Ectopic Expression of a Transmembrane Protein KaCyt b 6 from a Red Seaweed Kappaphycus alvarezii in Transgenic Tobacco Augmented the Photosynthesis and Growth

Abstract

Cytochrome b₆f complex is a thylakoid membrane-localized protein and catalyses the transfer of electrons from plastoquinol to plastocyanin in photosynthetic electron transport chain. In the present study, Cytochrome b₆ (KaCyt b₆ ) gene from Kappaphycus alvarezii (a red seaweed) was overexpressed in tobacco. A 935 base pair (bp) long KaCyt b₆ cDNA contained an open reading frame of 648 bp encoding a protein of 215 amino acids with an expected isoelectric point of 8.67 and a molecular mass of 24.37 kDa. The KaCyt b₆ gene was overexpressed in tobacco under control of CaMV35S promoter. The transgenic tobacco had higher electron transfer rate and photosynthetic yield over wild-type and vector control tobacco. The KaCyt b₆ tobacco also exhibited significantly higher photosynthetic gas exchange (P_N) and improved water use efficiency. The transgenic plants had higher ratio of P_N and intercellular CO₂. The KaCyt b₆ transgenic tobacco showed higher estimates of photosystem II quantum yield, higher activity of the water-splitting complex, PSII photochemistry, and photochemical quenching. The basal quantum yield of nonphotochemical processes in PSII was recorded lower in KaCyt b₆ tobacco. Transgenic tobacco contained higher contents of carotenoids and total chlorophyll and also had better ratios of chlorophyll a and b, and carotenoids and total chlorophyll contents hence improved photosynthetic efficiency and production of sugar and starch. The KaCyt b₆ transgenic plants performed superior under control and greenhouse conditions. To the best of our knowledge through literature survey, this is the first report on characterization of KaCyt b₆ gene from K. alvarezii for enhanced photosynthetic efficiency and growth in tobacco.

Introduction

The world population is growing and United Nation's reports expect it to reach 9.7 billion by 2050. The global climate changes and depletion of natural resources especially water and arable land affected natural ecosystem in various ways and these are the major challenges to meet the food security for growing population. The Food and Agriculture Organization (FAO) anticipated a requirement of 70% escalation in food production by 2050 to meet the supply for the growing population (FAO, 2016). The increase in crop yields can only meet the demands for food, feed, and energy for the growing population (Fedoroff et al., 2010; Reynolds et al., 2011). Photosynthesis fixes solar energy into NADPH and ATP (chemical energy) through electron transfer and employ this energy for biomass production. Photosynthetic efficiency is critical to growth, development, and yield in crop plants and it has been a promising target to enhance the plant productivity and yield (Miyagawa et al., 2001; Yamori, 2013; Yadav et al., 2018). The photosynthetic efficiency is an outcome of plant's ability to harvest the sun light, efficient transformation of solar energy into chemical energy, and amount of the chemical energy fixed as biomass (Yadav et al., 2018). In addition to improving crop biomass and yield, the enhanced photosynthesis would help to cope up with global warming through utilizing the main greenhouse gas that is, CO₂. Literature reports are available on attempts to improve the productivity and yield in crops by improving leaf photosynthesis (Murchie et al., 2009; Reynolds et al., 2009; Makino, 2011; Raines, 2011). In addition to traditional practices, different targets and approaches have been identified and reviewed to enhance the photosynthetic efficiency in crops (von Caemmerer and Evans, 2010; Raines, 2011; Reynolds et al., 2011; Parry et al., 2013; Singh et al., 2014; Mathan et al., 2016). Therefore, improving the photosynthetic efficiency can be an emerging route to improve the crop productivity to feed the growing population (Long et al., 2015).

In the recent past, genetic engineering has been widely used to enhance photosynthesis and growth of plants by overexpressing transgenes encoding for the enzymes, pathways, or transporters (Murchie and Niyogi, 2011; Karki et al., 2013; Parry et al., 2013; Price et al., 2013). Rice, potato, tobacco, Arabidopsis, and soybean were genetically engineered for improved CO₂ fixation and growth through introgression of genes encoding enzymes of Calvin–Benson cycle (Lefebvre et al., 2005; Feng et al., 2007; Rosenthal et al., 2011; Kandoi et al., 2016). Arabidopsis mutant for R-type anion channel (AtQUC1) showed enhanced photosynthesis, stomatal and mesophyll conductance, and accumulated higher contents of organic acid (Medeiros et al., 2016). Engineering of tobacco and soybean with cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) improved CO₂ assimilation and growth (Miyagawa et al., 2001; Köhler et al., 2017). Similarly overexpression of Brachypodium distachyon SBPase gene improved the biomass yield in wheat (Driever et al., 2017). The overexpression of the Rubisco large (LS) and small (SS) subunits with the Rubisco assembly chaperone RUBISCO ASSEMBLY FACTOR 1 (RAF1) resulted in ∼30% increase in Rubisco and 15% increase in CO₂ assimilation along with increased fresh weight (FW) in UBI-LSSS-RAF1 transgenic plants (Salesse-Smith et al., 2018). Engineering of tobacco with gene encoding CO₂-concentrating mechanism (CCM) from Synechococcus elongates improved the photosynthesis (Orr et al., 2020). The synthetically designed alternative photorespiratory pathways along with carbon concentrating cycles have also been hypothesized to enhance the crop productivity (Naseem et al., 2020).

The genetic engineering for increased photosynthetic electron transfer, NADPH, and ATP contents resulted in enhanced photosynthesis and growth of transformed plants (Hajirezaei et al., 2002; Chida et al., 2007; Rodriguez et al., 2007; Yadav et al., 2018). The overexpression of combination of Arabidopsis violaxanthin de-epoxidase (VDE), PSII subunit S (PsbS), and zeaxanthin epoxidase in tobacco resulted in enhanced photoprotection and improved photosynthesis (Kromdijk et al., 2016). Algal cytochrome c₆ enhanced photosynthesis and growth in Arabidopsis (Chida et al., 2007) and tobacco (Yadav et al., 2018). Rieske FeS protein overproduction improved the functioning of photosystem I (PSI) and PSII, and enhanced the maximum carbon assimilation in Arabidopsis (Simkin et al., 2017). Genetic engineering in tobacco for improved PsbS showed reduced loss of water molecule per CO₂ molecule assimilation that is, improved water use efficiency (WUE) (Głowacka et al., 2018). The cytochrome b₆/f complex, ATP synthase and Ferredoxin-NADP(+) reductase are major components of photosynthetic electron transfer chain. The cytochrome b₆/f complex and ATP synthase play a key role in the generation of NADPH and ATP for CO₂ fixation. The CO₂ fixation has been reported proportional to the Rubisco and Cyt b₆/f proteins (Onoda et al., 2005; Yamori et al., 2005). The chloroplast electron transport rates decreased more severely with reductions in cytochrome b₆/f complex as compared with ATP synthase activity in tobacco (Anderson et al., 1997; Ruuska et al., 2000) and this confirmed direct relationship of cytochrome b₆/f with photosynthetic electron transfer. Thus, cytochrome b₆/f is a key rate-limiting step (Rott et al., 2011) in chloroplast electron transport and is a potential target for enhancing photosynthetic performance of crops (Yamori et al., 2011).The algae are aquatic plants with huge variability and possess comparatively higher growth rate due to their better photosynthetic performance (Zou and Gao, 2010). Algae acclimatize to different light intensities by adjusting its photosynthetic functioning due to higher numbers of reaction centers (RCs) supporting the efficient electron transport (Sukenik et al., 1987). Algae offer a number of potential genes in genetic engineering of crops to improve photosynthetic efficiency, productivity, and biomass. In the recent past, cytochrome c₆ from Porphyra species (Chida et al., 2007), Ulva fasciata (Yadav et al., 2018), and CCM from Chlamydomonas reinhardtii (Nölke et al., 2019) were engineered in Arabidopsis and tobacco for betterment of photosynthesis. The cytochrome b₆ are important components of electron transfer and algae provides an advantage to study the mechanisms and regulation of photosynthetic electron flow. The cytochrome b₆ is a major subunit of cytochrome b₆/f complex and transfers electrons between the PSII and PSI RCs in photosynthetic electron transfer chain and contribute electrochemical gradient through pumping protons into the thylakoid space for ATP synthesis. Due to rate-limiting components in chloroplast electron transport, the cytochrome b₆ offers an opportunity in genetic engineering of crops to enhance the photosynthetic efficiency. Kappaphycus alvarezii (Doty) Doty ex P.C.Silva is a red seaweed and it exhibits average daily growth rate ranging from 3.64% to 13.98% (Rao et al., 2008). Considering higher daily growth rate and commercial acceptability, in the present study, K. alvarezii was used as a source of cytochrome b₆ gene. The overexpression of Cytochrome b₆ gene (KaCyt b₆ ) from K. alvarezii in plants was expected to achieve the enhanced photosynthesis through improved electron transfer and, thus, higher productivity in crop plants. The present investigation aimed at cloning KaCyt b₆ from K. alvarezii and its overexpression in transgenic tobacco for enhanced photosynthetic yield and growth. To the best of literature survey, it is the first report on characterization of a cytochrome b₆ protein from K. alvarezii in tobacco to improve the photosynthetic and growth performances. The T₁ transgenic tobacco plants showed higher electron transfer and CO₂ assimilation rate, and improved accumulation of photosynthetic pigments, soluble sugar, and starch contents. The present results showed KaCyt b₆ as a potential gene and photosynthetic electron transport as a potential target site for genetic engineering to enhance photosynthetic efficiency and crop yield. The study provided new insights in the improvement of photosynthetic and crop yield; and this would add to the development of molecular breeding strategies to meet the requirement of higher crop productivity for global food security.

