Overexpressing plant ferredoxin-like protein enhances photosynthetic efficiency and carbohydrates accumulation in Phalaenopsis

Crassulacean acid metabolism (CAM) is one of three major models of carbon dioxide assimilation pathway with better water-use efficiency and slower photosynthetic efficiency in photosynthesis. Previous studies indicated that the gene of sweet pepper plant ferredoxin-like protein (PFLP) shows high homology to the ferredoxin-1(Fd-1) family that belongs to photosynthetic type Fd and involves in photosystem I. It is speculated that overexpressing pflp in the transgenic plant may enhance photosynthetic efficiency through the electron transport chain (ETC). To reveal the function of PFLP in photosynthetic efficiency, pflp transgenic Phalaenopsis, a CAM plant, was generated to analyze photosynthetic markers. Transgenic plants exhibited 1.2-folds of electron transport rate than that of wild type (WT), and higher CO2 assimilation rates up to 1.6 and 1.5-folds samples at 4 pm and 10 pm respectively. Enzyme activity of phosphoenolpyruvate carboxylase (PEPC) was increased to 5.9-folds in Phase III, and NAD+-linked malic enzyme (NAD+-ME) activity increased 1.4-folds in Phase IV in transgenic plants. The photosynthesis products were analyzed between transgenic plants and WT. Soluble sugars contents such as glucose, fructose, and sucrose were found to significantly increase to 1.2, 1.8, and 1.3-folds higher in transgenic plants. The starch grains were also accumulated up to 1.4-folds in transgenic plants than that of WT. These results indicated that overexpressing pflp in transgenic plants increases carbohydrates accumulation by enhancing electron transport flow during photosynthesis. This is the first evidence for the PFLP function in CAM plants. Taken altogether, we suggest that pflp is an applicable gene for agriculture application that enhances electron transport chain efficiency during photosynthesis.


