JTCB Journal

1.      Abbreviations: 

Chl                        :               Chlorophyll

GC - MS                :               Gas Chromatography-Mass Spectrometry

GDC - H                :               Glycine Decarboxylase Subunit H

Gly                         :               Glycine

GOX                       :               Glycolate Oxidase

MDH                      :               Malate Dehydrogenase

NA                          :               Not available

NAD - ME              :               Malate Decarboxylase, NAD Dependent Malic Enzyme

NADP - ME            :               Malate Decarboxylase, NADP Dependent Malic Enzyme

PEPC                      :               Phosphoenolpyruvate Carboxylase

PPDK                     :               Pyruvate Orthophosphate Di Kinase

RbcL                      :               Rubisco Large Subunit

RbcS                      :               Rubisco Small Subunit

Rubisco                  :               Ribulose-1,5-bisphosphate carboxylase/oxygenase

Ser                          :               Serine

2.      Introduction 

Discovering the phenomenon of the lack of apparent photorespiration in the reproductive organs of the C₃ cereals [1] revealed also the fact that in cereals only the leaf registers C₃ photosynthesis. The rest of organs (ear, stem, leaf sheath, peduncle) have a CO₂ exchange kinetics similar to the maize C₄ leaf, also lacking apparent photorespiration [2]. These results suggested the assumption that cereal plants productivity is ensured by two mechanisms of carbon assimilation. One is of C₃ type, localized in the in the leaves and the second one localized in the ear photosynthetic active components, that has some elements of the C₄ photosynthesis that limits the CO₂ losses in post-illumination phase [3].

From another part it is known that majority of enzymes necessary for C₄ pathway are already present in C₃ plants, but in lower quantities and it is supposed that C₃ plants are preconditioned for C₄ metabolism [4,5]. In the last decade appeared many works that demonstrate the C₄ preconditioning assumption of the C₃ plants.

In the cells surrounding vascular bundles in tobacco the radioactive signal from labeled malate was found in soluble sugars and amino acids, suggesting that CO₂ originates from photosynthetic malate. It is considered that the genes involved in C₄ photosynthesis were recruited from orthologs present in C₃ species through modification of their spatial expression pattern and restriction to one cell type [6].

One of the main differences between C₃ and C₄ plants lies within the content and activity of PEPC. This is a specific C₄ enzyme, active in cytosol of mesophyll cells, that fix CO₂ in organic acids with four atoms of carbon: malate or aspartate. Nevertheless in the last decade several research teams reported about the presence and considerable activity of PEPC in C₃ plants: in the cells surrounding vascular bundles in tobacco [4] and Arabidopsis [6], in the ear of Tr. aestivum L. [7], assuming its role in CO₂ fixation. Plants with C₄ photosynthesis have much more PEPC than Rubisco and need much less Rubisco in order to fix the same quantity of CO₂ compared with C₃ plants. This results in a greater efficiency of N utilization [8,9].

By mRNA-Seq 5 different species from Flaveria genus were compared (C₃, C₄ and C₃-C₄ types of photosynthesis) in order to quantify the transcriptome differences in the leaf. In the C₄ Flaveria plants the biggest number of genes with reduced expression belonged to photorespiration group. Surprisingly was the fact that in C₃-C₄ intermediate species of Flaveria did not registered intermediate characteristics of genes expression levels: the expression level of photorespiration associated genes was even higher than in C₃ species of Flaveria, but the expression levels of Calvin-Benson cycle genes was C₃ similar [10]. Despite the fact that these species assimilate up to 50% of the carbon through C₄ pathway.

From literature data it is known that C₃ - C₄ intermediate species of Flaveria do not register intermediate characteristics for photosynthesis and photorespiration genes expression compared to C₃ and C₄ species. Moreover, the abundance of the majority of the transcripts associated with the photosynthesis and the photorespiration were greater than in the C₃ species, the expression of the majority of the genes of Calvin-Benson was C₃ like with exception of RbcL, the expression level of which was significantly lower than in the C₃ species [10].

Taking into account all mentioned above in this article was performed a comparative study of several key components that characterize the C₃ and C₄ metabolism: the PEPC activity; the relative contents of PEPC, RbcL and GDC-H; carbon isotopes discrimination; profiling of the main metabolites associated with photorespiration and the relative expression of RbcS, PEPC, GDC-H, GOX genes. All these investigations were performed for the flag leaf and ear components (glume, lemma and awn) in comparison to the maize leaf and tassel, in the same growing conditions and developmental stages.

