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
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
Citation: Baluar
N, Badicean D, Peterhaensel C,
Mereniuc 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-101. DOI: 10.29011/JTCB-101. 100001
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.
Keywords:
Carbon Metabolism;
Cereals Ear; Chloroplasts Ultrastructure; Compensation Point; CO2 Kinetics; C3 Plants; C4 Plants; Photorespiration
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; NADP - ME: Malate Decarboxylase, NADP Dependent Malic
Enzyme; PEPC: Fosfoenolpiruvat Carboxylase; PPDK: Piruvat Ortofosfat Dikinase; R:
Dark Respiration (Mitochondrial); Rubisco: Ribulozobifosfatcarboxylase
1. 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 ear`s 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 cereal`s 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).
2. Materials and Methods
2.1. 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.
2.2. 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.
2.3. 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.
3. 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.
3.1. 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.
3.2. 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.
3.3. 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 3a).
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 3c) making them representative for the flag
leaf of C3 cereals.
3.4. 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 3d,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 3d, 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.
3.5. 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 5c). 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).
3.6. 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).
3.7. 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].
4. 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.
5. Funding
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.
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 |
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 |
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