvSPP plants, this value was higher but still below 2% of d.m. (Fig. 10b). For all insoluble carbohydrates, such as hemicellulose, cellulose and lignin, their share in d.m. was greater in plants with HvUGPase and HvSPP overexpression than that of reference plants (Fig. 10c-e).

3.8. Heat of combustion and sequestered energy

For all F1 plants, the energy value (heat of combustion) was determined to compare the upper limit of the available thermal energy obtained from the complete combustion of one gram of given plant material (Fig. 11a). We observed that the energy value was slightly higher in plants overexpressing HvUGPase and HvSPP than in reference plants, probably due to the higher content of total carbohydrates in these plants, but the difference was in the range of 1.26–1.65%. Data on the energy value and dry mass of a given plant allowed calculation of the total energy sequestered by the aboveground part of the plant (Fig. 11b). A comparison of the total energy stored by the plant shows that the plants overexpressing HvUGPase and HvSPP sequestered nearly 20% more energy than the reference plants during the same growing period. This indicates the beneficial effects of the overexpression of the genes for enzymes in the photosynthetic sucrose synthesis pathway on the energy value of plants, even if the plants do not have an organ dedicated to the sequestration of newly bound carbon.

The photosynthetic sucrose synthesis pathway is the main route for the incorporation of newly bound carbon into plant life processes. Research into this metabolic pathway and the possibility of its modification is useful for improving both energetic plants and most crops. We used the Chrysanthemum rbcS promoter, a strong promoter specific for mesophyll cells, to overexpress genes encoding enzymes in the photosynthetic sucrose synthesis pathway, which allowed us to dissect the influence of a given gene on this pathway from its role in nonphotosynthetic tissues. The role of UGPase in nonphotosynthetic tissues and sink organs is associated either with UDP-glucose synthesis or UDP-glucose conversion to glucose 1-phosphate, as the enzyme is freely reversible. In accumulating organs such as tubers and grains, the enzyme participates in sucrose degradation pathways, providing carbon skeletons for starch [59, 62–65], whereas in tissues of intensive secondary cell wall formation, UGPase is involved in the synthesis of cellulose and hemicellulose [28, 42, 66]. Similarly, SPP activity is not limited to photosynthetic cells and was found in fruits and seeds at different stages of development, roots, shoot meristems and seedlings, flower buds, and trichomes [13, 20, 37, 66, 68]. In plants with ectopic overexpression of UGPase or SPP, one or more processes related to the accumulation of newly bound carbon may be enhanced or a new sink organ may be formed unpredictably. Atypical overexpression of the transgene can also cause unfavorable phenotypic changes, such as disturbances in flower development, and can even be lethal.

Therefore, in the pB-Chrbc vector, the promoter from the chrysanthemum rubisco small subunit gene was used, which is characterized by a high expression efficiency in N. tabacum leaves. While Outchkourov et al., 2003 [44] demonstrated its high efficiency at the protein level, our work showed that at the RNA level, this promoter is much more active than the promoter of the native gene encoding the L25 protein. Moreover, a comparison of its transcriptional activity in leaves and roots of young plants revealed its high leaf specificity (Table 2). Such specificity of rbcS promoters was shown for mono- and dicotyledonous plants [69, 70]. The very high transcriptional activity of this chrysanthemum-derived promoter in N. tabacum plants may result from conservation of the rbcS promoters of Asteraceae and Solanaceae plants, as well as the presence of numerous enhancers and their arrangement (Fig. 2, Supplementary Table S2) [44, 71]. The decrease in promoter activity between juvenile and mature leaves is associated with the developmental regulation of rbcS genes and was shown for rbcS genes from cotton, banana, rice and other plants [72–75], but even in fully developed leaves, the transgenes are expressed more than 10 times more efficiently than the corresponding native genes, Table 3.