3.      Materials and Methods

3.1.  Study Objects and Plant Growth Conditions 

C₃ plants of Tr. durum L. (variety Hederiforme 335), Triticale (variety Ingen 93) and Zea mays (line 459 and hybrid RF7xW47) served as study objects. All these genotypes were grown in the experimental fields of the Institute of Genetics, Physiology and Plants Protection of the Academy of Science of Republic of Moldova and in the greenhouse of the Umea Plant Science Center (Sweden). Greenhouse conditions were: 20°C/15°C - day/night temperatures, 50%-70% relative humidity, 16h photoperiod with light intensity of 700 μmol/m2/s. Cereal plants were grown in pots of 3 liter, in two rows (3cm between rows) and fertilized with Rika-S (N-P-K, 7-1-8) through an automated irrigation system. At the stage of 2-3 leaves, plants were vernalized during one month: 15°C/5°C day/night temperatures, 50%-70% relative humidity and 8h photoperiod. After verbalization plants were returned back to the greenhouse. Maize plants (hybrid W47xRf7) were grown at the same greenhouse conditions, but in 10-liter pots. Biological material from maize (leaf and tassel glumes) were collected at the beginning of tassel flowering. In case of Tr. durum and Triticale biological samples were collected at two developmental stages (earning and milk/waxy (ripening) from four different tissues: flag leaf, awn, lemma and glume). All samples were collected in three biological repetitions and each repetition was composed by pooled material from three individual plants grown in different pots. Samples collection started after 6-8h of illumination, when photosynthesis reaches its maximum capacity. Samples were ground manually in liquid nitrogen, using a pestle and mortar, until a fine powder was obtained and stored in freezer at -80°C. The same stock of ground material was used for all the downstream experiments. The field grown plants were used for gas exchange analysis, anatomical structure and chloroplast ultrastructure determination. Plants from the greenhouse were used for enzyme activity assays, western blots, gene expression analysis and metabolite profiling.

3.2.  Chlorophyll CONTENT, PEPC Activity and Western Blot

Chlorophyll quantification was done using UV/VIS Lambda 18 Perkin-Elmer spectrophotometer at the three wavelengths:  646.6nm, 663.6nm, 750nm. All calculations were done according to [11]. Total proteins extraction and PEPC activity was done according to [12], monitoring photometrically (at 340nm) the oxidation of NADH through coupling the carboxylase reaction with MDH, at optimal temperature of 30^°C, using HALO DB-30 UV-VIS (Dynamical) spectrophotometer. Protein extraction buffer contained 50mM HEPES-NaOH pH8, 10mM MgCl2, 5mM DTT, 1mM EDTA, 20% Glycerin, 1mM Pefabloc SC (Roche), 2% PVP. Reaction buffer contained 50mM HEPES-NaOH pH8, 5mM MgCl2, 5mM Glucose 6-phosphate, 1mM NaHCO3, 20% Glycerin, 0.2mM NADH, 6U/ml Malatdegydrogenase (MDH), pH8. In order to start the reaction to 850µl of reaction buffer was added 100µl protein extract and 50µl (2.5mM) PEP for positive reactions or water for controls. The activity of PEPC was calculated based on reaction rates recorded within a range where the increase in A340nm absorbance was linear. Protein concentrations were measured according to Bradford method using Bio-Rad kit in microplate assay and Spectra MAX 190 spectrophotometer. All isolated proteins were stored at -80ºC.

For Western blot the proteins were isolated from the same grinded material according to [13]. Protein extracts from different photosynthetic active components were resolved through SDS-PAGE [14] with different concentrations of acrylamide in order to ensure good protein separation. After electrophoresis proteins were electro-transferred on the nitrocellulose membrane [15]. For immunodetection polyclonal antibodies (Agrisera) were used in different dilutions: RbcL (1:5000), PEPC (1:500) si GDC-H (1:500). Coupled antibodies on membranes were detected through chemiluminescence using Amersham ECL Western blotting detection reagents and Fujifilm LAS-3000 Imager, according to manufacturer's recommendations.

3.3.  Total RNA Isolation and cDNA Synthesis

Total RNA was extracted from 20mg of grinded material using RiboZol (Ameresco) reagent, according to manufacturer's recommendations, in three biological repetitions. Isolated RNA concentration was checked at Nanodrop 2000 spectrophotometer and degradation level through gel electrophoresis (2% agarose, stained with Gel Red or Midori Green). After DNase treatment (DNA-free kit, Ambien) and RNA concentration re-measurement 1μg of total RNA was revers-transcribed using qScript cDNA Synthesis kit (Quanta), according to manufacturer's recommendations. Groups of five cDNA samples were pooled and used for negative RT reactions (reverse transcription without RT enzyme) - controls for DNA contamination in qPCR reactions. Isolated RNA samples were stored at -80^ºC and cDNA samples at -20^ºC.