Introduction
Many orchids are CAM (Crassulacean acid metabolism) plant species that are living in trees as epiphytes using CAM photosynthetic pathway to achieve a water-preserving strategy.In the review on the taxonomic occurrence of CAM, Winter et al. (1983) pointed out that Orchidaceae present the greatest uncertainty concerning the number of CAM plants.Orchidaceae family has more than 725 orchid genera with between 20,000 and 25,000 species (Dressler 1981) and is estimated to have 7000, mostly epiphytic, CAM species (Winter and Smith 1996).Orchidaceae family would account for almost 50% of all CAM plants (Zotz 2004).Little is known about the effects after boosting electron transport chain in the photosynthesis of CAM plants.It would be interesting to investigate the photosynthesis rate by changing the pattern of electron transport chain with transgenic technique in CAM plants, even it has been proven that a plant ferredoxin-like protein (PFLP) can promote C3 photosynthesis in rice (Chang et al. 2017).To reveal the partition of linear and cyclic electron transport flow in CAM plants and the function of the altered electron transport chain by the PFLP in the CAM pathway.Phalaenopsis was selected for this project not only for the popularity, but also for the future horticultural application.Phalaenopsis is a very popular ornamental plant all over the world due to their elegant appearance and extended longevity of their flowers.Due to the flowering period adjustment can be regulated solely by temperature (Zhang et al. 2018); Phalaenopsis is of great economic importance for the horticultural industry.
There are defined four phases in the CAM pathway (Osmond 1978).Phase I: CAM plants exhibit nocturnal CO 2 assimilated by phosphoenolpyruvate carboxylase (PEPC) to generate malic acid at night; Phase II: a transient phase in the early light period, CAM plant closes stomata and reach the maximum content of malic acid; Phase III: CO 2 is regenerated from malic acid by NAD + -linked malic enzyme (NAD + -ME) or NADP + -linked malic enzyme (NADP + -ME) into Calvin cycle during the light period (Day 1980;Arron et al. 1979).Phase IV, in the late afternoon, the uptake of CO 2 initially occurs.Therefore, the gas exchange rate, organic acids, and carbohydrates contents show a diurnal rhythm in CAM plants.Although CAM plants show nocturnal CO 2 fixation for highly water-use efficiency, the photosynthetic efficiency is slower than C3 and C4 plants (Nobel 1991).Recently, many reports have been studied on photosynthetic efficiency in C3 and C4 plants, but a few investigations have been published with CAM plants (Cui 2021;Eva et al. 2018;Li et al. 2023;Pradhan et al. 2022).
Ferredoxin (Fd) is an important electronic carrier in photosynthesis containing a 2Fe-2S center as the prosthetic group that is responsible for using photosynthetic electrons to generate NADPH for carbon dioxide assimilation (Blanco et al. 2013).These photosynthetic electrons are transferred from water to NADP + that are catalyzed by Fd-NADP + reductase Vol.: (0123456789) (Mulo 2011).This linear electron flow (LEF) is one of the mechanisms for photosynthetic electron transport chain (PETC) during photosynthesis, while Fd can alternatively transfer electrons back to the plastoquinone (PQ) pool for PETC leading to a cyclic electron flow (CEF) around PSI (Joliot et al. 2006;Shikanai 2007;Rochaix 2011).Fd donates electrons to Fd:NADPH reductase (FNR) to generate ATP and NADPH via LEF that are utilized in the Calvin cycle or photorespiratory cycle.Alternatively, Fd may return electrons to the thylakoid plastoquinone pool, forming a CEF (Goss and Hanke 2014).Two main functions of CEF have been reported (Heber and Walked 1992).One is the ATP supplied for the Calvin cycle or photorespiratory cycle, and the other fate is to dissipate excess photon energy by inducing nonphotochemical quenching (qN) at stress conditions (Golding et al. 2004;Khatri and Rathore 2022;Miyake et al. 2004Miyake et al. , 2005;;Patel et al. 2022).Previous reports that decreased Fd content can alter electron distribution leading to decreased photosynthesis rate and plant growth was retarded (Hanke and Hase 2008;Holtgrefe et al. 2003;Voss et al. 2008).CEF was stimulated as well as the qN of chlorophyll fluorescence was enhanced after overexpressing of Atferredoxin-2 (Atfd-2) in tobacco (Yamamoto et al. 2006) These studies only emphasized the function of Fd in the photosynthetic electron transport flow.
Fd can also indirectly regulate some enzyme activities during the Calvin cycle.Fd interacts simultaneously with both Ferredoxin-Thioredoxin reductase (FTR) and Thioredoxin (Trx) which is called the Ferredoxin-Thioredoxin system (Glauser et al. 2004;Schürmann and Buchanan 2008).In this system, Fd donates electrons to reduce Trx through FTR.FTR has two highly conserved motifs, Cys-Pro-Cys and Cys-His-Cys.These Cys residues are essential for the ligate Fe-S cluster and the redox active disulfide bridge (Schürmann and Buchanan 2001).Trx also has a highly conserved motif, Trp-Cys-Gly-Pro-Cys (Holmgren 1989).The disulfide bond is formed by two redox-active Cys residues, Through this mechanism, those Calvin cycle enzymes such as Fru-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, NADP-dependent malate dehydrogenase, phosphoribulokinase, glyceraldehyde-3-P dehydrogenase, and Rubisco activase are regulated (Brandes et al. 1996;Clancey and Gilbert 1987;Goyer et al. 1999;Khatri and Rathore 2019;Nishizawa and Buchanan 1981;Trost et al. 2006;Zhang and Portis 1999).Fd plays several roles in photosynthesis through regulating enzyme activities of the Calvin cycle indirectly.
In this report, the plant ferredoxin-like protein PFLP belonging to the Fd family, was originally isolated from sweet pepper by our research group, which shows high homology with Fd-1 from Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa, and Spinacia oleracea (Dayakar et al. 2003), and by overexpression of pflp in some many agricultural and horticultural crops, such as banana, rice, Oncidium orchid, and Phalaenopsis orchid, enhanced host resistance to virulent bacterial pathogens (Chan et al. 2005;Liau et al. 2003;Tang et al. 2001;Yip et al. 2011), the capacity of photosynthetic carbon assimilation (Chang et al. 2017), and salinity tolerance (Huang et al. 2020).Based on PFLP belongs to the Fd family, transformation of CAM plants could result elevated PFLP levels that may enhance PETC and further promote photosynthetic efficiency in CAM plants, too.To reveal a better comprehension for this aspect in the CAM pathway of photosynthesis, transgenic Phalaenopsis plants were generated with constitutive expression of pflp.The transgenic plants were evaluated with electron transport rate (ETR) and some other CAM photosynthetic features such as diurnal CO 2 uptake, organic acid contents, PEPC, and NAD + -ME were investigated to demonstrate that overexpressing pflp enhanced both photosynthetic efficiency and carbohydrates accumulation in the CAM pathway.

