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. 2023 Mar 2;21(6):1206–1216. doi: 10.1111/pbi.14030

Faster induction of photosynthesis increases biomass and grain yield in glasshouse‐grown transgenic Sorghum bicolor overexpressing Rieske FeS

Maria Ermakova 1,2,✉, Russell Woodford 1, Zachary Taylor 1,3, Robert T Furbank 1, Srinivas Belide 4, Susanne von Caemmerer 1
PMCID: PMC10214756  PMID: 36789455

Summary

Sorghum is one of the most important crops providing food and feed in many of the world's harsher environments. Sorghum utilizes the C4 pathway of photosynthesis in which a biochemical carbon‐concentrating mechanism results in high CO2 assimilation rates. Overexpressing the Rieske FeS subunit of the Cytochrome b 6 f complex was previously shown to increase the rate of photosynthetic electron transport and stimulate CO2 assimilation in the model C4 plant Setaria viridis. To test whether productivity of C4 crops could be improved by Rieske overexpression, we created transgenic Sorghum bicolor Tx430 plants with increased Rieske content. The transgenic plants showed no marked changes in abundances of other photosynthetic proteins or chlorophyll content. The steady‐state rates of electron transport and CO2 assimilation did not differ between the plants with increased Rieske abundance and control plants, suggesting that Cytochrome b 6 f is not the only factor limiting electron transport in sorghum at high light and high CO2 . However, faster responses of non‐photochemical quenching as well as an elevated quantum yield of Photosystem II and an increased CO2 assimilation rate were observed from the plants overexpressing Rieske during the photosynthetic induction, a process of activation of photosynthesis upon the dark–light transition. As a consequence, sorghum with increased Rieske content produced more biomass and grain when grown in glasshouse conditions. Our results indicate that increasing Rieske content has potential to boost productivity of sorghum and other C4 crops by improving the efficiency of light utilization and conversion to biomass through the faster induction of photosynthesis.

Keywords: sorghum, crop yield, C4 photosynthesis, light interception, electron transport, photosynthetic induction

Introduction

C4 plants utilize a specialized photosynthetic pathway in which a metabolic C4 cycle acts as a biochemical carbon concentrating mechanism (Hatch, 1987). The C4 cycle operates between mesophyll and bundle sheath (BS) cells, and Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco), the main enzyme of CO2 fixation, is localized to the BS (Kanai and Edwards, 1999). Atmospheric CO2 (in the form of HCO3 −) is first fixed in mesophyll cells by PEP carboxylase (PEPC) into a C4 acid (hence the term C4 photosynthesis). C4 acids diffuse to BS cells where they are decarboxylated to produce pyruvate and CO2, providing high CO2 partial pressure around Rubisco (Furbank and Hatch, 1987). Higher carboxylation efficiency of Rubisco in C4 plants allows better radiation‐use‐efficiency and increased biomass production, compared to C3 plants, in warm climates (Long, 1999; Sage and Zhu, 2011). Because of their superior productivity, C4 crops are becoming increasingly important for food and bioenergy security. The global production of C4 maize (Zea mays) often surpasses the two key C3 cereals, wheat and rice, and C4 miscanthus (Miscanthus × giganteus) and switchgrass (Panicum virgatum) are two of the currently leading dedicated biomass crops. This has created considerable interest in identifying and testing strategies to improve productivity of C4 crops (Sales et al., 2021; von Caemmerer and Furbank, 2016).

While C4 plants are more productive, running the C4 cycle requires additional input of energy. Whilst C3 plants need at least two mols of NADPH and three mols of ATP to fix 1 mol of CO2, C4 plants need two additional ATP molecules to regenerate PEP from pyruvate in mesophyll cells (Edwards et al., 2001; Hatch, 1987). NADPH and ATP are the products of light reactions of photosynthesis which include electron and proton transport in the thylakoid membranes of chloroplasts. NADPH is produced during linear electron flow as electrons originating from water split by Photosystem II (PSII) are transferred by the chain of cofactors, via Cytochrome b 6 f complex (Cytb 6 f) and Photosystem I (PSI), to NADP+. Cytb 6 f links oxidation of plastoquinol with the translocation of protons to the lumen, a space enclosed by the thylakoid membrane, by operating the Q‐cycle (Malone et al., 2021). The transmembrane proton gradient (ΔpH) established across the thylakoid membrane creates a proton motive force (pmf) that drives ATP production via the ATP synthase complex. In addition to linear electron flow, C4 plants are thought to run active cyclic electron flow to produce additional ATP (Ishikawa et al., 2016; Munekage and Taniguchi, 2016; Nakamura et al., 2013). Cyclic electron flow returns electrons from the reducing side of PSI back to the plastoquinone (PQ) pool to repeat plastoquinol oxidation by Cytb 6 f and build up additional pmf (Johnson, 2011). Thus, cyclic electron flow results in the net production of ATP but not NADPH. ΔpH controls PSII activity by regulating the energy‐dependent and quickly reversible form of non‐photochemical quenching (NPQ), q E (Li et al., 2002). NPQ is a common term for diverse reactions that help to reduce excitation energy reaching reaction centres of PSII (Malnoë, 2018). Establishing q E requires the PsbS protein that senses luminal pH and modifies the light‐harvesting complex II (LHCII) to dissipate a part of absorbed light as heat, as well as the conversion of violaxanthin to zeaxanthin (Johnson et al., 2009; Li et al., 2004).