3.4.  qPCR Analysis 

Samples processing and qPCR analysis were done according to MIQE guidelines [16]. Using NCBI database coding sequences of PEPC and Rubisco (small subunit) genes for Zea mays, Triticum sp. and coding sequences for GOX, GDC-H only for Triticum sp., (Table 1) were identified.

Primers where constructed using Primer3 software and synthesized commercially (Eurofins). The same primers were used for the analysis of Tr. durum and Triticale samples. The sequences of several potential reference genes were picked from the articles - 5 genes for maize and 3 for Tr. durum (Table 2), Triticale [17,18]. The experimental design was based on three biological repetitions and each biological repetition was processed in three technical repetitions. Amplification conditions were tested on a dilutions series of cDNA samples pool. PCR products were analyzed through 2% agarose gel electrophoresis in order to verify if only one fragment of expected size was amplified - crucial moments in qPCR.

For amplification we used EVA-Green kit (qPCR Eva Green qPCR Master mix (ABM)), half reactions and two step programs (40 de cycles: 94^°C - 60sec, 60^°C - 60sec) followed by melting curve analysis. Total volume of one reaction was 10µl and contained 1x EVA-Green reaction buffer, 10µM of each primer and 2µl of diluted cDNA (1:20). For standard curve we used a dilution series of pooled cDNA from each sample of the same genotype. All amplifications were run on Bio-Rad CFX70 machine and CFX Manager Software was used for data extraction. Obtained data were analyzed using the same method - determination of normalized relative expression level according to the formula:

RQ=(E_ref^Cq_ref)/(E_target^Cq_target), [19]

Where:

E - efficiency of amplification reactions for reference and target genes;

Cq - cycle of quantification for reference and target genes.

Obtained relative quantities were log transformed in order to have them normal distributed and analyzed statistically using Excel.

3.5.  Reference Genes Screening

To screen potential reference genes Norm Finder software was used. Due to the little number of samples in case of maize samples, the reference gene was selected with CFX manager software during qPCR data analysis. The best gene combination was MEP and FPGS (Table 2). In case of Tr. durum and Triticale the genes with lowest expression stability levels (M) having respectively the most stable expression levels (Table 3) were selected.

3.6.  Metabolites Analysis with GC-MS 

Metabolites analysis was performed on the Metabolomics Platform at the Umea Plant Science Center (Sweden). Samples for metabolomics were prepared and processed according to [20] and for data analysis SIMCA software was used.

3.7.  Total Carbon Isotopes Discrimination 

For this analysis only the samples of maize leaf and Triticale flag leaf, ear components were selected. Approximately 1mg of grinded material was dried out at 70^ºC for 18h and after that the relative abundance of ¹³C and ¹²C was determined using Isotope ratio mass spectrometer (Delta, Thermo Fisher Scientific) and Elemental analyzer (Flash EA 2000, Thermo Fisher Scientific). 

4.      Results and Discussion 

As mentioned previously the necessity to update research and discussions in the field of CO₂ assimilation mechanisms and carbon metabolism in the ears of C₃ cereals appeared as a consequence of the last results regarding the CO₂ exchange in the ear [3]. Our results (CO₂ exchange kinetics and compensation point, anatomical structure and chloroplasts ultrastructure in photosynthetic active components of the ear) demonstrated eloquently that in the ear of cereals (Tr. durum and Triticale) are present functional and structural elements of C₃ and C₄ types of photosynthesis. Based on these results were studied several key components of the carbon metabolism in the ear of cereals: the content and activity of key photosynthetic enzymes (Rubisco, PEPC, GDG-H); expression level of RbcL, PEPC, GDC-H and GOX; contend of the metabolites associated with photorespiration (glycolate, glycerite, serine and glycine).

