## "The Peculiarities of Carbon Metabolism in the Ears of C3 Cereals: CO2 Exchange Kinetics, Chloroplasts Structure and Ultrastructure in the Cells from Photosynthetic Active Compo¬nents of the Ear"

Nicolae Balaur1*, Dumitru Badicean1,2, Christoph Peterhaensel3, Lilia Mereniuc1, Veaceslav Vorontsov1, Dumitru Terteac4

1Institute of Genetics, Physiology and Plant Protection of Academy of Sciences of Republic of Moldova, Bioenergetics Laboratory, Republic of Moldova, Moldova

2Department of Plant Physiology, Umea University, Sweden

3Institute for Botany, Leibniz-University of Hannover, Germany

4Practical Scientific Institute of Horticulture and Food Technologies of Ministry of Agriculture and Food Industry, Chisinau, Republic of Moldova, Moldova

*Corresponding author: Nicolae Balaur, Institute of Genetics, Physiology and Plant Protection of Academy of Sciences of Republic of Moldova, Bioenergetics laboratory, Republic of Moldova, Maldova. Tel: +3732255696; Email: bn1939@yahoo.com

Received Date: 09 January, 2018; Accepted Date: 16 February, 2018; Published Date: 26 February, 2018

1.       Abstract

The ear of C3 cereals makes an important contribution to yield formation, but the mechanisms ensuring this phenomenon are not completely elucidated. Previously established peculiarities of carbon metabolism in the ear of cereals come in contradic­tion with the discovery of phenomenon of lack of apparent photorespiration in reproductive organs of the C3 plants. In this article are presented the CO2 exchange kinetics of the ear and compensation point; the structure and ultrastructure of chloro­plasts from glume, lemma and awn in comparison with flag leaf (Tr. durum, Triticale), leaf and tassel of C4 plants (Zea mays).

Was established, that CO2 exchange kinetics of the ear is similar to the leaf and tassel of maize plants. In open measurement system the ear does not register a compensation point, but in the close system - does. The lack of apparent photorespiration in the tassel and presence of a compensation point is evidenced also for the maize plants, where it is formed from the re­leased respired CO2. The glume, lemma and awn, similar to maize leaf, have two types of cells: mesophyll cells and cells ar­ranged around the vascular bundles, forming a “Kranz” type of crown. In these cells are present two types of chloroplasts: gra­nal in the mesophyll cells and granal-agranal in the Kranz type of cells, with well-developed granas and weak developed lamellas.

The obtained results demonstrate that in the cereals ear, concomitantly are functioning structural and functional elements of C3 and C4 types of photosynthesis.

2.       Keywords: Carbon Metabolism, Cereals Ear; Chloroplasts Ultrastructure; Compensation Point; CO2 Kinetics; C3 Plants; C4 Plants, Photorespiration

1.       Abbreviations:

A                             :               Intensity of Net Photosynthesis

AP                           :               Apparent Photorespiration

APP                        :               CO2 Assimilation after Light Turn off

FLC                        :               First Layer of Cells, from the Crown, Surrounding Vascular Bundles

SLC                        :               Second Layer of Cells, from the Crown, Surrounding Vascular Bundles

G                             :               Compensation Point

MDH                      :               Malate Dehydrogenase

PEPC                     :               Fosfoenolpiruvat Carboxylase

PPDK                     :               Piruvat Ortofosfat Dikinase

R                             :               Dark Respiration (Mitochondrial)

Rubisco                 :               Ribulozobifosfatcarboxylase

2.       Introduction

All photosynthetic organs in C3 and C4 plants fix carbon to produce sugars and other organic substances. Beside the flag leaf, photosynthesis of the ear has an important role in grain formation by contributing 40-50% to the carbon stored in grains [1-3]. Earlier studies even demonstrated that the ears contribution to barley grain formation reaches 76% [4]. All components of the ear contribute to its photosynthetic efficiency: awn, glume, lemma and palea [2, 5-8]. Assimilation of CO2 by the ear components vary, but only the awns respond for 40-80% of carbon metabolism from the entire ear, in different cereals [8,9]. Despite the important role of the cereals ear in yield formation, especially in drought conditions [8], the mechanisms of CO2 assimilation are not completely elucidated yet. During the last two decades many investigations of CO2 exchange in the ear and its photosynthetic active components were performed: CO2 compensation point [10,11], CO2 re-fixation [2,8,12,13], carbon isotopes discrimination [2,8], incorporation of labeled malate [10], C4 enzymes activities and analysis of the corresponding gene expression levels [14-16], drought resistance of the ear photosynthetic active components [7,8]. In these studies, and many others that today are updated, scientific opinion is oriented towards the conclusion that only C3 type of photosynthesis operates in the ear of cereals. In early investigations, based on compensation point and labeling experiments it was concluded that all ear components in general perform only C3 type of photosynthesis [10]. The percentage of labeled CO2 incorporated into C4 products (malate and aspartate) was less than 10% and it was suggested that C4 type of photosynthesis is not operational in these tissues. Despite these conclusions the authors cite many articles (23, published in the period of 1972-1993) regarding the evidence of structural and functional elements of C4 syndrome in the photosynthetic active components of the ear. Based on these early results it was proposed that the existence of an intermediate type of C3-C4 or C4 limited metabolism in the ear components of cereals is possible based on: 1) high activities of PEPC and PPDK; 2) labeled CO2 incorporation into C4 products; 3) CO2 exchange and anatomical characteristics of the ear; 4) lack of O2 photosynthesis inhibition in the rice ear; 5) the carbon isotope composition in the water-soluble fraction of ear components. After the above mentioned article [10] during the following 17 years of research on revealing the role of functional and structural C4 elements in photosynthetic active components of the ear, especially in the cereals, did not stop. The main focus is the evidence of possible mechanisms that might explain the main role of the ear and its components, high levels of CO2 assimilation compared to the flag leaf - key organ for carbon metabolism in C3 plants [8,9,13-16]. Based on these articles it is first of all important to mention the main role of awn in the synthesis of sugars, compared to the flag leaf [9]. This conclusion is based on photosynthetic intensity, chloroplasts ultrastructure and high PEPC levels, that in all checked developmental stages (6 stages, with maximum detected levels during grain filling) was higher in the awn compared to the flag leaf.