The vast majority of agriculturally important C4 crops, like maize, sorghum (Sorghum bicolor), sugarcane, miscanthus and several millets (e.g. Setaria italica), belong to the NADP‐ME subtype of C4 photosynthesis which employs NADP+‐dependent malic enzyme to decarboxylate the C4 acid malate in BS chloroplasts (Furbank, 2011). Due to drastic metabolic differences between mesophyll and BS cells in NADP‐ME plants, electron transport chains of the two cell types are also largely different: mesophyll cells predominantly run linear electron flow and BS cells – cyclic electron flow (Ermakova, Bellasio, et al., 2021; Munekage, 2016). To enable the production of NADPH and ATP in each cell type at the required ratio, plants need to tightly regulate the distribution of available light energy (Bellasio and Ermakova, 2022; Bellasio and Lundgren, 2016). Combined with the elevated ATP demand of C4 plants (Sage et al., 2011), this results in a slow operation of the C4 cycle at low irradiance, leading to a lower CO2 partial pressure in BS cells and a decreased efficiency of C4 photosynthesis (Furbank and Hatch, 1987; Kromdijk et al., 2010). Therefore, increasing radiation‐use‐efficiency is one of the primary strategies for increasing assimilation rates and productivity of C4 plants.

Constitutive overexpression of the Rieske FeS subunit of Cytb 6 f (hereafter Rieske), encoded by the nuclear petC gene was shown to increase abundance of the whole complex in both mesophyll and BS cells of a model NADP‐ME grass Setaria viridis (Ermakova et al., 2019). This resulted in a higher quantum yield of both photosystems and higher CO2 assimilation rates at high light and high CO2. However, the feasibility of using Rieske overexpression for improving crop productivity required further assessment. Here we test effects of increasing Rieske content on yield of the multipurpose C4 crop, sorghum. We show that glasshouse‐grown sorghum plants with increased Rieske abundance have faster induction of photosynthesis and produce more biomass and grain. Our results indicate that increasing Rieske content is a promising strategy for stimulating yield of sorghum and other C4 crops.

Results

Sorghum plants transformed with the construct for Rieske overexpression (see Materials and Methods for details) were selected based on kanamycin resistance and transferred to soil for growth in a glasshouse. Sixteen T0 plants were recovered and analysed for insertion number, transgene expression and leaf Rieske content. The ntpII insertions and transcripts of Brachypodium dystachion petC (BdpetC) were confirmed in 10 T0 plants (Figure 1a,b). Plants 25, 26, 29 and 32 showed relatively higher Rieske abundance per leaf area compared to wild type (WT) and to escape plants without the T‐DNA insertion (Figure 1a). The T1 progenies of those four plants were grown and analysed for insertion numbers and Rieske abundance. The homozygous T1 plants of lines 25 and 26 (4 insertions, Figure 1c) had higher Rieske leaf content compared to control plants (WT and null segregants). Furthermore, homozygous plants 25–11 and 26–11 showed relatively lower NPQ compared to control and other T1 plants when assayed at ambient light, in line with the NPQ phenotype reported in S. viridis overexpressing Rieske (Ermakova et al., 2019). The progenies of those two plants, as well as the progeny of the homozygous T1 plant 32–14 with increased Rieske leaf content (Figure S1), were used in further experiments, and are hereafter referred to as transgenic lines 25, 26 and 32.

Figure 1.

Figure 1

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Selection of transgenic sorghum lines overexpressing Rieske. (a) Immunodetection of Rieske in leaves of T0 plants. (b) Transcript abundance of B. dystachion petC (BdpetC) in T0 plants. (c) Immunodetection of Rieske in T1 progenies of lines 25 and 26. (a, c) Samples were loaded on leaf area basis, and the titration series of WT samples was used for relative quantification. Insertion numbers indicate a copy number of ntpII obtained by digital PCR. Asterisks indicate the plants which progenies were used in further experiments. (d) Quantum yield of non‐photochemical quenching (PhiNPQ) measured at ambient irradiance in T1 progenies of lines 25 and 26. WT and azygous plants were used as control. Each point represents a technical replicate.