4.1.  PEPC Activity in Photosynthetic Active Components of the Ear

One of the main differences between C₃ and C₄ plants lies within the content and activity of PEPC. This is a specific C₄ enzyme, active in cytosol of mesophyll cells, that fix CO₂ in organic acids with four atoms of carbon: malate or aspartate. Nevertheless in the last decade several research teams reported about the presence and considerable activity of PEPC in C₃ plants: in the cells surrounding vascular bundles in tobacco [4] and Arabidopsis [6], in the ear of Tr. aestivum L. [7], assuming its role in CO₂ fixation. Taking this into account the PEPC activity was determined in the flag leaf and ear components of C₄ (maize) and C₃ (Tr. durum, Triticale) plants. Obtained data were normalized against total chlorophyll content for each sample (Figure 1). PEPC activity in Tr. durum and Triticale was determined for earning phase and ripening phase, but for maize samples - beginning of tassel flowering. Recorded PEPC activity for maize (C₄), Tr. durum and Triticale (C₃) corresponds to data known in the literature [12]: PEPC activity in C₄ plant is 10-20 times higher than in C₃. For cereals ear components obtained values were greater than those for the flag leaf, with exception of awn, earning phase, were PEPC activity was similar to the flag leaf (Figure 2). In general, for earning phase, obtained values were greater compared to ripening phase. Maximum levels of PEPC activity was registered for Triticale lemma and for the glume, 2,04 and 1,4µmol/min/mg Chl respectively.

Similar results were obtained for Tr. Aestivum L., where for dough-development stage of the awn was registered PEPC activities (1.7 µmol mg-1 protein min-1) double higher than in the flag leaf [7]. Taking into account the high demand in carbohydrates and proteins for grain formation the authors concluded that PEPC in the ear is actively involved in the CO₂ fixation in the light and CO₂ respired in the dark or light. The presence of awns plays an important role in CO₂ assimilation because they increase considerable the photosynthetic active area of the ear. Also the high thermo-tolerance and drought resistance of the ear is explained through more abundant presence and activity of PEPC comparing to the flag leaf [21]. Despite the suggestions of other authors, we registered higher PEPC activity in the ear components comparing to the flag leaf. 

4.2.  Western blot of PEPC, RbcL and GDC-H Proteins 

In order to estimate the relative amount of some key enzymes in photosynthesis and photorespiration Western blot was performed using polyclonal antibodies (Agrisera, SE). It is known that Rubisco content in C₃ plants is much higher than in C₄ plants [9]. Our hypothesis was that in the ear components, lacking apparent photorespiration, there should exist a mechanism of CO₂ re-fixation that may result in lower amount of Rubisco. Obtained data partially confirmed that hypothesis. In Tr. durum and Triticale samples the most abundant protein was RbcL in both developmental stages, as expected for a C₃ plant (Figure 3). Nevertheless, for the flag leaf the amount of detected protein was much higher compared with the ear components. For the maize leaf (C₄) this protein was less abundant comparing to the rest of samples. 

Next detected protein was PEPC and its content in maize leaf was very abundant compared with RbcL, as expected for the C₄ plant. In case of Tr. durum and Triticale lower quantities of PEPC were detected, besides the fact that on the gel 100 µg total protein was loaded. The greatest band intensities for both types of cereals were obtained for the flag leaf and awn (Figure 3 A, B). One possible explanation may be due to the usage of the polyclonal antibody for maize PEPC, on the other hand it is possible to say that this protein is present and active in the ear components of C₃ cereals, especially in the awn. This fact confirms that PEPC enzyme with potential carboxylation function is present in C₃ plants, but in much lower concentration compared to C₄ plants (Figure 2 A, B).  The last protein that we detected was H protein of glycine-decarboxylase complex (GDC-H). Protein H plays a crucial role for the activity of entire GDC complex in mitochondria [22] and may be used as an indirect indicator of photorespiration.

4.3.  Carbon Isotope Discrimination in Triticale Ear Components 

Carbon isotopes ¹³CO₂ and ¹²CO₂ diffuse differentially through the leaf and because of Rubisco preferences for ¹²CO₂ plants with different type of carbon metabolism (C₃, C₄, C₃-C₄ and CAM) have a specific signature after isotopes discrimination [23]. In order to determine the specificity of CO₂ fixation metabolism in ear components total carbon isotopes discrimination was performed. We selected maize leaf as a C₄ control and Triticale flag leaf as a C₃ control. Clear differences were obtained for isotopes signature in maize leaf, that had less negative values compared to Triticale samples. For Triticale no difference was between the flag leaf and ear components (Table 4). Similar results were obtained for Flaveria species with intermediate type of metabolism (C₃ - C₄), where no difference was registered in carbon isotopes discrimination compared with C₃ flag leaf of Flaveria [10], despite the fact that intermediate Flaveria species fix up to 50% of carbon through C₄ pathway. Theoretically, lack of C₄ signature in the ear components does not necessarily mean complete absence of C₄ metabolism in respective photosynthetic tissues, taking into account also higher PEPC activity compared with the leaf (Figure 2). 