Vector construct, plant material, transformation and screening
The vector construct containing a CaMV35S promoter, a full-length sweet pepper pflp cDNA, and a nos poly(A) construct in binary vector pCAMBIA 1304 (Liau et al. 2003).This commercial pCAMBIA 1304 was originally constructed with marker genes for further post-transformation screening.There are green fluorescent protein (GFP), β-glucuronidase (GUS) fusion gene, and hygromycin phosphotransferase (hph) gene.
Vol:. ( 1234567890) The plant materials sampled for experiment were little plantlets of Phalaenopsis aphrodite subsp.formosana that were generated from protocorm-like bodies (PLBs) after transformation.PLBs are capable of developing into individual plants through somatic embryogenesis.This technique has been a routine tissue-based practice for micropropagation of orchid plants (Chen et al. 2019).The induction, proliferation, and regeneration of PLBs are the most advantageous methods for orchid mass propagation in the world floricultural market (Cardoso et al. 2020).PLBs were previously generated and maintained by sub-culturing and carefully separated by forceps in T2 medium (Chen et al. 2009) (supplement Table 1) and incubated at 28 °C with a 16 h photoperiod.
For transformation and screening, the newly formed PLBs from a 45-day-old culture were chopped, cultured in T2 medium, and incubated at 28 °C in the dark for 7 days.The constructed vector was transformed into Phalaenopsis PLBs via Agrobacterium-mediated transformation (Agrobacterium strain: EHA 105).By the hygromycin in culture media, the successful transformed plant materials were screened and selected for further experiments.The pre-cocultured PLBs in T2 medium were then infected with Agrobacterium in a liquid coculture medium (supplement table 2) containing 200 μM Acetosyringone (AS) for 20 min and then cocultured in coculture medium plates containing 200 μM AS and incubated at 28 °C in darkness for 2 days.The infected PLBs were suspension-cultured with liquid coculture medium containing 200 mg/L timentin and 200 mg/L cefotaxime for 6 h to prevent Agrobacterium overgrowth, then cultured in 1/4 MS medium plate (supplement table 3) supplemented with 200 mg/L timentin and 200 mg/L, cefotaxime, and 25 mg/L hygromycin in darkness for 30 days.After 30 days, the PLBs were transferred to a T2 medium with a 16 h photoperiod at 28 °C.Regenerated plants about 3-5 cm of leaf length and 3-4 leaves were eventually transferred to pots containing sphagnum moss and acclimatized for 3 months in the growth chambers.The growth chambers were set as a photoperiod of 16 h light (4:00-19:59, 100 μmol m −2 s −1 PAR) and 8 h dark (20:00-3:59), with 28 °C and 75-85% relative humidity.
Genomic PCR, reverse transcription PCR, and GUS stain Genomic DNA was extracted by Plant Genomic DNA Purification Kit (GeneMark, Taiwan).For genomic DNA amplification, 0.1 μg genomic DNA was used as a template, adding the following primer pairs: for the pflp gene, forward primer (5′-ACT GAA ACT TAT CAC ACC TGACG-3′) and reverse primer (5′-GGA TAA GCA ACA CAA GTT AGCAC-3′) (Chang et al. 2017).The DNA sample was denatured at 94 °Cfor 3 min followed by 35 amplification cycles (94 °C for 50 s, 55 °C for 50 s, 72 °C for 50 s) and finally 7 min at 72 °C.For RT-PCR detection, total RNA was isolated from the Phalaenopsis leaf using Trizol reagent (Molecular research center, USA) according to the RNA extraction manual.The total RNA was reverse transcripted into cDNA using ImProm-IITM Reverse Transcription System (Promega, USA) with oligo-dT primer, then 0.5 μg cDNA was used as the template for PCR.The PCR condition was similar to genomic PCR, but the amplification cycle was 28 cycles.For histochemical GUS staining, the leaf materials were soaked in GUS solution (0.1 M Na-phosphate buffer, pH 7.0, 1 mM X-Gluc) at 37 °C.After 2 days of incubation, samples were treated with 75% alcohol for depigment.Analysis of photosynthetic gas exchange The second leaf of seeding was used for photosynthetic gas exchange with an infrared gas analyzer system (LI-6400, Li-Cor Inc., Lincoln NE, USA).The gas exchange system was zeroed daily using anhydrous magnesium perchlorate to remove water and using soda lime to remove CO 2 from the air entering the cuvette.When the sample was measured, external air was removed of CO 2 and mixed with a supply of pure CO 2 to create a standard concentration of 400 μmol m −2 s −1 , the flow rate was 500 μmol m −2 s −1 , leaf temperatures were set at 28 °C and light intensity was maintained at 100 μmol m −2 s −1 PAR by a light emitting diode source (6400-02, Li-Cor Inc., Lincoln NE, USA) during the daytime.