T2 plants of the three selected transgenic lines and azygous control plants were grown over summer in a glasshouse with natural light. Abundances of photosynthetic proteins were analysed in leaf extracts loaded on leaf area basis by immunoblotting with specific antibodies (Figure 2a). Quantification of immunoblots demonstrated a significant, about 40%, increase of Rieske content in all three transgenic lines compared to control plants. In the BS protein samples, Rieske abundance in transgenic plants was increased to a similar extent suggesting matching levels of overexpression in both cell types (Figure S2). Relative abundance of other electron transport components, such as the D1 protein of PSII, AtpB subunit of ATP synthase, PsbS and Lhcb2 subunit of LHCII was largely unaltered in leaves of transgenic plants overexpressing Rieske (Figure 2b) as well as the relative leaf Chl content (Table 1). The content of PEPC and Rubisco large subunit (RbcL) did not differ between leaves of transgenic and control plants (Figure 2b).

Figure 2.

Figure 2

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Protein analysis and growth of sorghum lines overexpressing Rieske. (a) Immunodetection of photosynthetic proteins in leaves of control and transgenic plants: Rieske (Cytb 6 f), D1 (PSII core), AtpB (ATP synthase), PsbS (energy‐dependent NPQ), Lhcb2 (light‐harvesting complex II), RbcL (large subunit of Rubisco), PEPC (PEP carboxylase). Samples were loaded on leaf area basis, and the titration series of the Control sample #1 was used for relative quantification. (b) Quantification of immunoblots relative to control plants. Mean ± SE, n = 3 biological replicates. (c) Phenotype of plants 5 weeks after germination. (d–f) Height, leaf number and plant biomass (DW, dry weight), n = 5 biological replicates. (g, h) Total weight and number of seeds produced per plant, n = 18 biological replicates for Control (WT and azygous plants), n = 20 for Rieske‐OE (lines 25 and 26). Asterisks indicate statistically significant differences between transgenic and control plants based on one‐way ANOVA or t‐test (**P < 0.05 or *P < 0.1).

Table 1.

Properties of transgenic sorghum plants overexpressing Rieske

Control Line 25 Line 26 Line 32
Relative chlorophyll 60.1 ± 1.9 57.6 ± 1.9 60.9 ± 1.8 60.5 ± 1.8
Leaf thickness, mm 0.63 ± 0.05 0.63 ± 0.03 0.60 ± 0.03 0.63 ± 0.04
LMA, g (dry weight) m−2 119.42 ± 4.78 115.73 ± 4.08 113.92 ± 4.72 122.32 ± 2.65
Tiller number, plant−1 1.4 ± 0.27 2.4 ± 0.45 2.0 ± 0.36 2.0 ± 0.36
FV/FM 0.802 ± 0.002 0.802 ± 0.006 0.803 ± 0.003 0.810 ± 0.001*
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F V/F M, the maximum quantum efficiency of PSII; LMA, leaf mass per area.

Azygous plants were used as control. Mean ± SE, n = 5 biological replicates. Asterisks indicate statistically significant differences between transgenic and control plants (one‐way ANOVA and Dunnett's post hoc test at P < 0.05).

Sorghum plants with increased Rieske content grown in a glasshouse over summer were taller than the control plants at 5 weeks after germination (Figure 2c,d) and had more tillers (0.036 < P > 0.09, Table 1) and leaves (Figure 2e). Whilst the leaf thickness and leaf dry mass per area did not differ between the genotypes (Table 1), the total aboveground biomass of lines 26 and 32 at harvest was higher compared to control plants (Figure 2f). In another experiment, when plants were grown in a glasshouse during late summer‐autumn, Rieske‐OE plants of lines 25 and 26 had larger leaves compared to control plants (azygous and WT, Figure S3) and produced more seeds by weight and number than control plants (Figure 2g,h).

To understand why transgenic sorghum plants overexpressing Rieske accumulated more biomass, we analysed their photosynthetic properties. First, we conducted gas‐exchange and fluorescence analysis at different CO2 partial pressures and irradiances. No significant differences in CO2 assimilation rate or the effective quantum yield of PSII (PhiPSII) were detected between the plants with increased Rieske content and control plants at constant irradiance of 1500 μmol/m2/s and different CO2 partial pressures (Figure 3, left panels). At ambient CO2, CO2 assimilation rates and stomatal conductance were similar between the genotypes at all irradiances (Figure 3, right panels). The photochemical and non‐photochemical yields of PSI and PSII analysed at different irradiances were largely unaltered in transgenic plants compared to control plants (Figure 4), except for the quantum yield of PSI (PhiPSI) being higher and the non‐photochemical loss of PSI yield due to the acceptor side limitation (PhiNA) being lower in lines 25 and 32 at low irradiance of 95 μmol/m2/s. These results indicated that the steady‐state rates of electron transport and CO2 assimilation were largely unaffected in plants overexpressing Rieske. The maximum quantum efficiency of PSII (F V/F M), however, was significantly higher in plants of line 32 compared to control plants (Table 1).