4.4.  Relative expression level of some photosynthetic and photorepiratoric genes (RbcS, PEPC, GDC-H, GOX) 

The majority of enzymes necessary for C₄ pathway are already present in C₃ plants, but in lower quantities and it is supposed that C₃ plants are preconditioned for C₄ metabolism [3]. In the cells surrounding vascular bundles in tobacco the radioactive signal from labeled malate was found in soluble sugars and amino acids, suggesting that CO₂ originates from photosynthetic malate [6].

It is considered that the genes involved in C₄ photosynthesis were recruited from orthologs present in C₃ species through modification of their spatial expression pattern and restriction to one cell type [5]. 

In order to detect differences and similarities in CO₂ assimilation metabolism between ear components of C₃ cereals and maize leaf (C₄) we determined the relative expression levels of several key genes in C₃ and C₄ photosynthesis (Table 1). Relative expression data were normalized against a reference gene, individually detected for maize and cereals, using several candidate genes and Norm Finder software (Table 2, 3). 

In case of maize leaf and tassel the expression level of PEPC and RbcS was determined. In the leaf PEPC expression was 5.6-fold higher than RbcS (Figure 4). Plants with C₄ photosynthesis have much more PEPC than Rubisco and need much less Rubisco in order to fix the same quantity of CO₂ compared with C₃ plants. This results in a greater efficiency of N utilization [8,9]. In maize tassel the expression level of both genes was approximately at same level, but much lower compared with the leaf. For example, PEPC expression in the leaf was 4.9-fold higher than in the tassel. Although glumes of maize tassel are photosynthetic active and their chlorophyll content is very low (Figure 1). Relative gene expression data correspond to Western blot data for these proteins in maize, although in case of the proteins RbcL instead of RbcS was determined (Figure 3). 

For Tr. durum and Triticale, the relative expression of four genes was analyzed: RbcS, PEPC, GDC-H and GOX (Table 1). We chose these genes in order to characterize photosynthesis and the activity of photorespiratory cycle in the flag leaf and ear components.

For the ear components we obtained specific expression profiles for these genes. According to the data known in the literature the ear of Tr. aestivum has anatomical, ultrastructure and physiological advantages for CO₂ fixation compared with the flag leaf. These advantages are more evident especially at the ripening stages. Increased stomata number and PEPC activity demonstrates that the ear has a superior capacity to adapt to different ecological conditions [24]. From another part, different ear components have a specific role in photosynthesis or protection of the grain. Microarray analysis of oat ear components revealed that lemma and paleo express more defense related genes, but in the awn more genes associated with photosynthesis are expressed [25]. These data suggest that lemma and paleo have defense/protection functions, but awn has photosynthetic function.

In our experiments for Tr. durum the maximum expression levels were for RbcS and minimum for PEPC. Differences were 23-63 folds, depending on the sample (Figure 5).

For RbcS in the earning phase the relative expression was higher in the flag leaf comparative to the ear components, but at the ripening phase, for all the samples, the expression was approximately at the same level. The situation is different for PEPC - for majority of samples in both phases the expression level is equal or lower compared with the flag leaf. Greater expression than in the flag leaf was detected only for awn at the ripening phase (1,13 fold). Taking into account the fact that in C₃ plants many PEPC isoforms with other functions (housekeeping functions) are active than those of CO₂ fixation and that the designed primers may be complementary to many of them it is hard to conclude about the expression level of this gene in Tr. durum ear components.

For the expression of GDC-H and GOX genes in Tr. durum we registered intermediate values compared with RbcS and PEPC. In the leaf the GDC-H expression was greater than in the ear components for both phases, with only exception for the glume at the ripening phase (1,3-fold higher expressions than in the leaf). This means that photorespiratoric cycle is present and active at the considerable level but less active in the ear components than in the flag leaf (Figure 5). Protein H of GDC complex is not involved directly in decarboxylation of glycine but acts as a scaffold for the rest of the three subunits (P, T and L) and it was demonstrated that the increased activity of this protein increases the activity of entire GDC complex [22]. Elevated CO₂ flux through photorespiration cycle leads to the increased photosynthesis rates and 30% higher biomass accumulation. These results were obtained after overexpression of GDC-H from Flaveria pringley (C₃) in Arabidopsis [26]. The positive effect was explained through photosynthesis blocked by photorespiration metabolites and their catabolism increase reduces this block. Glycolate oxidase (GOX) is one of the key enzymes of photorespiration cycle, it is active in peroxisomes where oxidation of glycolate to glyoxylate occurs. In order to characterize how much active, the photorespiration cycle in ear components and in the flag leaf is - we determined the relative expression of this gene. Obtained data revealed that in all the ear components expression level of GOX was lower than in the flag leaf. During the earning phase in awn, lemma and glume it was 3, 8 and 5-fold lower than in the flag leaf, but at the ripening phase its expression level was 2, 3 and 2-fold lower respectively (Figure 5). 