Taking into account the cereals ear structure and spatial arrangement of its photosynthetic active components, the same works have studied re-fixation of CO2 released during dark respiration. It was supposed that CO2 re-fixation may be an adaptation mechanism, which improves photosynthesis and water use efficiency, especially in drought conditions, during grain formation and filling stages [8,9,13]. In a review [8] 95 scientific articles published during 1965-2007 were analyzed and the following conclusions were made regarding the photosynthetic activity of the ear: a) photosynthetic contribution of the ear for grain filling appears to be quite important, particularly when grain yield is source-limited (i.e., drought); b) awns, when present, seems to be the main photosynthetic organ of the ear, at least with regard to net fixation of atmospheric CO2; c) re-fixation of respired CO2 is a well-documented process in ears of C3 cereals and represents a potentially important contribution for total photosynthesis; d) green pericarp and inner bracts (lemmas) are probably the main sites for CO2 re-fixation: e) current evidence does not support the presence of C4 or CAM metabolism in ears of C3 cereals; f) the ear exhibits better photosynthetic performance under water stress conditions, resulting from a higher relative water content, and its capacity for osmotic adjustments compared to the flag leaf; g) delayed senescence (persistence of photosynthetic components) is a key process in maintaining ear photosynthesis, mainly under water stress.

The data presented by different authors show evidence of the contradictions regarding the mechanisms of CO2 assimilation in the photosynthetic active ear components. Basically all these mechanisms are divided into four types of approaches: 1) in the ear is active only C3 type of photosynthesis and C4 type is absent; 2) in the ear of cereals a higher level of PEPC is present comparative to the flag leaf; 3) because of morphological structure, spatial arrangement of ear components and presence of PEPC, PPDK the ear is able to re-fix respired CO2 at substantial rates; 4) photosynthetic active components of the ear have an important protection role against pathogens, high temperatures and water deficit especially during post-flowering and grain filling stages. These different approaches are the consequence of different used methods by different authors; studding the same morphological, physiological and biochemical indices of ear photosynthetic active components, but in different genotypes; under different ecological growing conditions; at different developmental stages of the cereals ear, etc.

In this context, the main aim of our article was to investigate the peculiarities of carbon metabolism in the ear of C3 cereals. This was achieved through the studies of CO2 exchange kinetics, compensation point, the structure and ultrastructure of chloroplasts in the same genotypes, growing conditions, developmental stages of the ear and using the same methods in comparison with the flag leaf of C3 cereals and maize tassel/leaf (C4 plants).

3.       Materials and Methods

3.1.  Study Objects and Plant Growth Conditions

C3 plants of Tr. durum L. (variety Hordeiforme 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. 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. The field grown plants were used for gas exchange analysis, anatomical structure and chloroplast ultrastructure determination.

3.2.  CO2 Exchange Kinetics and Compensation Point

Measurements of CO2 exchange kinetics in photosynthetic active organs (flag leaf and ear of C3 cereals, leaf and tassel of maize plants) were done at two developmental stages (earning and ripening) for C3 cereals and at the stage of seven leaves/tassel appearance for maize plants. All measurements were performed on the intact plants. Components of CO2 exchange (apparent photosynthesis, dark CO2 assimilation, respiration, apparent photorespiration, compensation point) were deter­mined using infrared gas analyzer (PP Systems, USA) integrated into the photosynthetic monitor PTM-48A (Bioinstrument S.R.L, Republic of Moldova) [17,18] in open and closed systems, according to manufacturer instructions.