Figure 3.

Figure 3

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Gas exchange and fluorescence analysis of control and transgenic sorghum plants overexpressing Rieske at different CO2 partial pressures (left panels, measured at 1500 μmol photons/m2/s) or irradiance (right panels, measured at ambient CO2). PhiPSII, the effective quantum yield of PSII; gsw, stomatal conductance to water vapour. Azygous plants were used as control. Mean ± SE, n = 5 biological replicates. No statistically significant differences were found between transgenic and control plants (one‐way ANOVA and Dunnett's post hoc test at P < 0.05).

Figure 4.

Figure 4

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Electron transport parameters estimated at different irradiance from leaves of control and transgenic sorghum plants overexpressing Rieske. PhiPSII, the effective quantum yield of PSII; PhiNPQ, the yield of non‐photochemical quenching; PhiNO, the yield of non‐regulated non‐photochemical reactions within PSII; PhiPSI, the effective quantum yield of PSI; PhiND, the non‐photochemical yield of PSI due to the donor side limitation; PhiNA, the non‐photochemical yield of PSI due to the acceptor side limitation. Azygous plants were used as control. Mean ± SE, n = 5 biological replicates.

Energisation of the thylakoid membranes in sorghum with increased Rieske content was tested by electrochromic shift signal and absorbance changes at 535 nm. By the end of 3‐min illumination intervals, pmf and proton conductivity of the thylakoid membrane (g H+, reflecting the speed of pmf dissipation and thereby reporting on ATP synthase activity) did not differ between the plants overexpressing Rieske and control plants at any irradiance (Figure 5a,b). To gain information about the kinetics of q E which follows the build‐up of ΔpH upon illumination, we recorded absorbance changes at 535 nm which reflect both zeaxanthin formation and the LHCII modifications induced by PsbS (Horton et al., 1991; Li et al., 2004). All three transgenic lines overexpressing Rieske established qE significantly faster than control plants upon the shift from dark to 1600 μmol/m2/s, indicating a faster build‐up of ΔpH, likely, due to a transiently increased Cytb 6 f activity (Figure 5c).

Figure 5.

Figure 5

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Analysis of the thylakoid membrane energisation in control plants and transgenic sorghum lines overexpressing Rieske. (a, b) Proton motive force (pmf) and proton conductivity of the thylakoid membrane (g H+) at different irradiances analysed using electrochromic shift signal. Mean ± SE, n = 4 biological replicates. (c) Absorbance changes at 535 nm upon the shift from darkness to 1600 μmol/m2/s. Arrow indicates the beginning of illumination. The absorbance at the beginning and end of the 3‐min illumination period was normalized to 0 and 1, respectively, to facilitate comparison of the kinetics. Averages of four biological replicates are presented. Azygous plants were used as control. Asterisks indicate intervals of statistically significant difference between transgenic and control plants (one‐way ANOVA and Dunnett's post hoc test at P < 0.05).

To capture the effects of transiently increased Cytb 6 f activity on assimilation, we next analysed photosynthetic induction, the process of activation of photosynthesis upon the dark–light transition, during the first 30 min of illumination of the overnight‐dark‐adapted plants with actinic light of 1000 μmol/m2/s1 (Figure 6). Since the steady‐state CO2 assimilation rate, stomatal conductance, PhiPSII and NPQ did not differ between genotypes (Figures 3 and 4), these parameters were normalized to 0 and 1 at the beginning and end of the 30‐min illumination, respectively, to facilitate comparison of the kinetics. During the induction of photosynthesis, CO2 assimilation rates increased faster in sorghum plants overexpressing Rieske compared to control plants, and between 18 and 26 min the rates were significantly higher in all three transgenic lines. The kinetics of PhiPSII induction was similar to the assimilation: transgenic lines reached the steady‐state faster and lines 25 and 26 had significantly increased PhiPSII, compared to control plants, between 18 and 26 min since the onset of illumination (Figure 6). Interestingly, plants overexpressing Rieske also showed more dynamic changes in NPQ: a faster NPQ built‐up during the first 7 min of illumination (with transiently higher NPQ levels in lines 25 and 26 compared to control plants) and a faster relaxation of NPQ (with transiently lower NPQ in line 32 compared to control plants). Importantly, although the stomatal conductance was significantly higher in all three transgenic lines, compared to control plants, between 16 and 23 min since the beginning of illumination (Figure 6), the detected increases of CO2 assimilation in these plants were not stimulated by the CO2 availability. This was evident from the unaltered ratios of intercellular to ambient CO2 partial pressures (C i/C a) during the second part on the induction (Figure 6).

Figure 6.

