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Immunolocalization of GLDP in bundle sheath and mesophyll cells of (A, B) Homolepis aturensis, (C, D) Steinchisma laxum, and (E, F) S. hians. C, chloroplasts; p, peroxisomes; black asterisk, mitochondria; white asterisk, mitochondria surrounded by chloroplast. Scale bars=500 nm.
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Photorespiratory glycine shuttling and decarboxylation in bundle sheath (BS) cells exhibited by C2 species is proposed to be the evolutionary bridge to C4 photosynthesis in eudicots. To evaluate this in grasses, we compare anatomy, cellular localization of glycine decarboxylase
(GDC), and photosynthetic physiology of a suspected C2 grass, Homolepis...
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... observed for H. aturensis and S. hians, a significantly greater number of mitochondria and peroxisomes are positioned at the centrip- etal BS pole in S. laxum, although chloroplasts are arranged equally around BS cells (Fig 1C, D; Supplementary Table S4). BS mitochondria are commonly surrounded by chloro- plasts in S. hians (Fig 3E), H. aturensis, and S. laxum in a pattern similar to previous reports for Steinchisma species (Brown et al., 1983). In contrast to centripetal chloroplasts displayed with their long axis parallel to the BS wall in S. laxum, the long axis of these chloroplasts in H. aturensis and S. hians are perpendicular to the BS wall (Figs 1, 2). ...
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... peroxisomes, and chloroplasts are abundant in mestome sheath cells of N. minor (Hattersley et al., 1986;Sage et al., 2014). Unlike H. aturensis, S. hians, and S. laxum, organelles are not polarized in their distribu- tion ( Supplementary Fig. S3), but are instead positioned around the mestome sheath cell periphery (Hattersley et al., 1986;Sage et al., 2014). ...
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... from quantification of gold particles conjugated to secondary antibodies that bind to anti-GLDP are sum- marized in Supplementary Table S5. GLDP is almost exclusively located in BS cells of H. aturensis and S. hians, and mestome sheath cells of N. minor (Fig. 3A, B, E, F; Supplementary Fig. S3). Both BS and M cells contained high levels of GLDP labeling in S. laxum (Fig. 3C, D). In com- parison with all other C 3 species examined, S. laxum had the highest GLDP labeling in BS mitochondria (Supplementary Table S5). These patterns in GLDP distribution in M and BS mitochondria of S. laxum and S. ...
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... from quantification of gold particles conjugated to secondary antibodies that bind to anti-GLDP are sum- marized in Supplementary Table S5. GLDP is almost exclusively located in BS cells of H. aturensis and S. hians, and mestome sheath cells of N. minor (Fig. 3A, B, E, F; Supplementary Fig. S3). Both BS and M cells contained high levels of GLDP labeling in S. laxum (Fig. 3C, D). ...
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... of gold particles conjugated to secondary antibodies that bind to anti-GLDP are sum- marized in Supplementary Table S5. GLDP is almost exclusively located in BS cells of H. aturensis and S. hians, and mestome sheath cells of N. minor (Fig. 3A, B, E, F; Supplementary Fig. S3). Both BS and M cells contained high levels of GLDP labeling in S. laxum (Fig. 3C, D). In com- parison with all other C 3 species examined, S. laxum had the highest GLDP labeling in BS mitochondria (Supplementary Table S5). These patterns in GLDP distribution in M and BS mitochondria of S. laxum and S. hians are similar to earlier reports on these species (Hylton et al., 1988). The gold density in C 3 species D. ...
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Imaging of mesophyll cell suspensions prepared from Arabidopsis has been pivotal for forming our current understanding of the molecular control of chloroplast division over the past 25 years. In this chapter, we provide a method for the preparation of leaf cell suspensions that improves upon a previous method by optimizing cellular preservation and...
Citations
... However, the limited capacity to accurately quantify chloroplast occupancy in the leaf has thus far hampered evaluation of the phenotypic consequences of any such manipulations. Most quantifications of chloroplast occupancy to date have been carried out using two-dimensional (2D) images obtained via light or electron microscopy (Pyke et al., 1994;Kub ınov a et al., 2014;Khoshravesh et al., 2016;Lee et al., 2021;Plackett & Hibberd, 2024), but where comparisons have been made, estimations of total chloroplast volume from 2D sections were significantly lower than those calculated from 3D reconstructions (Harwood et al., 2020). Furthermore, in the specific case of bundle sheath cells, 2D assessments were misleading because chloroplast distribution within the cell is very heterogeneous (Williams et al., 1989;Harwood et al., 2020). ...
There is an increasing demand to boost photosynthesis in rice to increase yield potential. Chloroplasts are the site of photosynthesis, and increasing their number and size is a potential route to elevate photosynthetic activity. Notably, bundle sheath cells do not make a significant contribution to overall carbon fixation in rice, and thus, various attempts are being made to increase chloroplast content specifically in this cell type.
In this study, we developed and applied a deep learning tool, Chloro‐Count, and used it to quantify chloroplast dimensions in bundle sheath cells of OsHAP3H gain‐ and loss‐of‐function mutants in rice.
Loss of OsHAP3H increased chloroplast occupancy in bundle sheath cells by 50%. When grown in the field, mutants exhibited increased numbers of tillers and panicles. The implementation of Chloro‐Count enabled precise quantification of chloroplasts in loss‐ and gain‐of‐function OsHAP3H mutants and facilitated a comparison between 2D and 3D quantification methods.