3.3.  Anatomical Structure and Chloroplasts Ultrastructure

For sections preparation the samples (leaf and tassel of maize plants; flag leaf, awn, lemma and glume for cereals) were fixed in glutaric aldehyde (2%) and post-fixed in osmium tetroxide (1%) [19]. Biological material was dehydrated in alcohol solutions (10%-100%) and embedded in Epon-812. All sectioning was done at BS-Y90A Tesla ultra-microtome. The sections for anatomical structure studies were stained in methylene blew (1%) and peronin-j (1%). Ultrafine sections were contrasted in Uranyl Acetate and Pb citrate 2% [20]. Anatomical structure of photosynthetic active organs was investigated on permanent preps using MezoPlant automated system (SIAMS, Russia). Chloroplasts ultrastructure was studied at the electronic microscope Tesla BS - 500.

4.   Results and Discussion

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 [18]. In this article, it was demonstrated that CO2 exchange kinetics in the ear of C3 plants, in post-illumination phase, do not show apparent photorespiration - a phenomenon evidenced for the first time in 2007 [21]. Based on these results, it was confirmed that in C3 cereals only the flag leaf performs C3 photosynthesis, the rest of the photosynthetic active organs (mainly the ear) in post-illumination phase showed similarities with CO2 exchange kinetics of the maize leaf (C4 plant). These results are in contradiction with those mentioned in the introduction, that in the ear the degree of C4 photosynthesis is equal to zero [10]. This essential contradiction, absence of apparent photorespiration similar to C4 leaf and absence of C4 metabolism in the ear as in C3 flag leaf, turns all research in that field towards a new beginning: taking into account known and important role of the ear in yield formation of C3 cereals [7], what is the ear of cereals from carbon metabolism point of view?

In order to get answer to this question we analyzed the same functional and structural indices that were reported previously in the literature. Our approach has three main peculiarities: 1) in order to have comparable results all analyses were performed on the same species, same photosynthetic active organs, at the same developmental stages, in the same ecological conditions; 2) to estimate similarities and differences in expression levels of C3 and C4 types of carbon metabolism in the ear comparative analyses with cereal flag leaf (C3) and leaf/tassel of maize (C4) were performed; 3) all measurements of CO2 exchange kinetics in leaf, ear and tassel were carried in open and closed systems, on intact plants.

4.1.  CO2 Exchange Kinetics for Leaves of Cereals and Maize in Open System

Exchange kinetics of CO2 is studied in two types of measurement systems: closed system, where the vegetative organ is separated from the plant and is placed in an isolated chamber that does not contact with atmospheric air; and open system, where vegetative organ is investigated on intact plant and atmospheric air freely circulates in the chamber [22]. The CO2 exchange kinetics of leaf was studied in open system using PTM - 48A monitor [17]. Advantage of this system is in differential and parallel measurement of all the components of CO2 exchange of the organs on the intact plants, while many of the methods used to study CO2 exchange are destructive [22,23].

Comparative study was done between C3 flag leaf, ear (Triticale, Tr. durum) and C4 leaf (maize) (Figures 1a, b). Figure 1a shows representative CO2 exchange profiles of Triticale flag leaves (1), Triticale ears (2) and maize leaves (3). After illumination, highest rates of net photosynthesis (A) were recorded for maize, intermediate rates for flag leaves, and lowest levels for ears. After darkening the samples, different CO2 exchange profiles are observed: for the flag leaf, a transient CO2 assimilation (APP) followed by a transient CO2 release (AP) was observed. The latter had been used as an estimate of photorespiration.

in previous studies [18]. In contrast, maize leaves did not show CO2 release in the APP phase, a well-known fact [24,25]. Interestingly, we observed the AP peak, but not the APP peak also for ears indicating that photorespiratory CO2 release is absent in these tissues. After the two transient peaks, CO2 release leveled out for all tissues and steady-state dark respiration (R) could be determined for all samples. R was highest for the ear sample and lower for the Triticale flag leaf and the maize leaf. Figure 1b shows a corresponding comparison of the Tr. durum flag leaf (1) and the ear (2). Again, the AP peak was observed in the leaf, but not for the ear sample.

The difference between the ear, flag leaf of cereals and maize leaf is highlighted through lower intensity of photosynthesis, CO2 assimilation in the dark, but on the other hand higher dark respiration. These peculiarities of CO2 exchange kinetics of the ear, as going to be demonstrated below, have a special impact on compensation point.

4.2.  CO2 Compensation Point of Maize and Cereals Leaves in Open System

Compensation point represents the CO2 concentration when a balance is established between assimilated and dissimilated CO2. For maize leaf (Figure 2, a, line 1) it was around 2 ppm, very reduced and corresponds to literature data for compensation point in C4 plants [25], confirming the absence of apparent photorespiration. For the flag leaf of C3 Triticale and Tr. durum plants compensation points were 52,5 ppm and 85 ppm respectively (Figure 2, a, lines 2 and 3), values corresponding to literature data for C3 flag leaf, where apparent photorespiration may be as high as 60% of the intensity of the photosynthesis [26]. The last three lines (4-6 in Figure 2 b) correspond to maize tassel and cereals ear respectively, where the compensation point is not registered - similar to maize leaf. These results indicate that in open system, in those photosynthetic active organs, where apparent photorespiration is not registered, the compensation point is also not registered and CO2 exchange in the organs lacking apparent photorespiration does not depend of CO2 concentration in the air.

