Rubisco small subunits of C4 plants, Napier grass and guinea grass confer C4-like catalytic properties on Rubisco in rice

ABSTRACT Overexpression of Rubisco small subunit (RbcS) of C4 plant, sorghum (sorghum bicolor) was shown to enhance the catalytic turnover rate (k cat) of Rubisco in rice (Oryza sativa). In this study, the effects of other Rubisco small subunits of C4 plants, Napier grass (Pennisetum purpureum) and guinea grass (Megathyrsus maximus) on kinetic properties of Rubisco in rice were studied. The expression levels of Napier grass RbcS (NgRbcS) and guinea grass RbcS (GgRbcS) proteins accounted for 41% and 45% of total RbcS, respectively in homozygous overexpression lines. The k cat and K m for CO2 (Kc) of Rubisco were increased in all transgenic lines. Interestingly, the k cat was markedly higher in NgRbcS homozygous line, whereas K c was notably higher in GgRbcS homozygous line. Although its effects depend on species, these results suggest that the introduction of C4 RbcS are effective approaches to alter the catalytic properties of Rubisco in rice. Abbreviation GgRbcS: guinea grass RbcS; K c: K m for CO2; k cat: catalytic turnover rate; NpRbcS: Napier grass RbcS; RbcL: Rubisco large subunit; RbcS: Rubisco small subunit; Sc/o: CO2/O2 specificity.


Introduction
The crop productivity should be substantially improved to meet the demand for food in the near future. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the initial CO 2 fixation of photosynthesis, which considered to be one of the major bottleneck of plant productivity (Parry et al., 2013). The photosynthetic rate at present atmospheric CO 2 level is largely limited by Rubisco because of its low catalytic turnover rate (k cat ) and competing oxygenase reaction, which initiates energy wasteful photorespiration in C 3 plants (von Caemmerer & Quick, 2000). Many important crops such as rice (Oryza sativa), potato (Solanum tuberosum) and soybean (Glycine max) are classified as C 3 plant. Unfortunately, the k cat of Rubisco in C 3 plants is substantially lower than that in C 4 plants (Ishikawa, Hatanaka, Misoo, & Fukayama, 2009;Sage, 2002). Thus, C 3 plants need to accumulate large amount of Rubisco in leaves, leading to low nitrogen use efficiency (Makino et al., 1992). In contrast, Rubisco from C 3 plants shows higher affinity for CO 2 (i.e. lower K m for CO 2 , K c ) to reduce oxygenation reaction (Sage, 2002). The enhancement of k cat usually decreases the affinity for CO 2 , leading to increase in photorespiration. However, the photorespiration will be reduced under elevated CO 2 condition. Hence, the enhancement of k cat as observed in C 4 -type Rubisco can give a benefit for the photosynthesis and plant productivity of C 3 plants in the future environment.
Rubisco is composed of the large subunit (RbcL) and small subunit (RbcS). RbcL is present in the chloroplast DNA, while RbcS is encoded by a multigene family in the nucleus (Dean, Pichersky, & Dunsmuir, 1989). RbcL contains most of important amino acids necessary for catalysis (Andersson & Backlund, 2008). In contrast, the function of RbcS is still obscure. However, we showed that the overexpression of Sorghum bicolor C 4 RbcS markedly enhanced the k cat of Rubisco in rice (Ishikawa, Hatanaka, Misoo, Miyake, & Fukayama, 2011). In addition, OsRbcS1, a member of rice RbcS gene family that is not expressed in photosynthetic cell also enhanced the k cat of Rubisco in rice (Morita, Hatanaka, Misoo, & Fukayama, 2014). These results clearly demonstrated that RbcS can be an important determinant of kinetic properties of Rubisco. Another functional group, cold-resistant plants also have acquired a high k cat Rubisco (Sage, 2002). However, RbcS from a cold resistant C 3 plant, timothy (Phleum pretense) could not enhance the k cat of Rubisco in rice (Fukayama et al., 2015). These results suggest that RbcS from high k cat Rubisco does not always enhance the k cat of low k cat Rubisco.
Although information of RbcS are still limited, we presumed that C 4 plants are likely to contain effective RbcS for the enhancement of k cat of C 3 Rubisco. Among Rubisco in C 4 plants, Rubisco in Napier grass (Pennisetum purpureum) and guinea grass (Megathyrsus maximus) showed markedly higher k cat than that from rice (Ishikawa et al., 2009). In this study, we isolated and overexpressed RbcS from Napier grass and guinea grass in rice, and found that these C 4 -type RbcS could be effective to improve the kinetic properties of Rubisco in rice.

Plant materials and growth conditions
Napier grass (cv. Merkeron) and guinea grass (cv. Natsuyutaka) were grown in the experimental field of Kobe University. Rice (cv. Nipponbare) and transgenic rice lines were grown in soil under natural light conditions in a temperature-controlled greenhouse (30°C day/ 25°C night). Rice seedlings at the leaf stage of 4.5 were transplanted into 1 L pots filled with paddy soil containing a chemical fertilizer (N:P:K = 8:8:8) at 0.3 g N per pot. At 10.5 leaf stage, the 9th leaf blades were sampled at 10:30-11:30 on sunny days, immediately frozen in liquid nitrogen and stored at −80°C until required.