Materials and Methods

Seaweed collection, RNA extraction, and cDNA synthesis

The present research work didn't involve any kind of human participants (cells or tissues) therefore this does not require any ethics committee approval. K. alvarezii thallus was collected from CSIR-CSMCRI seaweed experimental farm at Simbor (N 20°46′12.00′′; E 71°09′27.00′′), Una district, Gujarat. The samples were washed with autoclaved seawater, frozen in liquid nitrogen, and immediately processed for RNA isolation. The total RNA was extracted following cetyltrimethylammonium bromide (CTAB)-LiCl method (Zeng and Yang, 2002) with minor modifications. Briefly, 1 g thallus tissue was ground in liquid nitrogen in a prechilled pestle and mortar. The homogenate was transferred to a centrifuge tube containing prewarmed (65°C) 10 mL of CTAB buffer [2.0% (w/v) each CTAB and polyvinylpyrrolidone (PVP), 100 mmol Tris-HCl (pH 8.0), 25 mmol EDTA, 2.0 M NaCl, and 2.0% β-mercaptoethanol] and mixed well through vortexing. Samples were incubated at 65°C for 30 min with regular mixing. The samples were centrifuged at 11,000 rpm for 10 min at 18°C and supernatant was collected in a centrifuge tube. To this equal volume of chloroform:isoamyl alcohol (24:1 v/v) was added, mixed gently, and centrifuged at 13,000 rpm for 15 min at 4°C. The upper aqueous phase was collected in a fresh tube and the above step was repeated. To the upper phase, 1/3 volume of 8.0 M LiCl was added, mixed well, and incubated at −20°C overnight. The samples were centrifuged at 13,000 rpm for 15 min at 4°C and supernatant was discarded. The RNA pellet was washed twice with 80% ethanol. The pellet was dried and resuspended in 40 μL ultrapure RNase-free water and stored at −80°C. The quantity and quality of the extracted RNA was measured by NanoDrop spectrophotometer ND-1000 (NanoDrop Technologies) and agarose gel electrophoresis. The cDNA was synthesized using 1 μg of total RNA with ImProm-II™ Reverse Transcription System (Promega) and used as a template for PCRs.

Molecular cloning of full-length KaCyt b₆ gene

The sequences of algal Cyt b₆ genes were retrieved from NCBI database. Using the conserved sequences in the retrieved sequences, the degenerate primers for cloning of KaCyt b₆ gene were designed and purchased from Sigma-Aldrich (India). The KaCyt b₆ gene was partially amplified using degenerate primers (F 5′-GTGAACTAACATGGGTTACAGGAG-3′; R 5′-ATTTCTAATGGTGTAGCAAATGGA -3′) through PCR following the standard PCR program in a thermal cycler (Bio-Rad). The purified amplicon was cloned in pGEM^®-T vector (Promega Corporation) and sequenced (Macrogen, Inc., Seoul, South Korea). The KaCyt b₆ gene was made full length from both the ends through rapid amplification of cDNA ends (RACE). The 5′ end of the gene was completed following 5′-RACE protocol as per instructions of the manufacturer (Invitrogen, San Diego, CA). The gene-specific primer1 (GSP1; 5′-CTACGATGGGAACTGCTTCAGG-3′) and Superscript RTII were used to synthesize the first strand of cDNA. From reaction mixture, the RNA template was degraded using RNase H and the cDNA was purified using a column. The purified cDNA was subjected to terminal deoxynucleotidyl transferase reaction to add the homopolymer dC tail at 3′ end of the cDNA. The dC tailed cDNA was amplified with GSP2 (5′-CTGCCAAAATGACTCCTGTAACC-3′) and an abridged anchor primer (AAP; 5′-GGCCACGCGTCGACTAGTAC(G)₁₆-3′) supplied with the kit. For 3′ end amplification of KaCyt b₆ gene, the first strand of cDNA was synthesized with PK1 oligo dT adapter primer (AP1; 5′-CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGC(T)7-3′). Subsequently, the first strand of cDNA was amplified with 3GSP1 (5′-GGAGAACCAGCATGGCCAAATGA-3′) and PK2 (AP2; 5′-CCAGTGAGCAGAGTGACG-3′). Nested PCR with the above PCR product was performed using 3GSP2 (5′-GCCATCAGCAATAGGAGAACAAGC -3′) and PK3 (AP3; 5′-GAGGACTCGAGCTCAAGC-3′) primers. The amplified product of 5′- and 3′-RACE reactions were cloned and sequenced for confirmation. The full length of KaCyt b₆ sequence was obtained through alignment of the sequences of partially cloned fragment, 5′- and 3′-RACE products. Using aligned sequence, primers for full-length cloning of KaCyt b₆ gene were designed. The full-length KaCyt b₆ gene was cloned using Pfu and Taq DNA polymerase (2:1) (Invitrogen) and full-length primers (forward primer-5′-GAATAACCATGGGCAAAGTATATG -3′; reverse primer-5′-CGCTCGAGGAATTCCTACCAACCTTTTTC -3′). The amplified product was cloned into in pGEM-T vector and sequenced for confirmation. The KaCyt b₆ gene sequence was submitted to the GenBank database.

Bioinformatics analysis

The in silico analysis of KaCyt b₆ gene was performed using various online tools. The BLAST-N and P programs were used for homology comparisons and prediction of the conserved domains, respectively. The amino acid composition and physiological properties were predicted using ProtParam tool. The PSIPRED server and FoldIndex program were used for prediction of secondary structure of KaCyt b₆ and its folding characteristics, respectively. The membrane topology and subcellular localization were predicted with TMpred program and localizome server, respectively. The multiple sequence alignments of nucleotide and predicted protein sequences were carried out using ClustalW program. The evolutionary relationship among the KaCyt b₆ and different Cyt b₆ homologs from various algae and plant species was determined using MEGA 5 software (Tamura et al., 2011). The phylogenetic tree was constructed by the neighbor-joining method. Bootstrap analysis with 1000 replicates was conducted to optimize the confidence levels for the branches. The ORF in full-length cloned KaCyt b₆ gene was searched using ORF finder tool.

Construction of the plant expression vector and tobacco transformation

Full-length KaCyt b₆ was cloned using K. alvarezii cDNA as template and DNA polymerase with primers Cyt bF (5′-GTCGACATGGGCAAAGTATATGATTGG -3′) and Cyt bR (5′-CCGCTCGAGTTATAATGGTCCTGATATGCC -3′) containing Sal1 and Xho1 sites, respectively. The underlined portion represent the restriction site. The clones were sequenced and after confirmation these were cloned in binary vector. The full-length KaCyt b₆ ORF was cloned in between CaMV35S promoter and NOS terminator of pRT 101 vector. Furthermore, pRT 101 vector containing the entire cassette of CaMV35S-KaCyt b₆ gene and terminator was digested with PstI and the cassette was cloned into pCAMBIA1301 vector at the PstI site. The recombinant plasmid (pCAMBIA1301-CaMV35S-KaCyt b₆ gene with terminator) was mobilized into Agrobacterium strain LBA 4404 through the freeze/thaw method. The KaCyt b₆ gene was transformed in tobacco (Nicotiana tabacum cv. petit havana) through Agrobacterium-mediated cocultivation method following the standard protocol in our laboratory (Kumari et al., 2017; Yadav et al., 2018). The cocultivated explants were transferred onto shoot induction medium [MS basal medium (MS, 1962)+5 μM 6-benzylaminopurine (BAP), 1 μM indole-3-acetic acid (IAA), 300 mg l⁻¹ cefotaxime and 20 mg l⁻¹ hygromycin]. The regenerated shoots were inoculated on shoot elongation medium (MS basal +1 μM BAP, 300 mg l⁻¹ cefotaxime and 20 mg l⁻¹ hygromycin). The elongated shoots were transferred onto rooting medium (MS basal +0.2 μM IAA, 300 mg l⁻¹ cefotaxime and 20 mg l⁻¹ hygromycin). The cultures were incubated under culture room conditions [26°C ± 1°C, 60% relative humidity (RH), 12 h per day photoperiod with light intensity of 100 μmol photons m⁻² s⁻¹). The in vitro-grown putative positive transgenic tobacco plants were hardened and acclimatized in the containment facility. The seeds of positive tobacco plants were harvested for further gene function analysis.