Figure 6

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Induction of photosynthesis during the first 30 min of illumination with actinic light of 1000 μmol/m2/s in control plants and transgenic sorghum overexpressing Rieske. PhiPSII, the effective quantum yield of PSII; NPQ, non‐photochemical quenching; gsw, stomatal conductance to water vapour; C i/C a, the ratio between the intercellular and ambient CO2 partial pressures. The values of each parameter, except for C i/C a, were normalized to 0 and 1 at the beginning and end of the 30‐min illumination, respectively, to facilitate comparison of the kinetics. Azygous plants were used as control. Mean ± SE, n = 5 biological replicates. Asterisks indicate intervals of statistically significant difference between transgenic and control plants (one‐way ANOVA and Dunnett's post hoc test at P < 0.05).

Discussion

Sorghum is one of the most important crops in the world which serves as a source of food, fodder and fuel. Sorghum can withstand severe droughts allowing it to grow in regions where other major crops cannot be grown, like Sub‐Saharan Africa. However, in recent years, genetic progress in sorghum yield has stagnated and not kept pace with increasing demand (Ananda et al., 2020). Therefore, it is critical to develop new approaches for increasing sorghum productivity. Based on the crop model predictions, depending on the irrigation method, between 3.3% and 9.2% improvement in sorghum yield could be harnessed from improving photosynthesis (Wu et al., 2019). According to the biochemical model of C4 photosynthesis, assimilation at low CO2 is limited by PEPC and CA activities and mesophyll conductance to CO2, whilst assimilation at ambient and high CO2 is limited by Rubisco, electron transport or the regeneration rate of Rubisco's substrate (von Caemmerer, 2000, 2021; von Caemmerer and Furbank, 1999). Contribution of some of these factors to C4 photosynthesis and plant productivity was recently tested using transgenic approach in the model C4 plant S. viridis (Alonso‐Cantabrana et al., 2018; Ermakova et al., 2022; Osborn et al., 2016), and improvements of C4 photosynthesis were shown in S. viridis and Z. mays with increased Rieske and Rubisco content, respectively, as well as in S. viridis engineered with the CO2 permeable aquaporins targeted to mesophyll plasma membranes (Ermakova et al., 2019; Ermakova, Osborn, et al., 2021; Salesse‐Smith et al., 2018). Here we expanded on our previous results and assessed whether Rieske overexpression could improve productivity of sorghum.

Rieske overexpression in sorghum provided increased Cytb 6 f activity, confirmed by monitoring the build‐up of q E and dynamics of electron transport during the photosynthetic induction. A faster establishing of q E during dark–light transition in transgenic plants was likely prompted by a transiently larger ΔpH due to the increased Cytb 6 f activity (Figure 5c). However, by the end of 3‐min illumination periods, pmf, which is a sum of ΔpH and Δψ (the membrane potential), did not differ between genotypes (Figure 5a). This was consistent with the largely unchanged electron transport parameters and CO2 assimilation rates (Figures 3 and 4) – all indicating that steady‐state rates of electron transport were unaltered in sorghum plants overexpressing Rieske. Similar observations were made on tobacco plants with increased Rieske content (Heyno et al., 2022). In C3 plants, this phenomenon could be explained by a conserved relationship between ΔpH and NPQ which would reduce PSII activity in case if electron transport rate exceeds the capacity of dark reactions of photosynthesis to consume ATP and NADPH, typically due to a limited availability of CO2 (Kanazawa and Kramer, 2002). C4 photosynthesis, however, is thought to be less limited by CO2 due to the C4 cycle concentrating CO2 around Rubisco, and an increase of electron transport rate is projected to provide a proportional increase in assimilation (von Caemmerer and Furbank, 2016). Increasing Rieske content in S. viridis was sufficient to enhance electron transport rates, resulting in higher photosynthesis at non‐limiting CO2 and high light (Ermakova et al., 2019). One possible explanation for the difference observed between S. viridis and sorghum overexpressing Rieske is that C4 crops underwent a selection for photosynthetic traits during domestication which could have altered a balance between electron transport components. For example, translational efficiency of Lhca6, a subunit of the light‐harvesting complex I that facilitates the formation of PSI supercomplex involved in cyclic electron flow (Otani et al., 2018), was significantly enhanced during maize domestication (Zhu et al., 2021). Changes to the ratio of cyclic to linear electron flow capacity could likely result in additional to Cytb 6 f or different factors limiting electron transport. Better understanding of photosynthetic changes that C4 crops underwent during domestication will help to uncover additional targets for accelerating steady‐state electron transport and photosynthesis rates (Hu et al., 2018; Tao, George‐Jaeggli, et al., 2020; Zhu et al., 2021). Increased assimilation rates detected in Z. mays with increased Rubisco content at non‐limiting CO2 and high light could also be indicative of an altered relationship between electron transport and Rubisco limitations in C4 crops (Salesse‐Smith et al., 2018).