4.3.  CO2 Exchange Kinetics in Closed Measurement System

We also measured CO2 exchange of the different samples in closed systems. For the maize leaf, a constant reduction of CO2 in the closed chamber in the light by net photosynthesis (A) was observed down to a compensation point of approximately 2 ppm (Figure 3 a). In the dark phase, a constant linear increase in CO2 concentration in the chamber due to respiration (R) was detected. The curve for the Triticale flag leaf (b) and the Tr. durum flag leaf (c) differed in several respects: A was lower, and the compensation point higher as expected. The R curve was biphasic with a fast-initial increase in CO2 followed by a slower second phase. This is due to initial CO2 release by photorespiration and respiration followed by a phase with CO2 release only by respiration. We therefore interpret the difference between initial and secondary CO2 release as photorespiration. Interestingly, we observed a profile of the R curve that is similar to maize when determining gas exchange of Triticale (d) or Triticum (e) ears suggesting the absence of apparent photorespiration. In addition, the CO2 compensation point G was higher probably due to the low A of these samples.

This is similar to the CO2 assimilation in the light for the open system (Figure 1, a, line 3). The rest of the components were not registered, but however two new appeared. One in illumination phase (G) that indicates the compensation point and the second one characterizing the CO2 exchange in post-illumination phase (CO2 dissimilation - R), that is continuously increasing compared to the dark CO2 assimilation of the maize leaf (Figure 2, a, line 1). It is worth to mention an important peculiarity of the infrared gas-analyzer: it cannot distinguish the CO2 released from photorespiration from that released from respiration. Both CO2 fluxes are coming from mitochondria but from different chemical processes: photorespiration cycle and Krebs cycle. That is why the line in Figure 3 (a), that shows the CO2 exchange kinetics of maize leaf in post-illumination phase, actually reflects the level of CO2 released from respiration. This fact is based on two elements:1) it is known that maize leaf does not register apparent photorespiration and in post-illumination phase the CO2 burst is absent; 2) lack of apparent photorespiration in maize leaf results in a very reduced compensation point, also a well-known fact. In this way investigation of CO2 exchange of maize leaf in closed system registered in total three components: A, G around 2 ppm and R (Figure 3, a).

The flag leaf of cereals in closed system registers the same three components of CO2 exchange as maize leaf (Figure 3b), but on the curve, that characterize respiration (R) appears a flux of CO2 that changes line trajectory. This flux is the amount of CO2 released during apparent photorespiration, characteristic to C3 flag leaf (AP in Figure 1). In this way for cereals flag leaf in closed system are registered four components of CO2 exchange: A, G, AP and R. The same peculiarities were registered for the flag leaf of Tr. durum plants (Figure 3 c) making them representative for the flag leaf of C3 cereals.

4.4.  CO2 Compensation Point for the Ear of C3 Cereals in Closed System

The ear of Triticale and Tr. durum plants, similar to maize leaf, in closed system registers only three components of the CO2 exchange: A, G and R (Figure 3, d, e). Compared to the flag leaf of cereals the ear dos not register apparent photorespiration (AP). On the other hand the compensation point of the ear of Triticale and Tr. durum is around 142 ppm and 160 ppm respectively, similar high values for the ear was reported previously [10,11]. Comparative analysis of the ear, flag leaf of cereals and maize leaf evidenced one more peculiarity of the ear. The time to establish the equilibrium between CO2 assimilation and dissimilation (compensation point) is around 8 min, similar to maize leaf. The flag leaf of cereals reaches the compensation point in 25 min. The CO2 assimilation kinetics of the ear, having similarities with maize leaf, come in contradiction with the main peculiarity established in the literature - a photosynthetic active organ that does not register apparent photorespiration should have a reduced compensation point. And vice versa, the organ that registers high compensation point should have apparent photorespiration. In our case, for the ear that lack apparent photorespiration (Figure 2; Figure 3, d, e), was registered a compensation point that is twice as high as in the leaf and ten times higher than in the maize leaf.

Even 2% O2 concentration did not induce essential changes in the formation of compensation point of the ear (Figure 3, f). Calculation of regression curves for CO2 exchange in maize leaf and flag leaf, ear of Triticale and Tr. durum (Figure 4) demonstrated the absence of apparent photorespiration in the ear.

The CO2 exchange in the maize leaf (Figure 4a), the ear of Triticale (Figure 4c) and Tr. durum (Figure 4g) is described by linear equation of regression, demonstrating that in the post illumination phase, for these photosynthetic active organs, only one source of CO2 exists - respiration. In case of the flag leaf of Triticale (Figure 4b, d) and Tr. durum (Figure 4e, f) the angle change of the regression curve is approximated through two equations of linear regression, with a high degree of confidentiality (R2 > 0.9). The regression coefficients, before and after tilt angle change, differ essentially: for the flag leaf of Triticale - 0.628 and 0.466 respectively (the difference is 25.8%); for the flag leaf of Tr. durum - 0.987 and 0.287 respectively (the difference is 70.9%). These data show very clearly that in the C3 flag leaf two sources of CO2 release exists - photorespiration and respiration. But for the maize leaf and the ear of cereals only one source - respiration, based on which is formed the compensation point in the absence of apparent photorespiration. This conclusion is based on more in detail analysis of CO2 exchange kinetics of the maize leaf.