Molecular and β-glucuronidase analysis of transgenic tobacco

The putative transgenic tobacco lines were confirmed through β-glucuronidase (GUS) expression and PCR amplification of KaCyt b₆ gene, hygromycin (hptII) selection marker, and gus (uidA) reporter gene. The GUS expression in the leaves of tobacco plants was assessed using the GUS Reporter Gene Staining Kit (Sigma). GUS staining was performed by dipping the leaves of wild-type (WT), vector control (VC), and transgenic tobacco lines into GUS staining buffer, vacuum infiltration of the stained buffer-dipped leaves for 5 min, and finally incubation overnight at 37°C in the dark. The stained leaves were bleached with 80% ethanol and documented under microscope. Genomic DNA was isolated from WT, VC, and transgenic tobacco leaves following the CTAB method. Quality and quantity of the genomic DNA was determined by agarose gel electrophoresis and NanoDrop Spectrophotometer, respectively. The molecular analysis of the transgenic tobacco lines was performed through PCR amplification of the genomic DNA with primers specific to KaCyt b₆ , hptII, and uidA gene. The PCR product was analyzed on 1.2% agarose gel.

Physiological characterization of transgenic tobacco

The T₀ seeds of VC and transgenic lines (L₃ and L₅) overexpressing KaCyt b₆ were germinated on 0.75% agar-gelled hormone-free (HF) MS medium supplemented with 20 mg l⁻¹ hygromycin. Separately, the seeds of WT seeds were germinated on HF MS basal medium. Two-week-old seedlings of WT, VC, and transgenic tobacco lines were transplanted into soil and acclimatized for 2 weeks. After acclimatization of WT, VC, and transgenic tobacco lines, plant height, chlorophyll content, total soluble sugar, and starch were estimated at 15^th, 30^th, 45^th, and 60^th days. The photosynthetic gas exchange and chlorophyll fluorescence parameter were also measured on the 45^th day.

Determination of leaf area, water content, and photosynthetic pigments

The shoot length and leaf area of 6-week-old WT, VC, and transgenic tobacco lines were observed. The FW of leaves was measured and these leaves were oven dried at 90°C to record the dry weight (DW). The water content of leaves was calculated with (FW−DW)/(DW) × 100 and expressed as % of g⁻¹ DW. The photosynthetic pigments are indicators of relative greenness and photosynthetic efficiency in plants. The photosynthetic pigment contents were estimated in WT, VC, and T₁ transgenic tobacco lines grown under greenhouse condition. The photosynthetic pigment contents were extracted by homogenizing the leaf samples of WT, VC, and T₁ transgenic tobacco line in N,N-dimethylformamide (DMF) under diffused light. The 100 mg leaf samples were crushed in DMF and centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was collected and absorbance was read at 664, 664.5 647, and 461 nm using a UV-Visible spectrophotometer (SpectramaxPlus 384). Chlorophyll a and b, total chlorophyll, and carotenoid contents were calculated following Inskeep and Bloom (1985) and Chamovitz et al. (1993). The ratio of chlorophyll a and b (Chl a/b), and carotenoid/total chlorophyll contents (Caro/TC) were determined.

Estimation of soluble sugar and starch contents in tobacco overexpressing KaCyt b₆ gene

The fresh samples were ground to powder in liquid nitrogen and repeatedly extracted in 80% ethanol at 4°C. The ethanol extract was evaporated until dryness and residue was dissolved in deionized water for sugar estimation. The residual pellet left after ethanol extraction was used for starch content estimation. The residual pellet was digested in 52% perchloric acid and the digest was centrifuged at 10,000 rpm for 30 min. The supernatant was diluted with milliQ water and used for starch quantification. The soluble sugar and starch contents were determined following anthrone reagent (0.2% anthrone in ice-cold 95% sulfuric acid) method. The absorbance was read at 630 nm against a standard curve prepared with glucose. In the case of the starch estimation, the obtained value was multiplied by a factor of 0.9 to convert the sugar values into starch contents.

Gas exchange and chlorophyll fluorescence measurements in KaCyt b₆ tobacco

The gas exchange and chlorophyll fluorescence were simultaneously measured in leaves of control and transgenic tobacco using fluorometer attached to a portable infrared gas analyzer (Li-6400XT) with an open system (Li-Cor). Measurements were recorded on the dorsal surface of the fully developed leaves (fourth leaf from top) in 45 days old soil-transplanted WT, VC, and transgenic lines (L₃ and L₅) under ambient CO₂ conditions and at a constant irradiance of 1000 mmol photons m⁻² s⁻¹. The plants were dark adapted for 45 min using dark room facilities. The minimal (F₀) and maximal (F_M) fluorescence in the dark-adapted state was measured using a 0.1 μmol photons m⁻² s⁻¹ and saturating (>3000 μmol photons m⁻² s⁻¹) actinic light, respectively, for ≤1 s duration. The measurements in dark-adapted WT, VC, and transgenic tobacco lines were recorded under diffused light conditions. The plants were exposed under 1200 μmol m⁻² s⁻¹ for 2 h in a plant growth chamber (PGC-105; Percival Scientific) to achieve the steady-state fluorescence. Gas exchange measurements were carried out at 1000 μmol m⁻² s⁻¹ PPFD, ambient atmospheric CO₂ (380 μmol⁻¹ mol⁻¹), and 60–65% RH and 26°C block temperature. With gas exchange measurement net photosynthetic rate (P_N; μmol of CO₂ m⁻² s⁻¹), transpiration rate (Tr; mmol of H₂O m⁻² s⁻¹), intracellular CO₂ (C_i, μmol of CO₂ m⁻² s⁻¹), stomatal conductance (g_s, μmol of CO₂ m⁻² s⁻¹), and ratio of P_N and C_i were determined. WUE (μmol CO₂ mmol⁻¹ H₂O) was calculated as the ratio of photosynthesis and transpiration (P_N/T_r). With fluorescence measurements, minimal (F₀), maximal (F_M), variable (F_V) fluorescence, activity of the water-splitting complex (F_V/F₀), basal quantum yield of nonphotochemical processes in PSII (F₀/F_M), maximum efficiency of PSII (F_V/F_M), variable to maximal fluorescence (F_V’/F_M’), redox state of the plastoquinone pool/nonphotochemical quenching (1-qP), PSII quantum yield/PSII photochemistry/photosynthetic yield (φPSII), photochemical quenching (qP), non-qP (qN and NPQ), and electron transfer rate (ETR) were determined in WT, VC, and transgenic (L₃ and L₅) lines (Baker, 2008).

Data recording and statistical analysis

During the course of investigations, each experiment was performed thrice with five biological replicates. Data were subjected to one-way ANOVA for analysis of variance to determine the significance among mean values of control [WT/negative control (WT) and positive control (VC)] and transgenic tobacco plants using SIGMA plot 13. The statistical significance among the control (WT and VC) and transgenic tobacco plants was calculated at p ≥ 0.05. Data were presented as mean ± standard error and the statistically significant differences among mean values were denoted as different lower case letters.