Interestingly, the largest difference in electron transport and assimilation between plants overexpressing Rieske and control plants was detected during the induction of photosynthesis in the dark‐adapted leaves. Induction is a complex process that, in C3 plants, requires opening of stomata, activation of Rubisco and other enzymes and a build‐up of metabolite concentrations (Deans et al., 2019; Slattery et al., 2018). Faster activation of photosynthesis was identified as one of the desirable traits in crop plants promising up to 20% increase in total assimilation (Acevedo‐Siaca et al., 2020; Long et al., 2022). Activation of C4 photosynthesis is further complicated by the distribution of electron transport and metabolic reactions between mesophyll and BS cells and a necessity to coordinate activities of C4 and C3 cycles (Furbank and Taylor, 1995; Kromdijk et al., 2014). Our results support previous works suggesting that, due to the operation of carbon concentrating mechanism, activation of C4 photosynthesis is less limited by stomatal conductance compared to C3 photosynthesis (Furbank and Walker, 1985; Usuda and Edwards, 1984). Indeed, in our experiments, C i/C a never dropped below 0.25 (Figure 6) which is equivalent to C i ≥ 100 μmol/mol sufficient to saturate assimilation (Pignon and Long, 2020).

Photosynthetic induction in C4 plants is slower than in closely related C3 species and, in NADP‐ME dicot Flaveria bidentis, the inefficiency was attributed to an incomplete suppression of photorespiration caused by a slow build‐up of the C4 cycle intermediates (Arce Cubas et al., 2023; Sage and McKown, 2006). Alternatively, Rubisco activation was proposed as a primary cause of lost efficiency based on the increased leakiness of CO2 detected during the photosynthetic induction in NADP‐ME monocots, maize and sorghum, and metabolic modelling of NADP‐ME C4 photosynthesis (Wang et al., 2021, 2022). In this work, similar levels of PhiPSII between control and transgenic plants in the beginning of induction suggested unaltered linear electron flow rate (Figure 6), whilst the faster establishing of NPQ in plants with increased Rieske content indicated that more pmf was generated, likely, via cyclic electron flow. The increased CEF/LEF in the transgenic plants could support the presence of additional ATP sinks during the induction: both leakiness and Rubisco activation significantly increase ATP demand in mesophyll and BS cells, respectively (Farquhar, 1983; Robinson and Portis Jr., 1988; Streusand and Portis Jr., 1987). Since sorghum BS cells have low PSII abundance and activity (Meierhoff and Westhoff, 1993), NPQ and PhiPSII estimated by fluorescence analysis (Figure 6) predominantly report on electron transport in mesophyll cells. However, a larger pmf is likely being generated during the photosynthetic induction also in BS cells of sorghum with increased Rieske content, which is corroborated by a more active pmf build‐up in BS cells of S. viridis overexpressing Rieske (Ermakova et al., 2019). A faster build‐up of pmf could also promote a more alkaline stromal pH in BS cells required to activate Rubisco and fructose bisphosphatase (Flügge et al., 1980; Mott and Berry, 1986). Therefore, our results indicate that increased Cytb 6 f activity could stimulate CEF/LEF thereby providing more ATP to facilitate activation of Rubisco and the C3 cycle in BS cells and to counteract increased leakiness by supporting re‐fixation of CO2 in mesophyll cells. The faster relaxation of NPQ to the steady‐state level in sorghum plants with increased Rieske content could also contribute to the elevated PhiPSII and assimilation rate during the second part of the induction (Figure 6). Better understanding of dynamics of Rubisco (de)activation in C4 plants, which seems to differ between C3 and C4 and even within C4 species at least under low irradiance (Sage and Seemann, 1993), will be necessary to get an insight into molecular mechanisms of increased Cytb 6 f activity stimulating the photosynthetic induction in sorghum.

A cumulative effect of faster daily induction of photosynthesis could provide the increases in biomass and grain yield observed from the sorghum plants overexpressing Rieske. The transgenic plants produced more leaves, had larger leaves during the vegetative growth phase and accumulated more biomass by the end of growth season, compared to control plants (Figures 2, S3). Moreover, sorghum with increased Rieske content produced about 20% more grain by weight and number (Figure 2g,h) indicating that higher yield of transgenic plants was largely attributed to setting more seeds. Grain yield and grain number highly correlate in sorghum, and a supply of assimilates during the seed setting largely determines grain number (Craufurd and Peacock, 1993; van Oosterom and Hammer, 2008). Conceivably, an increase of CO2 assimilation during the photosynthetic induction could provide a surplus of assimilates signalling to plants to set more seeds. Therefore, our results show that increasing Rieske content in sorghum improves the light‐use‐efficiency and stimulates biomass production in glasshouse conditions, presenting a promising route for improving sorghum productivity. Further testing of increased Rieske content in elite sorghum lines and in field conditions with limited water supply is necessary to reveal the potential of this trait for developing higher‐yielding sorghum varieties. Exploring variation of Rieske content in sorghum variety panels (Tao, Zhao, et al., 2020) could inform breeding programs and help to introduce this trait into commercial hybrids.