4.5.  CO2 Compensation Point of Maize Leaf with Different Levels of Respiration

Because CO2 infrared gas analyzer cannot distinguish between photo respired and respired CO2 the work was focused on maize leaf that does not register apparent photorespiration but has a compensation point (Figure 2(1), 3(a)). Previously in our research, during measurements of compensation point of maize leaves, it was noticed that in some cases obtained values are greater than 10 µmol*mol-1 of CO2, previously shown in literature [25]. This peculiarity may be linked with dark respiration, because apparent photorespiration is absent. In Figure 5 for maize leaves were registered compensation points, higher than 2 ppm, with maximum value of 24 ppm. This comes in contradiction with the fact that in closed measurement, in post-illumination phase apparent photorespiration was not registered as in case of cereals flag leaf (Figure 3b), on the other hand compensation point is registered and increases with intensification of respiration (Figure 5d).

The highest compensation point corresponds to the highest level of regular respiration (24 ppm, Figure 5, c). The analysis of this correlation, between respiration and compensation point for maize leaf, demonstrates a strong positive correlation (R=0.81). As a result, for maize leaf lacking apparent photorespiration, the compensation point depends on concentration of CO2 released from dark respiration and not from apparent photorespiration that is missing in C4 plants. Taking all this into account one can make parallels with cereals ear, that manifest similarities with maize leaf in CO2 exchange kinetics: registers an intensity of apparent photosynthesis, has a compensation point (established in the same time frame as in maize leaf) and is lacking apparent photorespiration. According to the same analogy the compensation point in the ear is formed from one source of released CO2, from respiration. High values of compensation points for the ear (Figure 3d, e) correspond to the high levels of respiration (Figure 1, line 2).

4.6.  Anatomical Structure of Photosynthetic Active Organs in C3 and C4 plants

Comparative analysis of CO2 exchange kinetics of C3 cereals flag leaves, ear and maize leaf confirmed previously obtained results [18,21] that similar to maize leaf the ear does not register apparent photorespiration. Absence of apparent photorespiration in C4 leaf is ensured by C4 syndrome. It is based on a specialized anatomical structure (Kranz anatomy) that allows an efficient assimilation of CO2 released from decarboxylation of malate or aspartate [24,27]. In this context a comparative study was performed regarding the presence of structural elements of C4 syndrome in the photosynthetic active components of the ear of C3 cereals. From the literature it is known that C4 syndrome is structurally presented in a layer of cells surrounding vascular bundles (in maize leaf). In the flag leaf and ear components of cereals a prototype of crown exists, that is formed by two layers of cells (Figure 6).

The Kranz cell from maize leaf are bigger, contain more chloroplasts than mesophyll cells [24,27]. These peculiarities were evidenced also in the current study (Table 1). For maize leaf the volume of one Kranz cell is twice bigger than mesophyll cell and the volume, number of chloroplasts in one Kranz cell is higher than in mesophyll cell. The flag leaves of Triticale and Tr. durum plants have the same number of cells surrounding the vascular bundles but they lack above mentioned peculiarities. All indices of Kranz type cells in the flag leaf, containing chloroplasts, are smaller than the same indices in the mesophyll cells. Comparative analysis of these indices in the ear components (Table 1) demonstrated the same peculiarities as in maize leaf (Figure 6a, b, c): the same number of cells in the Kranz crown; the volume of one Kranz cell in glume and awn is bigger than in the mesophyll cells; number of the chloroplasts and the volume of one chloroplast are very similar to those in the maize leaf.

In the last decade more and more attention is paid to the role of the cells that form the crown around the vascular bundles and have photosynthetic function [28-31]. For a C4 leaf the distance between two vascular bundles, the number of mesophyll cells between them and the ratio between Kranz and mesophyll cells are much smaller compared with the C3 flag leaf. Comparing the density of vascular bundles in different Flaveria species (C3, C3-C4 and C4) it was demonstrated that increased density is an evolutionary criteria for appearance of the C4 photosynthesis [29]. Also it was suggested the necessity of genetic modification of anatomical structure of rice leaf (C3 plants) as a way for photosynthetic productivity increase [28]. Taking into account the similarities in CO2 exchange kinetics and anatomical structure between maize leaf and ear of cereals, demonstrated in the current study, we investigated the distances between vascular bundles. This parameter characterizes bundles' density per unit area and the number of mesophyll cells between them (Table 2).