Results

Molecular cloning of KaCyt b₆ cDNA and in silico analysis

In the present study, full-length Cyt b₆ gene was cloned from 14 different seaweeds comprising 4 red and 5 each of green and brown seaweeds and details of these cDNA sequences have been provided as Supplementary Table S1. Furthermore, based on natural growth rate and importance of K. alvarezii in global seaweed cultivation program, Cyt b₆ gene from K. alvarezii (KaCyt b₆ ) was functionally characterized in planta for enhanced photosynthesis and yield in plants through transgenic approach. Full-length KaCyt b₆ gene was cloned successfully from K. alvarezii through 5′ and 3′ rapid amplification of cDNA ends (RACE) approach. The KaCyt b₆ cDNA (GenBank accession no. KT184318) was 935 bp long. It had a 43 bp 5′ untranslated region (UTR), a 648 bp ORF, and a 244 bp 3′UTR (Supplementary Fig. S1a). Cloned ORF encoded a protein of 215 amino acids with an expected molecular weight of 24.37 kDa and an isoelectric point of 8.67. The amino acid sequence showed maximum (97%) identity with Grateloupia tenuistipitata Cyt b₆ protein. Hydropathicity analysis showed four transmembrane (TM) domains in KaCyt b₆ protein (Supplementary Fig. S1b). FoldIndex program revealed 3 disordered regions with 23 disorder-promoting amino acid residues in KaCyt b₆ protein (Supplementary Fig. S1c). Secondary structure prediction of KaCyt b₆ protein revealed 9 α-helixes and 1 strand in peptides (Supplementary Fig. S1d). Phyre2 software revealed 81% α-helixes, 0% β-sheets, and 43% TM helixes in secondary structure and predicted 5% disorder (Supplementary Fig. S1e). ScanProsite predicted four iron (heme)-binding (86, 100, 187 and 202) sites in KaCyt b₆ protein. Supplementary Figure S2a showed predicted 3D structure of KaCyt b₆. Localizome server predicted KaCyt b₆ to be a transmembrane protein (Supplementary Fig. S2b). KaCyt b₆ showed possibility of phosphorylation with protein kinase-(PK) C, PK-A, cyclin-dependent kinase 2 (cdc2), caseine kinase II (CKII), and an unspecific protein (un-sp). Highest score was 0.94 for un-sp at position T-61 and S-89. NetPhos 3.0 server search for overall phosphorylation site exhibited three serine, two threonine, and two tyrosine phosphorylation sites (Supplementary Fig. S2c).

Construction of KaCyt b₆ expression vector and tobacco transformation

The ORF of KaCyt b₆ was cloned in a binary vector pCAMBIA1301 under the control of constitutive promoter 35S (Fig. 1a). Tobacco regenerates from cocultured leaf discs on medium containing hygromycin were considered as putative KaCyt b₆ transgenic tobacco and these exhibited positive GUS histochemical assay. Soil-transplanted rooted plantlets of GUS-positive tobacco were grown until maturation in containment facility and seeds were harvested. Transgenic tobacco exhibiting positive PCR amplification of KaCyt b₆ gene, hptII, and UidA genes (Fig. 1b) were selected as confirmed transgenic tobacco. Amplification of 648 bp KaCyt b₆ ORF in transgenic tobacco confirmed its integration and overexpression under CaMV35S promoter. Seedlings of WT raised on MS medium and of VC and T₁ transgenic lines raised on hygromycin-supplemented MS medium (Fig. 1c) were used for further gene function analysis. T₁ seedlings of VC and transgenic tobacco exhibited GUS staining (Fig. 1d). These were subsequently confirmed through PCR amplification of KaCyt b₆, UidA, and hptII genes as that of T₀ tobacco. The transgenic lines L₃ and L₅ along with WT and VC were used for further transgenic analysis.

FIG. 1.

Genetic transformation of tobacco: Schematic diagram of the KaCyt b ₆—pCAMBIA1301 construct (a), molecular validation of genetic transformation by PCR amplification of KaCyt b₆ , hptII, and Uid A genes in control and transgenic tobacco (b), seed germination in T₁ transgenic lines (c), and GUS staining of WT, VC, and transgenic tobacco leaves (d). In gel pictures, the lane M denotes ladder, PC, WT, and lane 1–14 are transgenic lines. GUS, β-glucuronidase; PC, positive control; VC, vector control; WT, wild type. Color images are available online.

Transgenic tobacco overexpressing the KaCyt b₆ showed improved growth attributes

The transgenic tobacco overexpressing KaCyt b₆ gene exhibited comparatively higher leaf area, water contents, and plant growth as compared with WT plants. T₀ transgenic tobacco plants during acclimatization under containment facility exhibited enhanced growth as compared with WT and VC plants (Supplementary Fig. S3). Similarly, the T₁ transgenic tobacco showed enhanced growth under in vitro conditions (Fig. 2). Similar to T₀ transgenic tobacco, soil-transplanted T₁ transgenic tobacco also exhibited improved growth (Fig. 3a–d) and better phenotypic and photosynthetic appearance of leaves in terms of size (Fig. 3e–h) as compared with control plants during growth period. Shoot growth in T₁ transgenic lines was estimated significantly higher as compared with control plants (Fig. 4a). In consonance with growth, T₁ transgenic tobacco had higher contents of water (Fig. 4b). Transgenic tobacco overexpressing KaCyt b₆ grew normal and had comparatively bigger leaves and better flowering during their growth cycle (Supplementary Fig. S4). The present results clearly confirmed the enhanced growth of transgenic tobacco overexpressing KaCyt b₆ gene under controlled and greenhouse conditions.

FIG. 2.

Pictorial observation on plant growth under controlled conditions on 0.75% agar-gelled hormone-free MS medium after 15 days (a), 30 days (b), 45 days (c), and 60 days (d) of germination in control and L₃ and L₅ transgenic lines. Color images are available online.

FIG. 3.

Pictorial observations of plant growth (a–d) and leaf phenotypes (e–h) in soil-transplanted plants of WT, VC, and KaCyt b₆ transgenic tobacco lines (L₃ and L₅) after 15 days (a, e), 30 days (b, f), 45 days (c, g), and 60 (d, h) days of growth. Color images are available online.

FIG. 4.

Plant height during different time intervals (a) and leaf water contents (b) of 45 days old WT, VC, and KaCyt b₆ transgenic tobacco lines (L₃ and L₅) after 6 weeks of growth in soil. Color images are available online.

Photosynthetic pigments estimated higher in tobacco overexpressing KaCyt b₆

The transgenic tobacco overexpressing KaCyt b₆ had higher contents of total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid contents as compared with WT and VC tobacco. The contents of chlorophyll a and b were estimated higher in transgenic tobacco overexpressing KaCyt b₆ (Supplementary Fig. S5a, b). Similarly, the T₁ transgenic (L₃ and L₅) tobacco at different time intervals exhibited significantly higher contents of total chlorophyll and carotenoids as compared with WT and VC plants (Fig. 5a, b). In consonance with contents of chlorophyll and carotenoid contents, ratios of chlorophyll a and b (chlorophyll a/b), and carotenoids and total chlorophyll contents (carotenoids/total chlorophyll contents) were estimated significantly lower in KaCyt b₆ transgenic tobacco as compared with WT and VC tobacco (Fig. 5c, d).

FIG. 5.

Total chlorophyll contents (a), carotenoid contents (b), ratio of chlorophyll a and b (c) and ratio of carotenoid and total chlorophyll (d) in WT, VC, and KaCyt b₆ transgenic lines (L₃ and L₅) after 15 days, 30 days, 45 days, and 60 days of growth in soil. Color images are available online.

Overexpression of KaCyt b₆ in transgenic tobacco enhanced photosynthesis

In continuation of photosynthetic pigments, simultaneous photosynthetic gas exchange and chlorophyll fluorescence were studied in WT, VC, and transgenic tobacco overexpressing KaCyt b₆ . The P_N was significantly higher in KaCyt b₆ transgenic tobacco lines (L₃ and L₅) as compared with WT and VC tobacco under ambient conditions (Fig. 6a). The T₁ transgenic tobacco compared with control tobacco exhibited comparatively higher g_s (Supplementary Fig. S6a). The C_i was estimated comparatively lower in transgenic tobacco as compared with WT and VC tobacco (Supplementary Fig. S6b). However, the differences in the values of g_s and C_i of control and transgenic tobacco were nonsignificant. The P_N/C_i was calculated higher in KaCyt b₆ transgenic tobacco as compared with WT and VC tobacco (Fig. 6b). The E and WUE were recorded slightly higher in transgenic tobacco overexpressing KaCyt b₆ as compared with WT and VC tobacco, however, the increase was not statistically significant (Supplementary Fig. S6c, d). The F₀ and F_M were estimated lower in KaCyt b₆ transgenic tobacco lines than WT and VC plants (Supplementary Fig. S6e, f), however, the differences were nonsignificant. The F_V/F_M (Fig. 6c) and F_V/F₀ (Fig. 6d) were comparatively higher than control plants in transgenic tobacco lines. The F₀/F_M was comparatively lower in transgenic tobacco (L₃ and L₅) lines (Fig. 6e). The F_V’ did not vary significantly in transgenic and control tobacco plants (Supplementary Fig. S6g). The KaCyt b₆ transgenic tobacco lines exhibited comparatively higher F_V’/F_M’ as compared with WT and VC tobacco (Supplementary Fig. S6h). The KaCyt b₆ transgenic tobacco overexpressing KaCyt b₆ showed higher estimates of PSII photochemistry (ɸPSII), ETR, and qP as compared with control plants (Fig. 6f–h). The NPQ was estimated slightly lower in KaCyt b₆ transgenic tobacco than WT and VC plants (Supplementary Fig. S6i), however, the differences were nonsignificant. The estimates of 1–qP supported the lower estimates of non-qP in transgenic tobacco overexpressing KaCyt b₆ (Supplementary Fig. S6j). The KaCyt b₆ transgenic tobacco overexpressing KaCyt b₆ exhibited higher ETR (Fig. 6g) and photosynthetic yield as compared with control plants. The photosynthetic gas exchange and chlorophyll fluorescence measurements clearly showed the enhanced photosynthetic performance and ETR in transgenic tobacco overexpressing the KaCyt b₆ gene as compared with control tobacco (WT and VC) plants.