The transgenic plants overexpressing the HvUGPase and HvSPP transgenes under the ChrbcS promoter had similar phenotypes characterized by faster growth and earlier flowering than the reference plants. In plants overexpressing the transgene for UGPase, a faster growth or increased height phenotype was often observed, but due to the use of an ectopic promoter, the processes linking this phenotype with UGPase were obscured [40–42, 76]. In the case of the SPP gene, its association with plant height was demonstrated in N. tabacum with reduced expression of this gene, which resulted in reduced growth rates [77]. Here, using a promoter targeting the transgene expression to mesophyll cells, we have shown that the mere increase in efficiency of the photosynthetic sucrose synthesis pathway has a positive effect on plant growth rate and biomass accumulation. This may be due to the increased supply of sucrose, which is the main form in which newly bound carbon is transported over long distances to tissues or organs where it is intensively used. In the case of N. tabacum, a plant that does not have specific sequestering organs, the increased supply of sucrose is used to build cell walls faster, which is associated with faster growth. However, this does not explain why the transgenic plants start their generative phase, bloom, and set seeds earlier than the reference plants. The relationship between flowering time and the overexpression of genes for UGPase or other enzymes related to sucrose metabolism is known from earlier works. In those works, this relationship was suggested to result from the strengthening of the stalk’s capacity as an accumulating organ to dominate the transport of sucrose to the shoot apical meristem [78]. In our opinion, another explanation is possible, considering the role of sugars produced from photosynthesis as signaling molecules involved in regulatory mechanisms, including apical dominance, seed germination, senescence and the circadian cycle [79–84]. The regulation of the circadian clock by the rhythms of the photosynthetic endogenous sugars may explain the relationships between the increase in the efficiency of the sucrose synthesis pathway and the perception of the day length by plants, which can control the development of the storage organs or promote flowering [85, 86]. The differences in the phenotype of plants overexpressing the genes for UGPase and SPP between our work and previous research are probably due to the use of different promoters and different plants. It should be emphasized that in plants with the ChrbcP promoter, no disturbances in the development of flowers or leaves were observed, unlike in some previous works, which may be associated with a more precise modification of plant metabolism where the transgene expression was targeted to one type of tissue.

The main difference in the carbohydrate composition between plants overexpressing the HvUGPase and HvSPP genes and reference plants was a higher content of insoluble carbohydrates, such as cellulose, lignin and hemicellulose, and a lower content of soluble carbohydrates, such as monosaccharides and disaccharides. The greatest differences are shown for cellulose, the content of which in dry matter of transgenic plants was 2.18–2.43% higher than that in the reference plants. Moreover, the content of total carbohydrates in the transgenic plants was 1.71–3.16% higher than that in the reference plants. It seems that the differences in carbohydrate composition are due to a greater proportion of lignified material, mainly derived from stems, in the dry mass of transgenic plants than in the reference plants (Fig. 9). The small differences in the carbohydrate composition also indicate that the energy value of plants overexpressing HvUGPase or HvSPP is only slightly greater than that of the reference plants; the differences were in the range of 1.26–1.65% in favor of the transgenic plants. The main advantage of the transgenic plants over reference plants is most noticeable when the energy accumulated in the aerial part of the plant is compared. The transgenic plants sequestered 14.9–17.5% more energy than the reference plants in the same vegetative period. The increased expression of the genes encoding UGPase and SPP in the mesophyll cells resulted in faster growth and accelerated development of the N. tabacum plant. As a result of the increased supply of sucrose, the processes related to plant growth and the formation of a secondary cell wall, were enhanced without modifying other metabolic pathways. The main effect that has been achieved is the increase in energy stored in plants with overexpression of UGPase and SPP, which is the most important feature of plant used in energy crops.

Acknowledgments

This study was supported by: National Centre for Research and Development (NCBiR) under Grant no. PBS1/A8/9/2012 (Project Sormisol) and by the Polish Ministry of Education and Science under the SPUB (19/488367/SPUB/SP/2021) program. The authors would like to thank Dr. L. Graban from Department of Plant Breeding and Seed Production, The University of Warmia And Mazury for his help in the analysis of cell wall carbohydrates and energy value.

Funding

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author contribution statement

JP and MR conceived, designed and performed the experiments. MR, MF and JP analyzed the data. MR and JP wrote the manuscript and MF edited the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Increased energy sequestration in Nicotiana tabacum overexpressing UGPase and SPP genes in mesophyll.

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Isolation and cloning of H. vulgare cDNA for UGPase and SPP

2.2. pB-Ch binary vector construction and construct assembly

2.3. Stable transformation and generation of transgenic Nicotiana tabacum

2.4. Transgenic plant culture, growth analysis and sampling

2.5. Transgenic plant validation and analysis with PCR and RT-PCR

2.6. Digital droplet PCR (ddPCR)

2.7. Cell wall analysis – determination of mono- and disaccharides

2.8. Cell wall analysis – determination of cellulose, hemicellulose and lignin

2.9. Determination of the energy value of N. tabacum plants

2.10. Bioinformatics, phylogenetic analysis and programs

3. Results

3.1. Constructs for HvUGPase and HvSPP expression in mesophyll cells

3.2. Transgenic N. tabacum plants expressing HvUGPase and HvSPP

3.4. Chrbc-P promoter specificity and the expression level of transgenes

3.5. Expression of the transgenes and native genes for UGPase and SPP in mature leaves

3.6. The phenotypes of plants overexpressing HvUGPase and HvSPP

3.7. The composition of carbohydrates in plants overexpressing HvUGPase and HvSPP

3.8. Heat of combustion and sequestered energy

4. Discussion

Declarations

References

Supplementary Files

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