For Triticale samples the expression profile of analyzed genes in general is similar to Tr. durum, but there are several peculiarities. The maximum expression level was detected for RbcS but the lowest one for GOX or PEPC, depending on the sample. Registered differences between RbcS and PEPC were greater than those for Tr. durum: 600-1700-fold, depending on the sample (Figure 6). Greater PEPC expression than in the flag leaf was registered only for the glume at the earning phase (1,1 fold) and for awn at the ripening phase (1,4 fold). Similar to Tr. durum samples the RbcS expression level was greater in the flag leaf compared with the ear components with the exception of awn at the ripening phase (1,1-fold higher expression level than in the flag leaf, Figure 6).

Although differences in RbcS relative expression are not so great between samples, at protein level the differences are clearer: in the flag leaf there is more Rubisco than in ear components (Figure 3 A). For GDC-H gene lower expression levels than in the leaf were obtained for lemma and glume, earning phase (2,2 and 1,6-fold respectively). For awn at the earning phase and all ear components at the ripening phase the expression level of this gene was 1,4-1,6 fold higher than in the flag leaf. Greater expression of GDC-H may be associated with the presence of a glycine shuttle, that favor re-fixation of photorespiratoric CO₂. Recently the existence of a CO₂ concentration mechanism was proved, but previously it was only supposed to be present in the intermediate C₃-C₄ plants and it is considered to act as an intermediate step in the evolution of C₄ photosynthesis [10]. In Flaveria, C₃-C₄ intermediate plants, the genes associated with photorespiration were induced at higher levels even than in C₃ species and levels of glycine and serine (transport metabolites in CO₂ pump) were higher than in C₃ and C₄ species of Flaveria. Authors concluded that there exist three CO₂ fixation pathways acting in parallel in intermediate Flaveria species: NADP-ME, NAD-ME and glycine transport from mesophyll cells (where it cannot be de-carboxylate) into the cells surrounding vascular bundles. Here glycine is de-carboxylated and released CO₂ is re-fixed by Rubisco, all process acts as a CO₂ pump [10]. 

In case of GOX gene in all ear components the expression was lower compared to the flag leaf. Unlike Tr. durum in Triticale lemma and glume the expression of this gene was even lower than the PEPC expression (Figure 6). From literature data it is known that C₃ - C₄ intermediate species of Flaveria do not register intermediate characteristics for photosynthesis and photorespiration genes expression compared to C₃ and C₄ species. Moreover, the abundance of the majority of the transcripts associated with the photosynthesis and the photorespiration were greater than in the C₃ species, the expression of the majority of the genes of Calvin-Benson was C₃ like with exception of RbcL, the expression level of which was significantly lower than in the C₃ species [10]. 

4.5.  Metabolites Analysis Through GC-MS 

In order to study the metabolite profile in the samples of C₃ and C₄ plants, especially the relative content of photorespiration associated metabolites, we performed the GC-MS analysis. 82 different metabolites were identified in the studied samples, derived from flag leaf and ear components of the greenhouse grown Triticale, Tr. durum and maize plants. In our investigations we focused on four main metabolites know to be associated with the photorespiration:  glycolate, glycerite, glycine and serine. Glycolate is formed as a result of the photorespiration at the chloroplast level, is transported to the peroxisomes and metabolized to Glycine.  Glycine is shuttled back to mitochondria and metabolized to Serine that is shifted back to peroxisome and metabolized to Glycerite. The last one is transported to the chloroplast and fuels the Calvin Cycle.

The values presented in charts in Figures 7-8 are relative, based on the relative and untargeted GS-MS quantification.  In the first experiment we analyzed samples derived from greenhouse grown maize, Triticale and Tr. durum plants at the atmospheric CO₂ concentrations. We used the common stock of grinded samples for metabolites analyses.

For Tr. durum and Triticale samples the greatest amount of Glycolate was registered for the flag leaf compared with the ear components (Figure 7 A). Only in case of Tr. durum its relative amount was at the same level in the flag leaf and awn. The same trend was registered in case of the maize samples - approximately the same amount of glycolate in the leaf and in the tassel samples (Figure 7 E).