Chlorophyll fluorescence measurements
The chlorophyll fluorescence measurements were performed using a pulse-amplitude modulation fluorometer (PAM-210, Walz, Effeltrich, Germany).The second leaf of seedling was pretreated for 30 min in dark.
The minimal fluorescence (Fo) was determined under the radiation light that was not enough to induce any significant variable fluorescence.The maximal fluorescence value (Fm) was determined by a saturating pulse on the dark-adapted leaf and then maintained for 6 min under actinic light (Fs).Thereafter, a second saturating pulse was imposed to determine the maximal fluorescence value on the light-adapted leaf (Fm').The actinic light was turned off and the far-red light was turned on for about 3 s to obtain the minimal fluorescence level in the light-adapted state (Fo').Thecalculation of the fluorescence parameters was according to Rohacek and Bartak (1999).All measurements were made between 17:00 and 19:00.

Histological detection of starch grains
The leaf materials were fixed by Formalin Aceto Alcohol solution containing 3.7% (v/v) formaldehyde, 5% (v/v) acetic acid, and 50% (v/v) EtOH, then dehydrated by stepwise increasing the concentration of ethanol and xylene.Samples were embedded and cut at 5 μm on a microtome, the sections were stained with Lugol's iodine solution (6 mM iodine, 43 mM KI, and 0.2 N HCl) and examined using a light microscope (Primo-Star, Zeiss, Jena, Germany) to detect starch grains.

Metabolites analysis
For organic acid determination, the samples of 0.2 g of leaf material were boiled in distilled water (Callaway et al. 1997).Organic acids were analyzed by anion exchange chromatography on the Dionex AS-11 column (2 mm diameter) according to the manufacturer's application note 107 (Dionex Corporation, Sunnyvale, CA, USA) using 5-100 mM NaOH as gradient eluant and quantified by Dionex Electrochemical Detector (ED50).To reduce the background eluate conductivity, the detector was preceded by a suppressor system (ASRS ULTRAII-2 mm, Dionex Corporation, Sunnyvale, CA, USA).
For soluble sugar determination, the sample of 0.2 g of leaf material was homogenized and extracted by a hot 80% EtOH as previously described (Wang et al. 2000).Sugars were separated on HPAEC-PAD (Dionex Corporation, Sunnyvale, CA, USA) with Dionex CarboPac PA10 column (2 mm diameter) using 18 mM NaOH as eluant and quantified by Dionex Pulsed Amperometric Detector.The sediment was analyzed for starch by digesting with the mixture of pullulanase and amyloglucosidase, and the formed glucose was estimated by glucose oxidase and peroxidase method (Wang et al. 1993).

Statistical analysis
One-way ANOVA was used with a 5% LSD Means Test in cases of multiple comparisons.The statistical analysis was carried out using Cohort statistical analysis software (Ver.6, Cohort).Data presented are mean ± SE of replicates.