FIG. 6.

Net photosynthesis rate (a), ratio of photosynthesis and intercellular CO₂ (b), F_V/F_M (c), F_V/F₀ (d), F₀/F_M (e), and ɸPSII (f), ETR (g) and photochemical quenching (h) in WT, VC, and KaCyt b₆ transgenic lines (L₃ and L₅) after 45 days of soil transplantation. ETR, electron transfer rate. Color images are available online.

KaCyt b₆ tobacco accumulated higher amounts of sugar and starch

The transgenic tobacco overexpressing KaCyt b₆ gene accumulated higher contents of ultimate photosynthetic products. The accumulation of total soluble sugar and starch contents differed with age of plants. Compared with WT and VC tobacco, the KaCyt b₆ transgenic tobacco accumulated significantly higher contents of soluble sugar (Fig. 7a). In consonance with sugar contents and photosynthetic rate, transgenic tobacco accumulated significantly higher contents of starch as compared with control plants (Fig. 7b).

FIG. 7.

Total soluble sugar (a) and starch (b) contents in WT, VC, and KaCyt b₆ transgenic lines (L₃ and L₅) after 15 days, 30 days, 45 days, and 60 days of growth in soil. Color images are available online.

Discussion

Improved photosynthetic efficiency results enhanced crop productivity and yield. The conventional plant breeding has limitations to improve photosynthesis efficiency, however, past many years' breeding efforts maximized the plant architecture to support light capture. The different reports on enhancement of photosynthetic capacity through genetic engineering approach will be useful in the coming decades to increase the photosynthetic efficiency in plants.

In the present study, KaCyt b₆ was overexpressed in tobacco for enhanced photosynthesis and biomass yield through acceleration of photosynthetic electron transfer. The PCR results showed integration of KaCyt b₆ and its overexpression under CaMV35S promoter in transgenic tobacco. The Cytochrome b₆f complex is central to electron transport chain and plays essential role in cyclic and linear photophosphorylation. This creates a proton gradient to synthesize ATP and maintain the ratio of ATP/NADPH for carbon fixation. The higher ETR in tobacco overexpressing KaCyt b₆ indicated the improved electron transport from PSII to Calvin cycle (Kalaji et al., 2017). The higher F_V’/F_M’ and qP along lower NPQ in tobacco overexpressing KaCyt b₆ indicated prominence of linear electron flow and balance between electron transport and utilization of NADPH (Kalaji et al., 2014; Chang et al., 2017). The increased ETR and qP in transgenic plants maintained the oxidized state of Q_A and open PSII RCs (Stefanov et al., 2016). The lower 1-qP in KaCyt b₆ tobacco protected the PSII from overexcitation and thus reduced the degree of photoinhibition (Quigg et al., 2012; Kalaji et al., 2017). The expression of KaCyt b₆ in transgenic tobacco improved the transfer of electrons from plastoquinol to plastocyanin, thus electron transfer. The improved electron transfer supported higher quantum yield of PSII activity in KaCyt b₆ in transgenic tobacco. The transfer of higher light energy toward PSII center in KaCyt b₆ transgenic tobacco resulted in higher PSII activity (Ermakova et al., 2019). The lower F₀ (redox state of the PQ pool) indicated active reaction centers and higher flow of electron from LHCII to PSII reaction center (Kalaji et al., 2011, 2017). The lower F_M indicated enhanced photoprotection with an increase in NPQ (Bolhar-Nordenkampf et al., 1989). The higher F_V/F_M (maximum quantum yield of PSII photochemical activity) in transgenic lines can be correlated with higher pigment content, net photosynthetic rate, stomatal conductance, transpiration rate, efficient flow of electron from light-harvesting complex to PSII, PSII structural integrity, efficient interaction between PSII reaction centers and peripheral antennae, active oxygen-evolving complex, activation of photoprotective regulatory mechanisms and activation of photosynthetic electron transport chain (Wientjes et al., 2013; Sharma et al., 2015; Kalaji et al., 2017). The lower value of F_V/F_M in WT and VC indicated the amount of closed PSII RCs (Wientjes et al., 2013). Higher F_V/F₀ (water-splitting complex efficiency) indicated higher light energy conversion efficiency in transgenic lines (Zhao et al., 2017). Higher F_V/F₀ indicated improved electron transport to augment the water-splitting complex (Pereira et al., 2000). The present results showed that overexpression of KaCyt b₆ in tobacco might have led to the increased production of sugar and starch contents in the transgenic tobacco plants due to availability of sufficient reducing power for Calvin cycle in the form of NADPH and ATP. These observations are in agreement with Chida et al. (2007) and our previous study (Yadav et al., 2018) using UfCyt c₆ gene from Ulva fasciata for enhanced photosynthesis and biomass.

Pigment contents (total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid) are important physiological parameters directly affecting photosynthetic performance in plants. The dark green color of leaves and pigment estimation indicated higher contents of photosynthetic pigments in transgenic plant overexpressing KaCyt b₆ as compared with WT and VC tobacco. The higher photosynthetic pigments supported better light capture and directly improved the photosynthetic rate in KaCyt b₆ transgenic tobacco (Simkin et al., 2017). The higher carotenoid contents supported improved light-harvesting capacity in KaCyt b₆ tobacco (Simkin et al., 2017) and protected KaCyt b₆ tobacco from oxygen and excessive light-induced damages (Peixoto et al., 2002). The chlorophyll a/b ratio indicates the PSII/PSI contents and lower ratio indicates higher efficiency of PSII as compared with PSI in KaCyt b₆ tobacco. The lower chlorophyll a/b ratio in KaCyt b₆ tobacco indicated increased stacks of thylakoid membranes, density of light-harvesting complex and larger antenna size indicating the optimum utilization of lower internal intensity (Evans, 1987; Stefanov et al., 2016). The higher ɸPSII estimates indicated efficient utilization of light as photochemical energy (Kalaji et al., 2014). The lower NPQ indicated lower thermal dissipation in transgenic plants (Ermakova et al., 2019) and higher CO₂ assimilation rate (Kromdijk et al., 2016). The higher qP in transgenic lines indicated better separation of charges in reaction center, electron transport, PSII yield, and fraction of open PSII centers (Maxwell and Johnson, 2000; Guo et al., 2006; Mao et al., 2007; Kalaji et al., 2017). Photosynthesis in plants depends on stomatal and nonstomatal activities. Stomatal activity provides exchange of gases through transpiration and stomatal conductance to be fixed as carbohydrate. The transgenic tobacco has shown to have higher transpiration due to stomatal opening (Dąbrowski et al., 2017; Khatri and Rathore, 2019; Najar et al., 2019). The higher stomatal conductance further showed cooling effect and efficient gas exchange required for photosynthetic process and the same has been shown to be correlated with enhancement in net photosynthesis (Nascentes et al., 2018). In addition, the photosynthetic pigments are nonstomatal factors and results showed higher pigment contents in transgenic tobacco, which indicated better light harvest. The improved photosynthetic responses in transgenic lines indicated higher production and utilization of ATP and NADPH, which in turn enhanced Calvin cycle and promoted sugar and starch synthesis (Sun et al., 2002; Chang et al., 2017). The present results confirmed that the increased photosynthesis rate was coupled with an increase in the accumulation of carbohydrate contents. Sugar served as signaling molecules (Bolouri-Moghaddam et al., 2010; Smeekens et al., 2010) and as a compatible solute protected thylakoid membrane and improves the PSII efficiency. The accumulated sugar in leaf cell helped to maintain osmotic potential and enhanced the biomass accumulation.