Conclusion

In sorghum, effects of Rieske overexpression on stimulating electron transport were more apparent during the induction of photosynthesis suggesting that other photosynthetic components, besides Cytb 6 f, (co) limit the steady‐state electron transport. Nevertheless, faster activation of photosynthesis in the dark‐adapted sorghum overexpressing Rieske resulted in transiently increased CO2 assimilation rates and stimulated biomass and grain production, confirming that the photosynthetic induction limits productivity of C4 crops (Wang et al., 2021). Therefore, increasing Rieske content relative to other electron transport components is a promising way to boost crop yields to ensure future food and energy security.

Materials and methods

Generation and selection of transgenic plants

The gene construct for Rieske overexpression was created using the Golden Gate cloning system (Engler et al., 2014), as described in Ermakova et al. (2019). The first expression module containing the selection marker was occupied by neomycin phosphotransferase II (ntpII) driven by the Z. mays ubiquitine1 promotor. The second expression module contained the coding sequence of petC from Brachipodium dystachion (BdpetC) under the control of the Z. mays ubiquitine1 promotor. The construct was transformed into Agrobacterium tumefaciens strain AGL1 and then into Sorghum bicolor Tx430 according to Gurel et al. (2009). Transgenic plants were analysed for the ntpII copy number by digital PCR (iDNA genetics, Norwich, UK). Azygous plants were used as control in all experiments. Transcript abundance of BdpetC was estimated by qPCR as described in Ermakova et al. (2019). Electron transport at ambient light intensity and leaf properties (relative chlorophyll and leaf thickness) were assayed with MultispeQ (Kuhlgert et al., 2016) and analysed using the PhotosynQ platform (https://photosynq.com).

Plant growth conditions

Plants were grown in a naturally‐lit glasshouse at 28 °C day, 20 °C night and 60% humidity. Daily global solar exposure data for the main glasshouse experiment conducted during December 2021 – March 2022 are provided in Figure S4. Seeds were germinated in peat pots and, 1 week later, plants were transplanted into 8 L pots filled with potting mix supplemented with 2 g/L of slow‐release fertilizer (Osmocote, Scotts, Australia). Plants were watered daily and shuffled every 2–3 days to reduce any positional growth effects. The youngest fully expanded leaves of the 4–5 weeks old plants were used for all physiological analyses. Photos of plants, leaves and tiller count and height measurements were done during the fully emerged leaves stage, 5 weeks after germination.

Gas exchange and fluorescence analyses

Rates of CO2 assimilation were measured over a range of CO2 partial pressures and irradiances using a portable gas‐exchange system LI‐6800 (LI‐COR Biosciences, Lincoln, NE). Chlorophyll fluorescence was assessed simultaneously with a Fluorometer head 6800–01 A (LI‐COR Biosciences). Leaves were first equilibrated at 400 ppm CO2 in the reference side, 1500 μmol/m2/s, leaf temperature 28 °C, 60% humidity and flow rate 500 μmol/s. CO2 response curves were conducted under the constant irradiance of 1500 μmol/m2/s by imposing a stepwise increase of CO2 partial pressures from 0 to 1600 ppm at 3 min intervals. Light response curves were measured at 400 ppm in the reference cell under a stepwise increase of irradiance from 0 to 2000 μmol/m2/s at 3 min intervals. Red‐blue actinic light (90%/10%) was used in all measurements. The effective quantum yield of PSII (PhiPSII) was assessed at the end of each step upon the application of a multiphase saturating pulse (8000 μmol/m2/s) and calculated according to Genty et al. (1989).

Induction of photosynthesis was analysed on the overnight‐dark‐adapted plants. First, leaves were clipped into LI‐6800 chamber in darkness and the minimum and maximum levels of fluorescence were recorded upon the application of a saturating pulse. After that, leaves were illuminated with actinic light of 1000 μmol/m2/s1, and gas‐exchange and fluorescence parameters were recorded every 1 min for 30 min. NPQ was calculated according to Bilger and Björkman (1990). All parameters, except for C i/C a, were normalized to 0 and 1 at the beginning and end of the 30‐min illumination, respectively, to facilitate comparison of the kinetics.