Generation and selection of Phalaenopsis transformants
The transformed PLBs were screened and maintained in the media containing hygromycin.The plants for further experiments were regenerated successfully from these PLBs.All the regenerated plants were subjected to genomic PCR to confirm the presence of pflp transgene.At least 100 plants were confirmed after PCR results, and all these plants were pooled and grown for further analysis.The selected PCR results showed that pflp transgene has been integrated into the genome of transgenic plants (Fig. 1a).RT-PCR analysis was performed to detect pflp transgene expression levels.The transcript of pflp was detectable in the transgenic plant but not in WT (Fig. 1b).Due to the reason that the pflp expressing vector contains the gus reporter gene, GUS stain analysis was also used to detect transgene expression in protein levels.The result shows that transgenic plants exhibited GUS activity, while there was no signal detectable in WT (Fig. 1c).These results indicate both transcription and translation of pflp transgene were expressed in transgenic plants.After 3 months incubated in growth chambers, totally 75 plants without morphological abnormal phenotype were selected after the 3 months observation, and these 75 plants were pooled as candidates for further photosynthetic ability analysis.The phenotype of selected transgenic plants and WT were not different (Fig. 1d).

Overexpressing pflp enhances PETC in transgenic plant
The chlorophyll fluorescence of transgenic plants was analyzed to determine the PFLP effects on PETC.The ETR were elevated in both transgenic and WT plants in the initial phase, and the maximum ETR rates reached saturation was approximately 450 μmol m −2 s −1 PAR (Fig. 2a).In contrast, the transgenic plant presented an initial significantly increased ETR at 100 μmol m −2 s −1 PAR that was 1.2folds greater than that for WT (Fig. 2a).Especially at a higher light intensity (400-800 μmol m −2 s −1 PAR), transgenic plants showed much higher ETR than that of WT.The data suggested that transgenic plant has higher electron transport potential even in highintensity light.To confirm the higher ETR potential in transgenic plants was not due to the amount of the higher chlorophyll contents in transgenic plants, the chlorophyll contents were also analyzed and the result did not show a significant difference between transgenic plants and WT (Fig. 2b).On the other hand, the chlorophyll fluorescence parameters are shown in Table 1.The maximum quantum yield (Fv/Fm) appeared to be close to 0.8 in transgenic plants and WT.Moreover, qN also remained at similar levels, 0.766 ± 0.063 for WT and 0.797 ± 0.051 for transgenic plants, respectively.The actual quantum yield (ΦP) of the transgenic plant showed 1.7-folds higher than that of WT.It indicates a higher quantum efficiency of LEF in transgenic plants.In addition, the photochemical quenching (qP) of transgenic plants also showed 1.7-folds higher than that of WT.These results indicated that an increase in the efficiency of PETC is due to higher efficient electron transfer in LEF.

Comparing the CO 2 assimilation rate between transgenic plants and WT
The gas exchange rate is one of the photosynthetic feature markers to study the efficiency of carbon assimilation.CAM plant is characterized by nocturnal CO 2 fixation.Thus, diurnal CO 2 assimilation was investigated and compared between transgenic plants and WT.As shown in Fig. 3a, both WT and transgenic plants assimilated CO 2 in the late afternoon.The maximum CO 2 assimilation was recorded at the beginning of the night (22:00).In contrast to WT plants, transgenic plants showed a significant increase in CO 2 assimilation between 16:00 and 4:00.Transgenic plants started earlier to assimilate CO 2 than those of WT, the CO 2 assimilation rate of transgenic plants showed significantly increased 1.6-folds higher than those of WT at 16:00.At 22:00, the maximum CO 2 assimilation, the transgenic plant was up to 1.5folds compared with WT.These results demonstrated that transgenic plants started earlier and had higher CO 2 assimilation than those of WT.