For the maize leaf we found a relatively small distance between two vascular bundles - 49,4 ± 1,35µm, data corresponding to the literature [30]. For the flag leaf of cereals that parameter is characterized by greater distances, for Triticale - 90,7 ± 6,63µm and for Tr. durum - 84,9 ± 4,93µm. From ear components, lemma has the largest distance between the vascular bundles (Triticale - 115,1 ± 13,6µm, Tr. durum - 100,3 ± 14,9µm), similarly to maize tassel. In glume and awn these distances are comparable to those in maize leaf (Table 2).

From anatomical structure point of view the cell surrounding vascular bundles in photosynthetic active components of cereals ear, lacking apparent photorespiration, have similar characteristics to the Kranz cells from maize leaf and tassel (Figure 6).

4.7.  Chloroplasts Ultrastructure

It is known that C4 type of photosynthesis is manifested in the two cells types (mesophyll and “Kranz” cells). Two types of chloroplasts and enzymes are localized there: in mesophyll cells - granal chloroplasts and PEPC, in Kranz cells - lamellar chloro­plasts and Rubisco. As mentioned above in the photosynthetic active components of the ear (glume, lemma and awn) two types of cells are present: mesophyll cells and cells surrounding vascular bundles, forming a prototype of Kranz crown. This peculiarity imposed the necessity to study the ultrastructure of the chloroplasts from these two types of cells.

In Figure 7 is presented the ultrastructure of the most typical and representative chloroplasts from mesophyll and Kranz cells in maize leaf and cere­als flag leaf, ear components. A comparative analysis demonstrates that in glume, lemma and awn of Tr. durum and Triticale plants, similar to the maize leaf, two types of chloroplasts in two types of cells are located. The ultrastructure of chloroplasts from mesophyll cells in­cludes a well-organized membrane system of granas and lamellas, similar to chloroplasts from mesophyll cells of maize leaf (Figure 7 a, c, e, h, k). Chloroplasts ultrastructure in Kranz type of cells in glume, lemma and awn (Figure 7b, d, f, i, l) re­vealed a membrane system more similar to lamellar one, that is characteristic for chloroplasts from Kranz cells in maize leaf and tassel. In Kranz type of cells in ear components it is possible to distinguish two types of chloroplasts: one type with lamel­lar system of membranes (Figure 7f, l) and second one with more granas and less developed lamellas (Figure 7d, i). The chloroplasts from second layer of cells in the Kranz crown (Figure 6, FLC) are of granal type, with 4-5 granas, but lamel­las are missing almost completely (Figure 8).

Thus, was concluded that in mesophyll and Kranz type of cells from photosynthetic active components of the ear (glume, lemma and awn), similar to maize leaf, two types of chloroplasts are present: one type is granal, typical to mesophyll cells and the second one is lamellar, characteristic to Kranz type of cells. On the other hand, differently from maize leaf, in the Kranz cells of the ear components one more type of chloroplasts was detected - intermediate between granal and lamellar types, with weakly developed membrane system.

Summarizing all obtained results regarding anatomical structure and chloroplasts ultrastructure it is possible to conclude that around vascular bundles (MVB and SVB) of glume and awn (Figure 6 e, f, g, h) are present bigger cells than those from mesophyll cells. These cells form a crown that contains more and bigger chloroplasts (Table 1), arranged on all cell perime­ter. Arrangement of these chloroplasts in the Kranz type of cells in the ear components is similar to the C4 plants (Panicum maximum, Chloris gayana) in which de-carboxylation of aspartate and phosphoenolpyruvate is done by PEPCK [24]. These types of plants have granal chloroplasts in their Kranz cells. Chloroplasts from Kranz cells of maize leaf (C4 - NADP subtype, where malate and pyruvate are decarboxylase by NADP malate dehydrogenase) have lamellar membrane system. These chloro­plasts do not contain granas and are arranged on the perimeter of the cell wall opposite to the vascular bundle [24]. Compar­ing the literature and our results it is possible to conclude that in Kranz type of cells from the ear components, lacking apparent photorespiration, chloroplasts similar to those from C4 NADP-MDH subtype (lamellar chloroplasts) and PEPCK subtype (granal chloroplasts) are present.

Similar results regarding anatomical structure of Kranz cells and chloroplasts ultrastructure from these cells were described for plants with C3-C4 intermediate metabolism. It is considered that these plants have a decisive role in the understanding of the evolution of C4 plants [16,32-34].

5.       Conclusion

1.       The CO2 exchange kinetics of the ear registers only three components (intensity of apparent photosynthesis, CO2 assimila­tion in post illumination phase and respiration) from the total of four, registered in the flag leaf of cereals. The fourth component (apparent photorespiration) is not registered, similar to the tassel and leaf of maize plants. The cere­als ear, maize leaf and tassel do not register a compensation point in the open CO2 measurement system and the CO2 exchange does not depend of CO2 concentration in the air. In the closed measurement system, the ear does regis­ter a compensation point, similar to the cereals flag leaf, but much greater. By analogy with the maize leaf, that lack apparent photorespiration but register a compensation point depending on the CO2 released from the only source - respiration, the compensation point of the ear is probably formed also from the CO2 released from respiration. Be­cause the ear similar to the maize leaf does not register apparent photorespiration, needs the same amount of time to reach the compensation point and has similar CO2 exchange kinetics.