Glycine is the first amino acid that accumulates when the photorespiration cycle is active [23]. For triticale Triticale ear samples its content was higher compared with the flag leaf. The same is true for the maize samples - more glycine in the tassel compared with the leaf (Figure 7 F). In case of Tr. durum samples, the situation is different: in the ear components the content of Glycine is equal or lower than in the flag leaf (Figure 7 B). Interesting results were obtained for Serine. This amino acid is one the final products of photorespiration cycle obtained after decarboxylation of glycine in mitochondria by GDC complex. The content of serine in the ear components of Tr. durum and Triticale, with exception of lemma for Tr. durum, is greater than in the flag leaf (Figure 7 D). The same situation was registered for the maize samples - more serine in the tassel compared with the leaf (Figure 16 G). Glycerite, the last metabolite of photorespiration cycle, in the cereals samples was at the same level or more abundant in ear components than in the flag leaf (Figure 7 D). In case of maize samples, it was more abundant in the leaf, compared with the tassel (Figure 7 H). 

Taking into account the greater relative amount of Glycine, Serine and Glycerite in some of the ear components compared to the flag leaf it is possible to suppose the presence of a Glycine pump, similar to C₃ - C₄ intermediate species. This phenomenon may be explained through the presence of a glycine pump similar to C₃ - C₄ intermediate species of Flaveria, where glycine from mesophyll cells is transported to the cells surrounding vascular bundles and is de-carboxylated there [10]. This mechanism of CO₂ concentration is considered as the first step in C₄ evolution [27,28]. In our second experiment we decided to analyze the same four metabolites but in low/high CO₂ atmosphere where photorespiration was induced or suppressed.  For that purpose, from greenhouse grown Triticale plants, we cut the ears at ripening phase (bellow the flag leaf) and placed in a glass with water in a special adapted chamber, where the CO₂ content is maintained at a certain level.

Almost no differences were registered for Glycolate relative content, for analyzed flag leaf and ear samples in low and high CO₂ (Figure 8 A). In high CO₂ (suppressed photorespiration) Glycine was more abundant in flag leaf comparing to the ear components. In low CO₂ atmosphere (induced photorespiration) the relative amount of Glycine was greater for each sample, than in high CO₂, but however, the same trend was maintained- more Glycine in the flag leaf than in the ear components (Figure 8 B). Based on these data it is possible to conclude that photorespiratory cycle is present in the cereals ear components but is less active comparing to the flag leaf. 

In case of Serine we registered higher amounts in glume and much lower in the rest of samples for low and high CO₂. For Glycerite we registered the same tendencies as for Glycine: more abundant in low CO₂ conditions for the flag leaf comparative to the ear (Figure 8). 

5.      Conclusion 

1.       The active components of the ear registered increased levels of PEPC activity, with maximum values for glume and lemma. Determination of the relative amount of several key enzymes for photosynthesis and photorespiration (PEPC, RbcL, GDC-H) revealed and abundant level of RbcL in the flag leaf of cereals and of the PEPC in maize leaf. In the ear components were registered traces of PEPC and the amount of RbcL was lower compared to the flag leaf. In the maize leaf was not registered the presence of GDC-H, but these proteins were abundant in the flag leaf of cereals and in lower quantities in the ear components;

2.       Carbon isotopes discrimination registered a clear difference between the maize leaf and cereals ear, leaf. The ear components had the same signature as the flag leaf of cereals.

3.       GC-MS analysis of the metabolites associated with photorespiration (glycolate, glycerite, glycine and serine) revealed a higher content of serine in the ear components compared to the flag leaf. The same peculiarity was registered for glycine, but only for the ear of Triticale. Similar profile of serine and glycine was registered for the maize C₄ leaf and in the tassel the content of these metabolites was higher compared to the leaf;

4.       Determination of the relative expression level of RbcS, PEPC, GDC-H and GOX evidenced peculiarities for the photosynthetic active ear components. The most abundant expression was registered for RbcS and minimum for PEPC. The RbcS was more intense expressed in the flag leaf at earning phase of cereals, but in the waxy-milk phase its expression level was similar in the leaf and ear components. For PEPC higher expression was detected in awn, ripening phase. For the rest of ear components its expression level was lower comparing to the flag leaf. The GDC-H gene had more or less similar expression levels in the ear components and flag leaf in Tr. Ddurum, but for Triticale the highest expression level was detected for the awn. The highest GOX gene expression was detected in the flag leaf and lower levels of expression for the ear components. 