The results showed that enhancement of photosynthesis resulted in increased growth of transgenic tobacco overexpressing KaCyt b₆ under ambient climatic conditions. The improved growth of KaCyt b₆ transgenic tobacco can be directly correlated with higher rate of photosynthetic gas exchange and higher contents of soluble sugar and starch. The higher starch contents showed conversion of trapped solar energy as biomass in transgenic tobacco (Ambavaram et al., 2018). Similar results were reported in Panicum virgatum overexpressing PvBMY1 and PvBMY3 (Ambavaram et al., 2018), rice overexpressing Rieske FeS protein of cytochrome (Cyt) b₆/f complex (Yamori et al., 2016), and transgenic tomato plants overexpressing SBPase activity (Ding et al., 2016). In agreement with the present results, the cyt b₆/f complex may improve the CO₂ assimilation rate around the PSII (Yamori et al., 2016), hence biomass and yield. Similarly, Arabidopsis overexpressing Rieske FeS showed improved photosynthesis, biomass, and seed yield (Simkin et al., 2017). In the present case, the enhanced levels of sugar and starch content could be correlated with higher photosynthetic rate (Yin et al., 2015) and growth of KaCyt b₆ transgenic tobacco. The higher sugar content in KaCyt b₆ tobacco contributed to enhanced antioxidant defense potential, osmotic adjustment, maintenance of cellular structure and redox potential, plant metabolism, and development (Sperdouli and Moustakas, 2012). The increased soluble sugar maintains positive turgor pressure, which prevented dehydration and directly helped in leaf and growth expansion (Hasanuzzaman et al., 2017). The higher contents of starch can be correlated with the efficient supply of ATP and NADPH, which are utilized as energy molecules in the Calvin cycle (Chida et al., 2007) and supported higher synthesis of sugar and starch, hence growth of transgenic tobacco. The results of gas exchange and fluorescence measurements suggested that the overexpression of KaCyt b₆ in transgenic tobacco could be responsible for the increased photosynthetic efficiency in transgenic tobacco as compared with control (WT and VC) tobacco. The results clearly supported the fact that the photosynthetic electron transport rate can be improved through genetic engineering of the cyt b₆/f complex and this is one of the potential target site for enhancing photosynthetic efficiency and crop productivity.

Conclusion

Cytochrome b₆ (KaCyt b₆ ) gene from a red seaweed (K. alvarezii) was transformed and overexpressed under the control of CaMV35S promoter in transgenic tobacco. Compared with control tobacco (WT and VC) plants, the transgenic tobacco overexpressing KaCyt b₆ exhibited higher electron transport rate, photosystem II quantum yield, activity of the water-splitting complex, PSII photochemistry, qP, photosynthetic gas exchange, WUE, and photosynthetic yield. Transgenic tobacco exhibited higher contents of photosynthetic pigments and better ratios of photosynthetic pigments, which supported higher photosynthetic efficiency. The improved photosynthetic performance in transgenic tobacco supported higher production of sugar and starch contents. As a result, KaCyt b₆ transgenic tobacco exhibited comparatively enhanced growth under controlled and greenhouse conditions.

Footnotes

Acknowledgments

CSIR-CSMCRI PRIS-15/2020. S.K.Y. and K.K. are thankful to the Academy of Council of Scientific and Industrial Research (AcSIR), Ghaziabad (INDIA) for registration in PhD program.

Disclosure Statement

No competing financial interests exist.

Funding Information

Authors thankfully acknowledge the Council of Scientific and Industrial Research (CSIR), Government of India (CSC0116) for XII^th five-year plan project and Gujarat State Biotechnology Mission (GSBTM) (80G2DT/GAP 2080) Government of Gujarat for financial support. K.K. acknowledges financial support from CSIR, Government of India, in the form of CSIR-SRF.

Supplementary Material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

Supplementary Figure S6

Supplementary Table S1

References

Ambavaram

M.M.

, Ali

, Ryan

K.P.

, Peoples

, Snell

K.D.

, and Somleva

M.N.

(2018). Novel transcription factors PvBMY1 and PvBMY3 increase biomass yield in greenhouse-grown switchgrass (Panicum virgatum L.). Plant Sci, 273, 100–109.

Anderson

J.M.

, Price

G.D.

, Chow

W.S.

, Hope

A.B.

, and Badger

M.R.

(1997). Reduced levels of cytochrome bf complex in transgenic tobacco leads to marked photochemical reduction of the plastoquinone pool, without significant change in acclimation to irradiance. Photo Res, 53, 215–227.

Baker

N.R.

(2008). Chlorophyll fluorescence: a probe of photosynthesis in vivo . Annu Rev Plant Biol, 59, 89–113.

Bolhar-Nordenkampf

H.R.

, Long

S.P.

, Baker

N.R.

, Oquist

, Schreiber

, and Lechner

E.G.

(1989). Chlorophyll fluorescence as a probe of the photosynthetic competence of leaves in the field: a review of current instrumentation. Funct Ecol, 3, 497–514.

Bolouri-Moghaddam

M.R.

, Le Roy

, Xiang

, Rolland

, and Van den Ende

(2010). Sugar signalling and antioxidant network connections in plant cells. FEBS J, 277, 2022–2037.

Chamovitz

, Sandmann

, and Hirschberg

(1993). Molecular and biochemical characterization of herbicide-resistant mutants of cyanobacteria reveals that phytoene desaturation is a rate limiting step in carotenoid biosynthesis. J Biol Chem, 268, 17348–17353.

Chang

, Huang

H.E.

, Cheng

C.F.

, Ho

M.H.

, and Ger

M.J.

(2017). Constitutive expression of a plant ferredoxin-like protein (pflp) enhances capacity of photosynthetic carbon assimilation in rice (Oryza sativa). Transgenic Res, 26, 279–289.

Chida

, Nakazawa

, Akazaki

, Hirano

, Suruga

, Ogawa

. et al. (2007). Expression of the algal cytochrome c₆ gene in Arabidopsis enhances photosynthesis and growth. Plant Cell Physiol, 48, 948–957.

Dąbrowski

, Kalaji

M.H.

, Baczewska

A.H

, Pawluśkiewicz

, Mastalerczuk

, Borawska-Jarmułowicz

, et al. (2017). Delayed chlorophyll a fluorescence, MR820, and gas exchange changes in perennial ryegrass under salt stress. J Lumin, 183, 322–333.

10.

Ding

, Wang

, Zhang

, and Ai

(2016). Changes in SBPase activity influence photosynthetic capacity, growth, and tolerance to chilling stress in transgenic tomato plants. Sci Rep 6, 32741.

11.

Driever

S.M.

, Simkin

A.J.

, Alotaibi

, Fisk

S.J.

, Madgwick

P.J.

, Sparks

C.A.

et al. (2017). Increased SBPase activity improves photosynthesis and grain yield in wheat grown in greenhouse conditions. Philos Trans R Soc B Biol Sci 372, 20160384.

12.

Ermakova

, Lopez-Calcagno

P.E.

, Raines

C.A.

, Furbank

R.T.

, and von Caemmerer

(2019). Overexpression of the Rieske FeS protein of the Cytochrome b6f complex increases C4 photosynthesis. Commun Biol 2, 314.

13.

Evans

J.R.

(1987). The relationship between electron transport components and photosynthetic capacity in pea leaves grown at different irradiances. Funct Plant Biol, 14, 157–170.

14.

FAO. (2016). Food and Agriculture: Key to Achieving the 2030, Agenda for Sustainable Development. Job No. I5499 (Food and Agriculture Organization of the United Nations,, Rome, 23).

15.

Fedoroff

N.V.

, Battisti

D.S.

, Beachy

R.N.

, Cooper

P.J.M.

, Fischhoff

D.A.

, Hodges

C.N.

, et al. (2010). Radically rethinking agriculture in the 21st century. Science, 327, 883–884.

16.

Feng

, Wang

, Li

, Tan

, Kong

, Li

, et al. (2007). Overexpression of SBPase enhances photosynthesis against high temperature stress in transgenic rice plants. Plant Cell Rep, 26, 1635–1646.

17.

Głowacka

, Kromdijk

, Kucera

, Xie

, Cavanagh

A.P.

, Leonelli

, et al. (2018). Photosystem II Subunit S overexpression increases the efficiency of water use in a field-grown crop. Nature Commun 9, 868.

18.

Guo

H.X.

, Liu

W.Q.

, and Shi

Y.C.

(2006). Effects of different nitrogen forms on photosynthetic rate and the chlorophyll fluorescence induction kinetics of flue-cured tobacco. Photosynthetica, 44, 140–142.

19.

Hajirezaei

M.R.

, Peisker

, Tschiersch

, Palatnik

J.F.

, Valle

E.M.

, Carrillo

, et al. (2002). Small changes in the activity of chloroplastic NADP⁺-dependent ferredoxin oxidoreductase lead to impaired plant growth and restrict photosynthetic activity of transgenic tobacco plants. Plant J, 29, 281–293.

20.

Hasanuzzaman

, Davies

N.W.

, Shabala

, Zhou

, Brodribb

T.J.

, and Shabala

(2017). Residual transpiration as a component of salinity stress tolerance mechanism: a case study for barley. BMC Plant Biol 17, 107.