2.       Comparative analysis of the structure of photosynthetic active components of the ear (glume, lemma, awn), lacking appar­ent photorespiration, has registered two types of cells, similar to the maize leaf. The one is mesophyll cells and se second one is the cells surrounding the vascular bundles, forming a “Kranz” crown. These Kranz cells have similar structural characteristics with the Kranz cells surrounding the vascular bundles in the maize leaf and tas­sel, much bigger than the mesophyll cells, containing greater and bigger number of chloroplasts. The distance be­tween the vascular bundles in glume and awn, that characterize their density on a unit area, is twice smaller than in the flag leaf and similar to the distance in maize leaf. In the mesophyll cells and “Kranz” type of cells of ear compo­nents (glume, lemma and awn) similar to the maize leaf are located two types of chloroplasts. In the mesophyll cells are located the granal type of chloroplasts, containing well developed system of granas and lamellas. In the “Kranz” type of cells is located the “Granal-agranal” (lamellar) type of chloroplasts: the “granal” type contains well devel­oped granas and weak developed lamellas, but in the “agranal” type the situation is vice-verso.

3.       The obtained results indicate that in the ear of cereals function concomitant, but at different level, structural and func­tional elements of C3 and C4 photosynthesis. The CO2 exchange kinetics is similar to the leaf and tassel of maize plants; lack of apparent photorespiration and presence of the compensation point, formed from the respired CO2, by analogy with maize leaf; presence of two types of chloroplasts with an intermediate ultrastructure between granal and agranal types.

6.       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.

Figures 1(a-b): Typical and representative CO2 exchange kinetics of C3 (Triticale (a), Tr. durum (b)) and C4 (maize (a)) in open measure­ment system. Presented curves are the result of CO2 concentration measurement, per second, by infrared gas ana­lyzer, where the air enters from illuminated chamber for 30sec (A) and darkened chamber for 180sec (APP, AP, R). Light and dark conditions are ensured by automatic open/close of transparent and dark chambers. The measurements were done on flag leaves or ear from field grown plants. A - Net photosynthesis; APP - dark CO2 assimilation (post-illumination phase); AP - apparent photorespiration (CO2 flux during post-illumination phase); R - respiration; C0 - atmospheric CO2 concentra­tion; 1 - flag leaf of Triticale and Tr. durum at earning; 2 - ear of Triticale and Tr. durum at earning; 3 - maize leaf; s - sec­onds.

Figures 2(a-b): CO2 exchange in open measurement system depending on internal CO2 concentration (at the exit from measure­ment chamber). The measurements were done on flag leaves or ear from field grown plants. (a) maize leaf; 2 - Triticale flag leaf; 3 - Tr. durum flag leaf. (b) 4 - ear of Triticale; 5 - ear of Tr. durum; 6 - maize tassel. G - compensation point.

Figures 3(a-f): CO2 exchange kinetics, compensation point in closed CO2 measurement system and the expression of apparent pho­torespiration in the leaf, at 21% O2: a) maize; b) Triticale; c) Tr. durum and the ears d) Tr. durum; e) Triticale; f) Triti­cale at 2% of O2. The measurements were done on leaves and ears from field grown plants. A - net photosynthesis; G - Compensa­tion Point; R - Regular Respiration; AP -Apparent Photorespiration; s - seconds

Figures 4(a-g): CO2 exchange kinetics in closed measurement system, in post-illumination phase. a) maize leaf; b), d) Triticale flag leaf; c) Triticale ear; e), f) Tr. durum flag leaf and ear (g). The measurements were done on leaves or ear from field grown plants. Y - equation for linear regression; R2 - coefficient of determination; s – seconds.

Figures 5(a-d): Dependence of compensation point and respiration values in the maize leaf. The numbers on bars in chart d) repre­sent the values of compensation point for the different leaves (presented in charts a-c) that demonstrate the correlation be­tween compensation point and intensity of the respiration. The measurements were done on leaves from field grown plants. A - net photosynthesis; G - compensation point; R -respiration; s - seconds. In chart d) is presented the average of 3 biologi­cal repetitions ± 95% confidence interval.

Figures 6(a-h): The structure of cells crown surrounding the vascular bundle in the photosynthetic active organs lacking apparent photorespiration. a) Kranz cells surrounding vascular bundle in the maize leaf (C4 plants); b) Kranz cells surrounding second­ary vascular bundle in the maize leaf; c) Kranz cells surrounding vascular bundles in the photosynthetic active compo­nents of the maize tassel; d) ”Kranz” type of cells surrounding vascular bundles in the Triticale leaf (C3 plant); e, f) ”Kranz” type cells surrounding vascular bundles in the Triticale glume (C3 plant); g, h) ”Kranz” type cells surrounding vascu­lar bundles (primary and secondary) in the Triticale awn (C3 plant); MVB - main vascular bundle, SVB - secondary vascular bundle, K - ”Kranz” cells, FLC - first layer of cells, SLC - secondary layer of cells.