Determination of the full length mRNA sequence of RbcS
Total RNA was isolated from leaf blade of Napier grass and guinea grass using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). The first strand cDNA was synthesized from the total RNA using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, Otsu, Japan). RT-PCR was performed using primers designed at conserved region of RbcS. The PCR product was cloned into the pGEM-T Eazy vector (Promega, Madison, WI), and their nucleotide sequences were determined. Based on these nucleotide sequences, 5ʹRACE and 3ʹRACE were performed using a SMART RACE cDNA amplification kit (BD Bioscience, San Jose, CA) as described previously (Fukayama et al., 2015). Based on the sequences of RACE products, the coding sequences of Napier grass RbcS (NgRbcS) and guinea grass RbcS (GgRbcS) were amplified by RT-PCR. Primers used for RT-PCR, 5ʹRACE and 3ʹRACE were listed in Table S1.

Plasmid construction and transformation of rice
The coding sequences with almost full length 3ʹUTR of NgRbcS and GgRbcS were cloned into the binary vector pIG121Hm containing the chlorophyll a/b-binding protein (Cab) promoter of rice. These constructs were introduced into rice (cv. Nipponbare) via Agrobacteriummediated gene transfer. Hygromycin-resistant transgenic rice plants were regenerated and grown in soil. The level of transgene expression was screened by SDS-PAGE after staining with Coomassie blue as described previously (Fukayama et al., 2015). The transgenic lines showing higher expression levels were used for further analysis.

Determination of kinetic properties of Rubisco
Leaves were homogenized in extraction buffer containing 100 mM Bicine-NaOH, 1 mM EDTA, 5 mM MgCl 2 , 2 mM NaH 2 PO 4 , 0.4% (w/v) BSA, 5 mM DTT, 4 mM aminon-caproic acid and 0.8 mM benzamidine, pH 8.0, using a chilled mortar and pestle with a small amount of quartz sand. The homogenate was then centrifuged at 15,000 × g for 2 min at 4°C. Rubisco in the supernatant was activated by pre-incubation with 15 mM MgCl 2 and 10 mM NaHCO 3 on ice for 15-20 min. Rubisco activity was determined at 28°C using [ 14 C] NaHCO 3 (specific activity, 37 MBq mmol −1 ) by assaying the incorporation of 14 C into acid-stable products, as described previously (Ishikawa et al., 2009).
The k cat of Rubisco (mol mol −1 s −1 ) was calculated as ratio of in vitro maximum Rubisco activity to Rubisco catalytic sites. For determination of K c , Rubisco activities were measured at six different NaH 14 CO 3 concentrations (0.5-15 mM) as mentioned above. The K c was calculated from the Hanes-Woolf plot.
To determine Rubisco CO 2 /O 2 specificity (S c/o ), the CO 2 compensation points were measured by the gas exchange rates using an open gas-exchange system (LI-6400, Li-Cor, Lincoln) under the condition of leaf temperature of 28°C, a photosynthetic photon flux density (PPFD) of 1,200 μmol quanta m −2 s −1 , a leafto-air vapor pressure difference of 1.0-1.2 kPa and four different O 2 partial pressures ranging from two to 20%. The Sc/o was estimated by the slope of the regression lines of the dependence of CO 2 compensation point on the O 2 concentration (Laisk & Loreto, 1996).