Electron and proton transport

Fluorescence parameters informing on the activity of PSII and absorbance at 820 nm reflecting the formation of oxidized P700, the reaction centre of PSI, were analysed simultaneously by the Dual‐PAM‐100 (Heinz Walz, Effeltrich, Germany). Measurements were done using red actinic light and 300‐ms saturating pulses of 10000  μmol/m2/s. Leaves were first dark‐adapted for 30 min to record the minimal and maximal levels of fluorescence and calculate FV/FM, the maximum quantum efficiency of PSII. Then, a saturating pulse was applied after a pre‐illumination with strong far‐red light to record the maximal level of P700+ signal and, after the pulse, the minimal level of P700+ signal. Next, leaves were illuminated for 10 min with an actinic light of 378  μmol/m2/s. After that, photosynthetic parameters were assessed over a range of irradiances from 0 to 2043  μmol/m2/s at 2 min intervals by applying a saturating pulse at the end of each step. The effective quantum yield of PSII (PhiPSII), the yield of non‐photochemical quenching (PhiNPQ) and the yield of non‐regulated non‐photochemical reactions within PSII (PhiNO) were calculated according to Kramer et al. (2004). The effective quantum yield of PSI (PhiPSI), the non‐photochemical yield of PSI caused by the donor side limitation (PhiND) and the non‐photochemical yield of PSI caused by the acceptor side limitation (PhiNA) were calculated according to Klughammer and Schreiber (2008).

The electrochromic shift signal (ECS) was monitored as the absorbance change at 515–550 nm using the Dual‐PAM‐100 equipped with the P515/535 emitter‐detector module (Heinz Walz). The absorbance signal at 535 nm was monitored simultaneously. Leaves were first dark‐adapted for 40 min and the amplitude of ECS induced by a single turnover flash was recorded. Dark‐interval relaxation kinetics of ECS was then recorded after 3‐min intervals of illumination with red actinic light of increasing irradiance. Proton motive force (pmf) was estimated from the amplitude of the rapid decay of ECS signal upon light–dark transition, normalized for the ECS induced by the single turnover flash. Proton conductivity of the thylakoid membrane through ATP synthase was calculated as an inverse time constant obtained by the fitting of first‐order ECS relaxation kinetics after Sacksteder and Kramer (2000).

Protein isolation and Western blotting

BS strands were isolated following the procedure of Ghannoum et al. (2005). Total protein extracts were isolated from 0.7 cm2 frozen leaf discs or from BS strands as described in Ermakova, Bellasio, et al. (2021), loaded on leaf area or protein basis and separated by SDS‐PAGE as described in Ermakova et al. (2019). Proteins were then transferred to a nitrocellulose membrane and probed with antibodies against various photosynthetic proteins in dilutions recommended by the producer: Rieske (AS08 330; Agrisera, Vännäs, Sweden), D1 (AS10 704; Agrisera), AtpB (AS05 085; Agrisera), PsbS (AS09 533; Agrisera), Lhcb2 (AS01 003; Agrisera), RbcL (Martin‐Avila et al., 2020), PEPC (Ermakova, Arrivault, et al., 2021). Quantification of immunoblots was performed with Image Lab software (Biorad, Hercules, CA).

Statistical analysis

The relationship between mean values of different groups was tested by one‐way ANOVAs with Dunnett's or Tukey's post‐hoc test or by two‐tailed heteroscedastic t‐test, as indicated in figure legends.

Conflict of interest statement

The authors declare no conflict of interests.

Author contributions

SvC, ME and RTF designed the research; SB generated transgenic plants; ME, RW, SB and ZT performed research; ME and RW analysed data; ME wrote the paper with input from all authors.

Supporting information

Figure S1 Immunodetection of Rieske in the T1 progeny of line 32.

Figure S2 Immunodetection of Rieske in protein samples isolated from bundle sheath strands.

Figure S3 Length and width of top fully expanded leaves from WT, azygous and Rieske overexpressing plants.

Figure S4 Daily global solar exposure data for Canberra.

PBI-21-1206-s001.docx (499.7KB, docx)

Acknowledgements

We thank Spencer Whitney for the gift of RbcL antibody and Siena Mitchell, Alexandra Williams, Ayla Manwaring, Kelly Chapman and James Samuel Nix for technical assistance. This work was performed under the collaborative project agreement between the Australian National University and CSIRO Food & Agriculture and supported by the Australian Research Council Centre of Excellence for Translational Photosynthesis (CE140100015) and by the seed grant from the Research School of Biology of the Australian National University. Open access publishing facilitated by Monash University, as part of the Wiley ‐ Monash University agreement via the Council of Australian University Librarians.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Immunodetection of Rieske in the T1 progeny of line 32.

Figure S2 Immunodetection of Rieske in protein samples isolated from bundle sheath strands.

Figure S3 Length and width of top fully expanded leaves from WT, azygous and Rieske overexpressing plants.

Figure S4 Daily global solar exposure data for Canberra.

PBI-21-1206-s001.docx (499.7KB, docx)

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