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Figures 7(a-l): Ultrastructure of chloroplasts from mesophyll and Kranz type cells of C3 and C4 plants (second layer of cells - SLC). a) Mesophyll cells of maize leaf; c) cereals flag leaf; e) cereals glume; h) cereals lemma; k) cereals awn. b) Kranz type cells of maize leaf; d) cereals flag leaf; f) cereals glume; i) cereals lemma; l) cereals awn.

Figure 8: Ultrastructure of chloroplasts from Kranz type cells surrounding the vascular bundles in ear of cereals (first layer of cells - FLC). Chl - chloroplast; m - mytocondria; g - grana; a - starch.

 Variety Studied organ Nr of Kranz cells Volume of one cell, µm3 x10 Nr of chloroplasts in one cell Volume of one chloroplast, µm3 Kranz meso­phyll Kranz meso­phyll Kranz mesophyll earning phase (tassel appearance) Zea mays, Line 459 leaf 8 ± 2 22,8 10,2 10 ± 2,12 7 ± 2,02 58,4 ± 1,32 30,0 ± 2,07 Earning Phase Tr.durum,L. Hordeiforme 335 Flag leaf 9 ± 1 11,3 23,1 7 ± 3,07 13 ± 2,12 18,9 ± 0,78 23,9± 1,07 Glume 4 ± 3 7,8 5,0 4 ± 1,87 8 ± 2,34 15,6 ± 1,32 16,0 ± 0,98 Lemma 4 ± 2 6,0 5,6 3 ± 2,01 5 ± 1,09 14,7 ± 0,46 15,7 ± 0,47 Awn 7 ± 2 6,2 6,8 6 ± 2,24 9 ± 1,43 13,3 ± 0,43 18,6± 0,62 Ripening Phase Tr.durum,L. Hordeiforme 335 Flag leaf 9 ± 2 12,2 24,2 8 ± 3,21 13± 2,22 20,1 ± 0,52 24,0 ± 0,98 Glume 5 ± 2 8,0 5,7 5 ± 1,79 8 ± 2,17 16,7 ± 0,73 17,8 ± 0,73 Lemma 5 ± 1 6,8 6,4 4 ± 2,17 6 ± 0,32 16,1 ± 0,43 18,1 ± 0,43 Awn 8 ± 2 6,4 7,8 7 ± 2,43 9 ± 1,22 14,2 ± 0,94 19,3 ± 0,94 Earning Phase Triticale, Ingen 93 Flag leaf 8 ± 2 11,2 22,9 7 ± 0,78 16 ± 2,18 23,8 ± 0,21 29,4 ± 0,42 Glume 8 ± 2 6,2 2,5 5 ± 0,82 6 ± 0,89 15,7 ± 0,72 16,2 ± 0,67 Lemma 6 ± 2 3,0 2,2 5 ± 1,23 4 ± 1,02 15,0 ± 0,39 15,4 ± 0,72 Awn 10 ± 2 3,2 5,2 5 ± 1,08 7 ± 1,52 20,2 ± 0,72 23,2 ± 0,43 Ripening Phase Triticale, Ingen 93 Flag leaf 8 ± 2 11,9 23,2 8 ± 1,23 18 ± 2,03 21,1 ± 1,08 31,7 ± 0,38 Glume 9 ± 2 7,1 3,0 5 ± 1,89 7 ± 0,94 17,5 ± 0,89 18,8 ± 0,52 Lemma 6 ± 2 3,6 2,7 6 ± 2,04 5 ± 1,08 15,3 ± 0,72 15,7 ± 0,34 Awn 10 ± 2 3,8 5,5 6 ± 1,47 8 ± 1,23 21,3 ± 0,43 25,2 ± 0,77

Table 1: Comparative analysis of cells surrounding the vascular bundles and mesophyll cells of maize, Tr. durum and Triti­cale flag leaf and ear components.

 Variety photosynthetic active organs (average ± standard deviation) Leaf Tassel Glume Lemma Awn Zea mays, Line 459 49,4 ± 1,35 162 ± 27,31 - - - Triticale, Ingen 93 90,7 ± 6,63 - 49,6 ± 5,58 115 ± 13,59 58,8 ± 6,32 Tr.durum L., Hordeiforme 335 84,9 ± 4,93 - 62,4 ± 5,94 158,3 ±14,94 58,9 ± 6,81

Table 2: Comparative analysis distances between vascular bundles in photosynthetic active organs of C3 and C4 plants (µm).

Citation: Baluar N, Badicean DPeterhaensel CMereniuc L, Vorontsov V, et al. (2018) The Peculiarities of Carbon Metabolism in the Ears of C3 Cereals CO2 Exchange Kinetics, Chloroplasts Structure and Ultrastructure in the Cells from Photosynthetic Active Components of the Ear. J Tissue Cult Bioeng: JTCB-101DOI: 10.29011/JTCB-101. 100001