Effects of increasing CO 2 assimilation on organic acid contents in transgenic plants
In CAM, the uptake of CO 2 would generate organic acids into vacuole for temporary storage during the nighttime, and convert to CO 2 into Calvin cycle at day (Bonner and Bonner 1948;Day 1980).To detect the diurnal rhythm of organic acids contents in a transgenic plant, malic acid, and citric acid contents were monitored in the four classical phases of CAM, driven by changes in carbon metabolism (Males and Griffiths 2017) at midday (12:00, phase III), dusk (20:00, phase IV), midnight (00:00, phase I), and daybreak (04:00, phase II) also according to Osmond's report (1978).Figure 3b showsmalic acid content with a diurnal rhythm in transgenic plants and WT, the highest level of malic acid was reached at phase II (04:00) and the minimum was found at phase IV (20:00).In contrast, the content of malic acid in transgenic plants exhibited 30% and 20% greater than these of WT at phase I (00:00) and phase II (04:00), respectively.Therefore, the higher accumulation of malic acid in transgenic plants results from increasing CO 2 assimilation.
Many CAM plants also accumulate citric acid in addition to malic acid (Borland and Griffiths 1988;Herppich et al. 1995;Lüttge 1988).For Phalaenopsis, Fig. 3c shows a diurnal rhythm of citric acid content which was similar to the pattern of malic acid.In contrast, the content of citric acid in transgenic plants exhibited 1.3-folds greater than that of WT at phase II (04:00).The higher content of citric acid in transgenic plants also responds to increasing CO 2 assimilation (Fig. 3c) as citric acid served as one of the precursors.These results indicate that the CO 2 assimilation increasing could yield higher organic acid contents for temporary storage in transgenic plants at night.
The activities of phosphoenolpyruvate carboxylase (PEPC) and NAD + -linked malic enzyme (NAD + -ME) Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme that fixes nocturnal CO 2 in CAM.It takes CO 2 to catalyze PEP carboxylation that yields oxaloacetate (OAA) and inorganic phosphate.OAA can be reduced to malic acid and stored in the vacuole (Chollet et al. 1996).In transgenic plants, the activity of PEPC appeared to significantly increase from phase III (12:00) to phase IV (20:00) (Fig. 4a).Especially at phase III (12:00), the PEPC activity of transgenic plants up to 5.9-folds compared with WT.During the daytime, malic acid is transported to the chloroplast from the vacuole and then decarboxylated by mitochondrial NAD + -ME.For biochemical support, the enzyme activity of NAD + -ME was analyzed.As shown in Fig. 4b, the activity of NAD + -ME in transgenic plants exhibited 1.4-folds greater activity than that of WT at phase IV (20:00) (Fig. 4b).These data indicated that transgenic plants contain higher PEPC and NAD + -ME activities than WT that could release more CO 2 from organic acid into the Calvin cycle during daytime.Moreover, the activity of cytosolic NADP + -ME were not detected both in WT and transgenic plants (data not shown).

The impact of overexpressing pflp on carbohydrate accumulation in transgenic plants
The CO 2 absorbed by plants finally is converted to carbohydrates through photosynthesis.Glucose, fructose, and sucrose were the major soluble carbohydrates in plant leaves.At phase III (12:00), all the content of soluble carbohydrates reached a maximum.The transgenic plant showed significantly higher soluble carbohydrates than for WT (Fig. 5).The content of glucose, fructose, and sucrose in the transgenic plant was 1.2, 1.8, and 1.3-folds higher than those in the WT, respectively.The distribution of starch grains was observed in vegetative tissues through Lugol assay.Starch stains with Lugol's solution displayed the blue-purple color and were found mainly in mesophyll cells.In transgenic plants, the number of starch grains was significantly more than for WT (Fig. 6a and b).At phase IV (20:00), the starch amounts of transgenic plants and WT were showed the highest level, and it was increased approximately 1.4-folds in the transgenic plant compared with WT (Fig. 6c).Apparently, these data indicated that overexpressing pflp plants exhibited higher carbohydrate accumulation.