Results
In this study, we studied the effects of RbcS from two C 4 plants, Napier grass and guinea grass on Rubisco kinetics in rice. Because mRNA sequences of RbcS from Napier grass and guinea grass were not found in published genome database, RT-PCR was carried out using primer set designated at a conserved region of RbcS among Poaceae and obtained ten and seven different sequences from Napier grass and guinea grass, respectively. The full length mRNA sequences of Napier grass RbcS (NgRbcS) and guinea grass RbcS (GgRbcS) showing highest frequency in the RT-PCR products were determined by 5ʹRACE and 3ʹRACE. The phylogenetic tree analysis of deduced amino acid sequence showed that NpRbcS and GgRbcS classified into different clade from sorghum RbcS in C 4 plants (Figure 1(a)). Deduced amino acid sequences of NgRbcS and GgRbcS share 74.4 and 76.7% identity with rice RbcS, respectively. These identities were slightly higher than sorghum RbcS which shares 73.4% identity with rice RbcS (Ishikawa et al., 2011). Amino acid sequence of βA-βB loop has been suggested to be an important determinant of Rubisco kinetic properties (Spreitzer, 2003). Comparing the amino acid sequences of βA-βB loop, NgRbcS and GgRbcS contain two and three different amino acids with rice RbcS, and two and one different amino acids with sorghum RbcS, respectively (Figure 1(b)).
These differences in amino acids can expect different effects on Rubisco kinetic properties. Semi quantitative RT-PCR analysis indicated that NpRbcS and GgRbcS were highly expressed in leaf blade (Figure 2), suggesting that these RbcSs are an usual photosynthetic RbcS and not an unusual non-photosynthetic RbcS reported in rice (Morita et al., 2014;Morita, Hatanaka, Misoo, & Fukayama, 2016).
NgRbcS and GgRbcS were overexpressed in rice. Transgenic plants showed the band of foreign C 4 RbcS as well as rice RbcS in SDS-PAGE with Coomassie blue staining (Figure 3). Transgenic lines showing higher expression level were used for subsequent analyses. These transgenic lines exhibited a normal growth behavior and fertility. The expression levels of NgRbcS and GgRbcS relative to total RbcS were 41% and 45%, respectively in homozygous lines (Figure 3). The expression levels of transgene in these lines were similar level compared to those of previous reports overexpressing sorghum RbcS or timothy RbcS (Fukayama et al., 2015;Ishikawa et al., 2011).  The kinetic properties of Rubisco expressing NgRbcS and GgRbcS were analyzed using these transgenic lines ( Figure 4). All transgenic plants showed higher k cat and K c of Rubisco than non-transgenic rice (WT). In particular, the k cat was significantly increased in homozygous NpRbcS line (SNN) which was comparable to a high expression line of sorghum RbcS (SS10). In contrast, K c was greatly increased in homozygous GgRbcS line (SGG). These findings suggest that the effects of RbcS on the Rubisco kinetics would differ between NpRbcS and GgRbcS. Carboxylation efficiencies (k cat /K c ) of WT, SS10, SNN and SGG were 0.184, 0.180, 0.189, and 0.147, respectively. Because the expression level of foreign RbcS in SNN was lower than those in SS10 and SGG, these results imply that NpRbcS can be most effective to improve the kinetic properties of Rubisco for carboxylation.
The S c/o in transgenic lines overexpressing NgRbcS or GgRbcS were marginally lower than that in WT, whereas these differences were not statistically significant. These results suggest that the incorporation of NpRbcS and GgRbcS to Rice Rubisco would not cause large-scale effects on the S c/o .

Discussion
In this study, we showed that C 4 RbcS from Napier grass and guinea grass as well as sorghum can confer C4-like high k cat type catalytic properties on Rubisco in rice. In contrast, the introduction of RbcS from pea (Pisum sativum L.) into Arabidopsis thaliana (Getzoff, Zhu, Bohnert, & Jensen, 1998) and timothy RbcS into rice (Fukayama et al., 2015) did not significantly affect on the kinetic properties of Rubisco. First of all, the k cat values of Rubisco in C 4 plants tested in this study were markedly higher than C 3 plants, whereas the k cat values of Rubisco in pea and timothy were not as high as C 4 plants. Considering these observations, the introduction of RbcS from plants showing markedly higher Rubisco k cat is considered to be effective to change the catalytic properties of Rubisco in C 3 plants. Although the amino acid sequences of RbcSs from C 4 plants used in this study shared high identity among them, these showed different effects on the kinetics of Rubisco in rice (Figure 4). Spreitzer (2003) reported that βA-βB loop of RbcS can be an important region affecting the kinetics of Rubisco. All effective RbcSs to enhance the k cat of Rubisco in rice contain Ser55 which is substituted for His in rice (Figure 1). However, the amino acid sequences of this region are quite similar among these three C 4 plants. Although the effects of RbcS on Rubisco kinetics were different between Napier grass RbcS and guinea grass RbcS as shown in this study (Figure 4), these amino acid sequences in βA-βB loop differ only one amino acid, namely, Arg56 in Napier grass is substituted for Thr in guinea grass. There should be a lack of information to conclude that this marginal difference has different effects on Rubisco. It is also possible that the difference in amino acid sequence other than βA-βB loop may also affect the kinetics of hybrid Rubisco consisting of rice RbcL and C 4 RbcS.

RbcS
In this study, we demonstrated that RbcS from C 4 plants containing high k cat type-Rubisco would be effective to enhance the k cat and carboxylation efficiency of rice Rubisco. Among C 4 plant RbcSs, Napier grass RbcS can be most effective to improve these parameters for photosynthesis. However, we analyzed the kinetics of chimera Rubiscos consisting of rice RbcS and C 4 RbcS. To make an accurate assessment of the effects of C 4 RbcSs, rice RbcS should be knocked down or knocked out in our transgenic plants. At the same time, this way will lead to the reduction of Rubisco content. To improve the photosynthetic nitrogen efficiency and capacity under elevated CO 2 condition, we presume that the reduction of Rubisco content to an appropriate level as well as the increase in k cat will be necessary. We consider that our transgenic plants expressing C 4 RbcS are useful for the elucidation of the structure-function relationship of Rubisco and finally the improvement of photosynthetic capacity of C 3 plants in the future environment.

Disclosure statement
No potential conflict of interest was reported by the authors.