21.

Inskeep

, and Bloom

P.R.

(1985). Extinction coefficients of Chlorophyll a and b in N,N-dimethylformamide and 80% acetone. Plant Physiol, 77, 483–485.

22.

Kalaji

H.M.

, Bosa

, Kościelniak

, and Żuk-Gołaszewska

(2011). Effects of salt stress on photosystem II efficiency and CO₂ assimilation of two Syrian barley landraces. Environ Exp Bot, 73, 64–72.

23.

Kalaji

H.M.

, Schansker

, Brestic

, Bussotti

, Calatayud

, Ferroni

, et al. (2017). Frequently asked questions about chlorophyll fluorescence, the sequel. Photosynth Res, 132, 13–66.

24.

Kalaji

H.M.

, Schansker

, Ladle

R.J.

, Goltsev

, Bosa

, Allakhverdiev

S.I.

, et al. (2014). Frequently asked questions about in vivo chlorophyll fluorescence: practical issues. Photosynth Res, 122, 121–158.

25.

Kandoi

, Mohanty

, Govindjee, and Tripathy

B.C.

(2016). Towards efficient photosynthesis: overexpression of Zea mays phosphoenolpyruvate carboxylase in Arabidopsis thaliana . Photosynth Res, 130, 47–72.

26.

Karki

, Rizal

, and Quick

W.P.

(2013). Improvement of photosynthesis in rice (Oryza sativa L.) by inserting the C₄ pathway. Rice 6, 28.

27.

Khatri

, and Rathore

M.S.

(2019). Photosystem photochemistry, prompt and delayed fluorescence, photosynthetic responses and electron flow in tobacco under drought and salt stress. Photosynthetica, 57, 61–74.

28.

Köhler

I.H.

, Ruiz-Vera

U.M.

, VanLoocke

, Thomey

M.L.

, Clemente

, Long

S.P.

, et al. (2017). Expression of cyanobacterial FBP/SBPase in soybean prevents yield depression under future climate conditions. J Exp Bot, 68, 715–726.

29.

Kromdijk

, Głowacka

, Leonelli

, Gabilly

S.T.

, Iwai

, Niyogi

K.K.

, et al. (2016). Improving photosynthesis and crop productivity by accelerating recovery from photoprotection. Science, 354, 857–861.

30.

Kumari

, Udawat

, Dubey

A.K

, Haque

M.I.

, Rathore

M.S.

, and Jha

(2017). Overexpression of SbSI-1, a nuclear protein from Salicornia brachiata confers drought and salt stress tolerance and maintains photosynthetic efficiency in transgenic tobacco. Front Plant Sci 8, 1215.

31.

Lefebvre

, Lawson

, Zakhleniuk

O.V.

, Lloyd

J.C.

, Raines

C.A.

, and Fryer

(2005). Increased sedoheptulose-,7-bisphosphatase activity in transgenic tobacco plants stimulates photosynthesis and growth from an early stage in development. Plant Physiol, 138, 451–460.

32.

Long

S.P.

, Marshall-Colon

, and Zhu

X.G.

(2015). Meeting the global food demand of the future by engineering crop photosynthesis and yield potential. Cell, 161, 56–66.

33.

Makino

(2011). Photosynthesis, grain yield, and nitrogen utilization in rice and wheat. Plant Physiol, 155, 125–129.

34.

Mao

L.Z.

, Lu

H.F.

, Wang

, and Cai

M.M.

(2007). Comparative photosynthesis characteristics of Calycanthus chinensis and Chimonanthus praecox . Photosynthetica, 45, 601–605.

35.

Mathan

, Bhattacharya

, and Ranjan

(2016). Enhancing crop yield by optimizing plant developmental features. Development, 143, 3283–3294.

36.

Maxwell

, and Johnson

G.N.

(2000). Chlorophyll fluorescence-a practical guide. J Exp Bot, 51, 659–668.

37.

Medeiros

D.B.

, Martins

S.C.

, Cavalcanti

J.H.F.

, Daloso

D.M.

, Martinoia

, Nunes-Nesi

, et al. (2016). Enhanced photosynthesis and growth in atquac1 knockout mutants are due to altered organic acid accumulation and an increase in both stomatal and mesophyll conductance. Plant Physiol, 170, 86–101.

38.

Miyagawa

, Tamoi

, and Shigeoka

(2001). Overexpression of a cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in tobacco enhances photosynthesis and growth. Nat Biotechnol, 19, 965–969.

39.

Murashige

, and Skoog

(1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant, 15, 473–497.

40.

Murchie

E.H.

, and Niyogi

K.K.

(2011). Manipulation of photoprotection to improve plant photosynthesis. Plant Physiol, 155, 86–92.

41.

Murchie

E.H.

, Pinto

, and Horton

(2009). Agriculture and the new challenges for photosynthesis research. New Phytol, 181, 532–552.

42.

Najar

, Aydi

, Sassi-Aydi

, Zarai

, and Abdelly

(2019). Effect of salt stress on photosynthesis and chlorophyll fluorescence in Medicago truncatula . Plant Biosyst, 153, 88–97.

43.

Nascentes

R.F.

, Carbonari

C.A.

, Simões

P.S.

, Brunelli

M.C.

, Velini

E.D.

, and Duke

S.O.

(2018). Low doses of glyphosate enhance growth, CO₂ assimilation, stomatal conductance and transpiration in sugarcane and eucalyptus. Pest Manag Sci, 74, 1197–1205.

44.

Naseem

, Osmanoglu, Ö., and Dandekar

(2020). Synthetic rewiring of plant CO₂ sequestration galvanizes plant biomass production. Trends Biotechnol, 38, 354–359.

45.

Nölke

, Barsoum

, Houdelet

, Arcalís

, Kreuzaler

, Fischer

, et al. (2019). The integration of algal carbon concentration mechanism components into tobacco chloroplasts increases photosynthetic efficiency and biomass. Biotechnol J 14, e1800170.

46.

Onoda

, Hikosaka

, and Hirose

(2005). Seasonal change in the balance between capacities of RuBP carboxylation and RuBP regeneration affects CO₂ response of photosynthesis in Polygonum cuspidatum . J Exp Bot, 56, 755–763.

47.

Orr

D.J.

, Worrall

, Lin

M.T.

, Carmo-Silva

, Hanson

M.R.

, and Parry

M.A.

(2020). Hybrid cyanobacterial-tobacco Rubisco supports autotrophic growth and procarboxysomal aggregation. Plant Physiol, 182, 807–818.

48.

Parry

M.A.J.

, Andralojc

P.J

, Scales

J.C.

, Salvucci

M.E.

, Carmo-Silva

A.E.

, Alonso

, et al. (2013). Rubisco activity and regulation as targets for crop improvement. J Exp Bot, 64, 717–730.

49.

Peixoto

H.P.

, Da Matta

F.M.

, and Da Matta

J.C.

(2002). Responses of the photosynthetic apparatus to aluminum stress in two sorghum cultivars. J Plant Nutr, 25, 821–832.

50.

Pereira

W.E.

, de Siqueira

D.L.

, Martínez

C.A.

, and Puiatti

(2000). Gas exchange and chlorophyll fluorescence in four citrus rootstocks under aluminium stress. J Plant Physiol, 157, 513–520.

51.

Price

G.D.

, Pengelly

J.J.L.

, Forster

, Du

, Whitney

S.M.

, von Caemmerer

, et al. (2013). The cyanobacterial CCM as a source of genes for improving photosynthetic CO₂ fixation in crop species. J Exp Bot, 64, 753–768.

52.

Quigg

, Kotabova

, Jarešová

, Kaňa

, Šetlík

, Šedivá

, et al. (2012). Photosynthesis in Chromera velia represents a simple system with high efficiency. PLoS One 7, e47036.

53.

Raines

C.A.

(2011). Increasing photosynthetic carbon assimilation in C3 plants to improve crop yield: current and future strategies. Plant Physiol, 155, 36–42.

54.

Rao

P.V.S.

, Kumar

K.S.

, Ganesan

, and Thakur

M.C.

(2008). Feasibility of cultivation of Kappaphycus alvarezii (Doty) Doty at different localities on the Northwest coast of India. Aquacult Res, 39, 1107–1114.

55.

Reynolds

, Bonnett

, Chapman

S.C.

, Furbank

R.T.

, Manès

, Mather

D.E.

, et al. (2011). Raising yield potential of wheat. I. Overview of a consortium approach and breeding strategies. J Exp Bot, 62, 439–452.

56.

Reynolds

, Foulkes

M.J.

, Slafer

G.A.

, Berry

, Parry

M.A.J.

, Snape

J.W.