Discussion
The pflp transgenic Phalaenopsis lines were generated and PCR-confirmed.The photosynthetic activities were evaluated with non-transgenic WT.Compared with WT, the pflp transgenic plant exhibits higher ETR and CO 2 uptake, and posed higher PEPC activity which promotes CO 2 conversation to malic acid.Higher NAD + -ME activities were observed to release much more CO 2 from organic acid for Calvin cycle in the pflp transgenic plants.Moreover, photosynthetic products such as starch, glucose, fructose, and sucrose are highly accumulated in the transgenic plant than these of WT.In this report, we confirmed that overexpressing a pflp in a CAM Phalaenopsis not only enhanced photosynthetic efficiency but also consequently increased carbohydrate accumulation.
The electron transfer from plastoquinone to cytochrome b6f is considered to be the rate-limiting step in PETC (Junge 1977) due to a structure of cytochrome b6f complex provides a basis for control the rate-limiting electron transfer step of oxygenic photosynthesis associated with the plastoquinol/quinone exchange pathway (Ness et al. 2019).Chida et al. (2007) reported that enhancement of electron transporter could increase ETR even it is not the ratelimiting step.In our previous work pointed out that PFLP shows high homology to Fd-1which is located downstream of cytochrome b6f in PETC (Dayakar et al. 2003).In this study, analysis of chlorophyll fluorescence indicated that pflp transgenic plant could enhance ETR.The parameter ΦP reflecting the actual quantum yield of PSII was higher in the transgenic plants.It confirms that PSII efficiency is enhanced in the transgenic plant.Additionally, the other parameter qP represents means excitation energy using for photosynthesis, also significantly increased in the transgenic plant than that of WT.Therefore, overexpression of pflp in the CAM plant improved electron transfer efficiency in PETC.Moreover, high light intensity could induce higher ETR.But previous studies indicated the maximum of photosynthetic efficiency is reached at about 130-180 μmol m −2 s −1 PAR in Phalaenopsis (Ota et al. 1991;Lootens and Heursel 1998;Lee and Guo 2000).Lee and Guo (2000) also reported a light saturation point at about 200 μmol m −2 s −1 PAR.If the light intensity is over 200 μmol m −2 s −1 PAR, the photoinhibition will be induced in Phalaenopsis (Fig. 2a).Our data indicate that transgenic plants presented significantly increased ETR, with 20% and 30% more than for WT at 100 and 200 μmol m −2 s −1 PAR conditions, respectively.Therefore, no matter unsaturated or saturated light conditions, PFLP could enhance ETR in the transgenic plant.Yamamoto et al. (2006) reported that overexpression of Atfd-2 in tobacco displayed a normal rate of photosynthesis, and stimulate CEF to enhance qN.Their results indicated that over reduced Fd prefers to return the plastoquinines to form CEF, but does not reduce FNR to generate NADPH through LEF.Therefore, Atfd-2 transgenic plant might not have enough NADPH to support more CO 2 to fix in the Calvin cycle.For the maximum quantum yield (Fv/ Fm) is one of the healthy markers in plants and will decrease under stress conditions (Rohacek and Bartak 1999).In this study, it appeared to be close to 0.8 in both transgenic plants and WT plants indicating both plants were not under stresses during the experiments.Moreover, the parameter qN in the pflp transgenic plant did not show a significant difference with WT.It is implied that transgenic plants may display a normal CEF.The parameters ΦP and qP showed significantly increased in the transgenic plant than that of WT.These results strongly suggest that driven LEF to enhance ETR is prefer in PFLP rather than the CEF.The pflp transgenic plant improved photosynthesis efficiency through stimulation of LEF.
In the CAM plants, Chen et al. (2008) reported that NAD + -ME should play an important role in the decarboxylation of malic acid for Phalaenopsis.NAD + -ME participation is required for carbohydrate synthesis to release CO 2 into the Calvin cycle.The NAD + -ME presented higher activity in the transgenic plant than that of WT during the daytime.Due to the high activity of NAD + -ME, the content of malic acid was decreasedsignificantly in the transgenic plant than that of WT at phase III (12:00).Some reports indicated PEPC belongsto an allosteric enzyme and the activity is decreased by malic acid (Carter et al. 1995;Nimmo 2000).It is reasonable that transgenic plants showed significantly higher activity of PEPC than WT in phase III (12:00) resulted from higher malic acid level than that of WT.As for the higher content of citric acid in transgenic plants also responds to increasing CO 2 assimilation, whenever CO 2 uptake is increased, Oxaloacetate might also be elevated then citrate level increased.The nocturnal citric acid accumulation is not associated with net fixation of CO 2 , as provision of acetyl-CoA for the citrate synthase reaction via oxidative decarboxylation of pyruvate itself releases CO 2 .Nevertheless, both citric acid and malic acid provide CO 2 for carbohydrate assimilation for photosynthesis.With the high activity of PEPC, pflp transgenic plant converts more intercellular CO 2 to malic acid in mesophyll cells.Therefore, transgenic plants presented higher CO 2 assimilation rate than WT after 12 h.As the consequence, higher contents of carbohydrates such as glucose, fructose, sucrose, and starch were observed in the pflp transgenic plant than these of WT.Our result showed that PFLP can enhance the carbon fixation in CAM transgenic plants.Therefore, we proposed two possible mechanisms to elevate carbohydrates synthesis by PFLP.Firstly, the pflp transgenic plant exhibits higher PETC under both saturated and unsaturated light conditions.It indicates that transgenic plants can utilize more light energy to produce carbohydrates.Secondly, it has been known that many enzymes involve in the Calvin cycle can be regulated by the Ferredoxin-Thioredoxin system (Glauser et al. 2004;Schürmann and Buchanan 2008).Overexpressing of pflp in the transgenic plant may enhance these enzyme activities to synthesis more carbohydrates.Further studies are necessary to make sure the relationship between PFLP and enzymes involved in the Calvin cycle.
Genetic engineering has proven to be a powerful tool in enhancing photosynthesis efficiency to solve the world food crisis problem (Dubey et al. 2021;Patel and Mishra 2021;Stefanov et al. 2022;Yadav et al. 2020).Different genetic strategies have been proposed to increase carbohydrate products, including overexpressing catalytic enzymes in the Calvin cycle or enhancing electron transfer of photosynthesis (Miyagawa et al. 2001;Lefebvre et al. 2005;Chida et al. 2007;Tula et al. 2020;Yadav et al. 2018).In this study, by overexpressing pflp, previously cloned from a sweet pepper, in the transgenic CAM plants, the photosynthesis rate and sugar productions were elevated accordingly through enhancing LEF.This report provides the first evidence for the PFLP function in CAM plants, a novel ferredoxin proten is capable of alternating electron paritioning by increasing linear electron flow in the photosystem I.