6.      General Conclusion

The obtained results demonstrate that the ear of cereals, covering 10-70% from all plant photosynthesis, cannot be considered as an organ with only C₃ type of photosynthesis. There are present also structural and functional elements of C₄ photosynthesis, that may ensure the re-fixation of the CO₂ released from respiration and photorespiration. 

As a proof is the lack of apparent photorespiration, but also the presence of an active photorespiration cycle. This complex mechanism that combines elements of C₃ and C₄ photosynthesis, with different expression levels, place the ear in an intermediate zone, similar to C₃ - C₄ plants. The lack of apparent photorespiration and the concomitant presence of the photorespiration cycle in the ear may serve as pomp that ensure with CO₂ the pentose phosphate cycle and increase the efficiency of photosynthesis. 

It is possible to suppose that during the evolution of C₄ plants this pomp was substituted by the C₄ cycle, where the CO₂ fixation is performed in the mesophyll cells by PEPC, an enzyme with only carboxylation capacity, different from Rubisco. These peculiarities may open new opportunities for biotechnological use of evidences mechanisms in order to increase the photosynthesis efficiency and productivity of cereals. 

7.      Funding

This work was supported by Institutional Project 11.817.04.04F, financed by Academy of Science of Republic of Moldova and grant nr. 5386 from Science and Technology Center in Ukraine; also, partially by Visbi Postdoctoral fellowship to DB.

Figure 1: Chlorophyll content in maize (C₄) and Tr. durum, Triticale (C₃) samples harvested at the two developmental stages. The average of 3 biological repetitions ± 95% confidence interval is presented.

Figure 2: Activity of PEPC in Tr. durum, Triticale at two developmental stages and maize samples. The average of 3 biological repetitions ± 95% confidence interval is presented.

Figure 3: Western-blot analysis of PEPC, RbcL and GDC-H proteins in maize, Triticale (A) and Tr. durum (B) samples. In case of the RbcL detection, for maize samples, 30µg total protein was loaded and for cereal samples - 4 µg; in case of PEPC for maize samples 30µg total protein was loaded and for cereal samples - 100 µg; in case of GDC-H detection for all samples 30µg total protein was loaded. The gels were resolved on SDS-PAGE (8% for RbcL, PEPC and 18% for GDC-H), transferred on nitrocellulose membrane and probed with polyclonal antibodies. The membranes were developed using ECL standard reagent.

Figure 4: Normalized relative expression level of PEPC and Rubisco genes in maize samples (tassel and leaf) collected at the tassel flowering stage, in log2 scale. The average of 3 biological repetitions ± 95% confidence interval is presented.

Figure 5: Normalized relative expression level of Rubisco, PEPC, GDC-H and GOX genes in Tr. durum samples, in log2 scale. The samples were collected from different photosynthetic active tissues at 2 different developmental phases (earning and ripening). The average of 3 biological repetitions ± 95% confidence interval is presented.

Figure 6: Normalized relative expression level of Rubisco, PEPC, GDC-H and GOX genes in Triticale samples, in log scale. The samples were collected from different photosynthetic active tissues at 2 different developmental phases (earning and ripening). Is presented the average of 3 biological repetitions ± 95% confidence interval.

Figures 7(A-H): Relative content of four main metabolites associated with photorespiration (glycolate, glycine, serine and glycerite) in C₃ cereals (Triticale and Tr. durum) and maize (C₄), determined through untargeted GS-MS analysis. The material was harvested from greenhouse grown plants at ripening phase. Is presented the average of 3 biological repetitions ± 95% confidence interval.

Figures 8(A-D): Relative content of four main metabolites associated with photorespiration (glycerite, glycolate, glycine and serine) in Triticale ears acclimated in low CO₂ (150 ppm, 22^ºC, 80% humidity) and high CO₂ (2000 ppm, 22^ºC, 80% humidity) chamber. The plants were grown in greenhouse conditions and harvested ears at ripening stage were placed in a glass with water, in special adapted chamber with low/high CO₂ atmosphere. Metabolites analysis was performed through untargeted GS-MS analysis. The material was harvested from greenhouse grown plants at ripening phase. Is presented the average of 3 biological repetitions ± 95% confidence interval.

Table 1: The sequence of used primers, amplicon size and reaction efficiencies for the detection of RbcS, PEPC, GDC-H and GOX1.

Table 2: Primers sequences for reference genes, amplicon size and reaction efficiencies.

Table 3: Expression stability levels of candidate reference genes for maize, Tr. durum and Triticale.

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