, et al. (2009). Raising yield potential in wheat. J Exp Bot, 60, 1899–1918.

57.

Rodriguez

R.E.

, Lodeyro

, Poli

H.O.

, Zurbriggen

, Peisker

, Palatnik

J.F.

, et al. (2007). Transgenic tobacco plants overexpressing chloroplastic ferredoxin-NADP(H) reductase display normal rates of photosynthesis and increased tolerance to oxidative stress. Plant Physiol, 143, 639–649.

58.

Rosenthal

, Locke

, Khozaei

, Raines

, Long

, and Ort

(2011). Over-expressing the C₃ photosynthesis cycle enzyme sedoheptulose-1–7 bisphosphatase improves photosynthetic carbon gain and yield under fully open air CO₂ fumigation (FACE). BMC Plant Biol 11, 123.

59.

Rott

, Martins

.d.F., Thiele

, Lein

, Bock

, Kramer

D.M.

, et al. (2011). ATP synthase repression in tobacco restricts photosynthetic electron transport, CO₂ assimilation, and plant growth by over acidification of the thylakoid lumen. Plant Cell, 23, 304–321.

60.

Ruuska

S.A.

, Andrews

T.J.

, Badger

M.R.

, Price

G.D.

, and von Caemmerer

(2000). The role of chloroplast electron transport and metabolites in modulating rubisco activity in tobacco. Insights from transgenic plants with reduced amounts of cytochrome b/f complex or glyceraldehyde 3-phosphate dehydrogenase. Plant Physiol, 122, 491–504.

61.

Salesse-Smith

C.E.

, Sharwood

R.E.

, Busch

F.A.

, Kromdijk

, Bardal

, and Stern

D.B.

(2018). Overexpression of Rubisco subunits with RAF1 increases Rubisco content in maize. Nat Plants, 4, 802–810.

62.

Sharma

D.K.

, Andersen

S.B.

, Ottosen

C.O.

, and Rosenqvist

(2015). Wheat cultivars selected for high Fv/Fm under heat stress maintain high photosynthesis, total chlorophyll, stomatal conductance, transpiration and dry matter. Physiol Plant, 153, 284–298.

63.

Simkin

A.J.

, McAusland

, Lawson

, and Raines

C.A.

(2017). Overexpression of the RieskeFeS protein increases electron transport rates and biomass yield. Plant physiol, 175, 134–145.

64.

Singh

, Pandey

, James

, Chandrasekhar

, Achary

V.M.M.

, Kaul

, et al. (2014). Enhancing C3 photosynthesis: an outlook on feasible interventions for crop improvement. Plant Biotechnol J, 12, 1217–1230.

65.

Smeekens

, Ma

J.K.

, Hanson

, and Rolland

(2010). Sugar signals and molecular networks controlling plant growth. Curr Opin Plant Biol, 13, 274–279.

66.

Sperdouli

, and Moustakas

(2012). Interaction of proline, sugars, and anthocyanins during photosynthetic acclimation of Arabidopsis thaliana to drought stress. J Plant Physiol, 169, 577–585.

67.

Stefanov

, Yotsova

, Rashkov

, Ivanova

, Markovska

, and Apostolova

E.L.

(2016). Effects of salinity on the photosynthetic apparatus of two Paulownia lines. Plant Physiol Biochem, 101, 54–59.

68.

Sukenik

, Bennett

, and Falkowski

(1987). Light-saturated photosynthesis-limitation by electron transport or carbon fixation?. Biochim Biophysi Acta, 891, 205–215.

69.

Sun

, Gibson

K.M.

, Kiirats

, Okita

T.W.

, and Edwards

G.E.

(2002). Interactions of nitrate and CO₂ enrichment on growth, carbohydrates, and Rubisco in Arabidopsis starch mutants. Significance of starch and hexose. Plant Physiol, 130, 1573–1583.

70.

Tamura

, Peterson

, Stecher

, Nei

, and Kumar

(2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol, 28, 2731–2739.

71.

von Caemmerer

, and Evans

J.R.

(2010). Enhancing C3 photosynthesis. Plant Physiol, 154, 589–592.

72.

Wientjes

, van Amerongen

, and Croce

(2013). Quantum yield of charge separation in photosystem II: functional effect of changes in the antenna size upon light acclimation. J Phys Chem B, 117, 11200–11208.

73.

Yadav

S.K.

, Khatri

, Rathore

M.S.

, and Jha

(2018). Introgression of UfCyt c₆ , a thylakoid lumen protein from a green seaweed Ulva fasciata Delile enhanced photosynthesis and growth in tobacco. Mol Biol Rep, 45, 1745–1758.

74.

Yamori

(2013). Improving photosynthesis to increase food and fuel production by biotechnological strategies in crops. J Plant Biochem Physiol 1, 113.

75.

Yamori

, Kondo

, Sugiura

, Terashima

, Suzuki

, and Makino

(2016). Enhanced leaf photosynthesis as a target to increase grain yield: insights from transgenic rice lines with variable Rieske FeS protein content in the cytochrome b6/f complex. Plant Cell Environ, 39, 80–87.

76.

Yamori

, Noguchi

, and Terashima

(2005). Temperature acclimation of photosynthesis in spinach leaves: analyses of photosynthetic components and temperature dependencies of photosynthetic partial reactions. Plant Cell Environ, 28, 536–547.

77.

Yamori

, Takahashi

, Makino

, Price

G.D.

, Badger

M.R.

, and von Caemmerer

(2011). The roles of ATP synthase and the cytochrome b6/f complexes in limiting chloroplast electron transport and determining photosynthetic capacity. Plant Physiol, 155, 956–962.

78.

Yin

, Yu

, Zhao

, Sun

, Ma

, et al. (2015). The influence of light intensity and photoperiod on duckweed biomass and starch accumulation for bioethanol production. Bioresour Technol, 187, 84–90.

79.

Zeng

, and Yang

(2002). RNA isolation from highly viscous samples rich in polyphenols and polysaccharides. Plant Mol Biol Rep 20, 417.

80.

Zhao

L.S.

, Li

, Wang

Q.M.

, Song

X.Y.

, Su

H.N.

, Xie

B.B.

, et al. (2017). Nitrogen starvation impacts the photosynthetic performance of Porphyridium cruentum as revealed by chlorophyll a fluorescence. Sci Rep 7, 8542.

81.

Zou

, and Gao

(2010). Photosynthetic acclimation to different light levels in the brown marine macroalga, Hizikia fusiformis (Sargassaceae, Phaeophyta). J Appl Phycol, 22, 395–404.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.29 MB

0.12 MB

0.24 MB

0.22 MB

0.21 MB

0.23 MB

Ectopic Expression of a Transmembrane Protein KaCyt b 6 from a Red Seaweed Kappaphycus alvarezii in Transgenic Tobacco Augmented the Photosynthesis and Growth

Abstract

Introduction

Materials and Methods

Seaweed collection, RNA extraction, and cDNA synthesis

Molecular cloning of full-length KaCyt b6 gene

Bioinformatics analysis

Construction of the plant expression vector and tobacco transformation

Molecular and β-glucuronidase analysis of transgenic tobacco

Physiological characterization of transgenic tobacco

Determination of leaf area, water content, and photosynthetic pigments

Estimation of soluble sugar and starch contents in tobacco overexpressing KaCyt b6 gene

Gas exchange and chlorophyll fluorescence measurements in KaCyt b6 tobacco

Data recording and statistical analysis

Results

Molecular cloning of KaCyt b6 cDNA and in silico analysis

Construction of KaCyt b6 expression vector and tobacco transformation

Transgenic tobacco overexpressing the KaCyt b6 showed improved growth attributes

Photosynthetic pigments estimated higher in tobacco overexpressing KaCyt b6

Overexpression of KaCyt b6 in transgenic tobacco enhanced photosynthesis

KaCyt b6 tobacco accumulated higher amounts of sugar and starch

Discussion

Conclusion

Footnotes

Acknowledgments

Disclosure Statement

Funding Information

Supplementary Material

References

Supplementary Material

Molecular cloning of full-length KaCyt b₆ gene

Estimation of soluble sugar and starch contents in tobacco overexpressing KaCyt b₆ gene

Gas exchange and chlorophyll fluorescence measurements in KaCyt b₆ tobacco

Molecular cloning of KaCyt b₆ cDNA and in silico analysis

Construction of KaCyt b₆ expression vector and tobacco transformation

Transgenic tobacco overexpressing the KaCyt b₆ showed improved growth attributes

Photosynthetic pigments estimated higher in tobacco overexpressing KaCyt b₆

Overexpression of KaCyt b₆ in transgenic tobacco enhanced photosynthesis

KaCyt b₆ tobacco accumulated higher amounts of sugar and starch