Fig. 2
Fig. 2 The capacity of PETC in pflp transgenic plants and WT. a The electron transport rate as a function of actinic light intensity of WT (close circles) and transgenic plants (open circles).Data are means ± SE of 16 replicates.b Chlorophyll content in WT (black bar) and transgenic plants (white bar).All these transgenic plants were selected from a pool of 75 transgenic plants.Data are means ± SE of 5 replicates.Asterisks indicate the mean values are significantly different between WT and transgenic plants (P < 0.05)

Fig. 3
Fig. 3 Comparing the carbon source between WT and transgenic plants.a The diurnal patterns of CO 2 assimilation rate of WT (close circles) and transgenic plants (open circles).Data are means ± SE of 8 replicates.b Malic acid and c citric acid in WT (black bar) and transgenic plants (white bar) with diurnal rhythm.Phase III: 12:00 as midday; Phase IV: 20:00 as evening; Phase I: 00:00 as midnight; Phase II: 04:00 as daybreak.All these transgenic plants were selected from a pool of 75 transgenic plants.Data are means ± SE of 3 replicates.Asterisks indicate the mean values are significantly different between WT and transgenic plants (P < 0.05)

Fig. 4 Fig. 5
Fig. 4 The diurnal oscillation of fixed CO 2 enzyme activity.a PEPC, b NAD + -ME activities in WT (black bar) and transgenic plants (white bar).Data are means ± SE of 5 replicates.All these transgenic plants were selected from a pool of 75 transgenic plants.Asterisks indicate the mean values are significantly different between WT and transgenic plants (P < 0.05)

Fig. 6
Fig. 6 Starch distribution in leaves of a WT and b transgenic plants.Seedling leave were sampled at 20:00 as evening (phase IV) and stained with Lugol's solution for starch grains.Scale bar = 200 μm.c Changes in starch content of WT (black bar) and transgenic plants (white bar) with diurnal rhythm.All these transgenic plants were selected from a pool of 75 transgenic plants.Data are means ± SE of 4 replicates.Asterisks indicate the mean values are significantly different between WT and transgenic plants (P < 0.05)

Table 1
Chlorophyll fluorescence analysis of PSII efficiency in WT and transgenic plants The values represent the mean ± SE of 16 individual plants.Asterisks indicate the mean values are significantly different between WT and transgenic plants (P < 0.05) Fv/Fm The maximum quantum yield.ΦP The actual quantum yield.qP Photochemical quenching.qN Nonphotochemical quenching Vol.: (0123456789)