Collectively, our observations revealed that a mechanism operates in bundle sheath cells to restrict chloroplast occupancy as cell dimensions increase. That mechanism is unperturbed in Oshap3H mutants but loss of OsHAP3H function leads to an increase in chloroplast numbers. The use of Chloro‐Count also revealed that 2D quantification is compromised by the positioning of chloroplasts within the cell.
... It has a C 3 -type δ 13 C value (Smith and Brown, 1973), but the concentration of chloroplasts within large bundle-sheath cells led Christin et al. (2013) to suggest that the species is a C 3 -C 4 intermediate. This was confirmed by Khoshravesh et al. (2016), who reported a CO 2 compensation point (Box 1) for H. aturensis that was in between that of C 3 plants (typically 50 ppm CO 2 ) and that of C 4 plants (close to 0 ppm CO 2 ). Furthermore, the authors provided evidence for the operation of a C 2 -metabolic cycle in H. aturensis involving photorespiratory glycine (Lundgren, 2020; Box 1). ...
C4 photosynthesis is one of three metabolic pathways found in the photosynthetic tissues of vascular plants for assimilation of atmospheric carbon dioxide (CO2). Like crassulacean acid metabolism (CAM) photosynthesis, C4 photosynthesis has evolved from the ancestral C3 pathway. C4 photosynthesis is an adaptation that enhances CO2 assimilation in warm, high-light environments. On Barro Colorado Island (BCI), C4 photosynthesis (including variations of it) occurs in close to 60 her?baceous species from six families: Hydrocharitaceae, Cyperaceae, Poaceae, Euphorbia?ceae, Amaranthaceae, and Portulacaceae. Plants are typically found in clearings and not inside the forest, and about one-third of the C4 species are non-native. With at least 42 C4 species, Poaceae is the greatest contributor to the C4 flora of BCI. One grass species, Homolepis aturensis (Kunth) Chase has C3–C4 intermediate characteristics termed C2 photosynthesis. Portulaca oleracea L. (Portulacaceae) is a C4 plant with the remark?able capacity to also exhibit CAM photosynthesis under conditions of water-deficit stress. Hydrilla verticillata (L.f.) Royle (Hydrocharitaceae) is a submerged species that lacks Kranz-anatomy typical of fully developed C4. The species usually exhibits C3 photosynthesis but induces a C4 CO2 concentrating pathway when CO2 concentration in the water declines.
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... The species Sedobassia sedoides (Chenopodiaceae) has been classified as a C3-C4 intermediate species with C2 type of photosynthesis based on its anatomical features, gas exchange analysis, and the immunolocalization of glycine decarboxylase (GDC) [28]. C2 photosynthesis is a carbon concentration mechanism (CCM) that reassimilates CO2 released via photorespiration [29][30][31]. C2 photosynthetic activity depends on the photorespiratory decarboxylation of glycine via the GDC complex in bundle sheath cells. C2 metabolism is hypothesized to provide plants with several advantages over C3 species, including an expanded ecological niche in warmer and drier areas, improved net carbon assimilation (via CCM) and stress tolerance. ...
The adaptation of plants to combined stresses requires unique responses capable of overcoming both the negative effects of each individual stress and their combination. Here, we studied the C3-C4 (C2) halophyte Sedobassia sedoides in response to elevated temperature (35 °C) and salinity (300 mM NaCl) as well as their combined effect. The responses we studied included changes in water–salt balance, light and dark photosynthetic reactions, the expression of photosynthetic genes, the activity of malate dehydrogenase complex enzymes, and the antioxidant system. Salt treatment led to altered water–salt balance, improved water use efficiency, and an increase in the abundance of key enzymes involved in intermediate C3-C4 photosynthesis (i.e., Rubisco and glycine decarboxylase). We also observed a possible increase in the activity of the C2 carbon-concentrating mechanism (CCM), which allowed plants to maintain high photosynthesis intensity and biomass accumulation. Elevated temperatures caused an imbalance in the dark and light reactions of photosynthesis, leading to stromal overreduction and the excessive generation of reactive oxygen species (ROS). In response, S. sedoides significantly activated a metabolic pathway for removing excess NADPH, the malate valve, which is catalyzed by NADP-MDH, without observable activation of the antioxidant system. The combined action of these two factors caused the activation of antioxidant defenses (i.e., increased activity of SOD and POX and upregulation of FDI), which led to a decrease in oxidative stress and helped restore the photosynthetic energy balance. Overall, improved PSII functioning and increased activity of PSI cyclic electron transport (CET) and C2 CCM led to an increase in the photosynthesis intensity of S. sedoides under the combined effect of salinity and elevated temperature relative to high temperature alone.
... A few grasses and eudicot species, including most C3-C4 intermediates, conduct C2 carbon fixation in which (a) rubisco in the mesophyll oxygenates RuBP, producing 2-phosphoglycolate and 3-phosphoglycerate or pyruvate; (b) reactions in mesophyll organelles convert 2-phosphoglycolate to glycolate, then to glyoxylate, and finally to glycine; (c) glycine diffuses from the mesophyll to bundle sheath cells; (d) glycine decarboxylase decarboxylates glycine and releases CO2 in the bundle sheath cells; (e); the Calvin-Benson-Bassham cycle in the bundle sheath re-fixes this CO2 to hexose sugars; and (f) serine recycles back to the mesophyll [73][74][75] . The net effect is that C2 plants oxygenate substantial amounts of RuBP despite having a CO2 concentrating mechanism. ...
Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase), the most prevalent protein on the planet 1,2 , catalyzes two competing chemical reactions. One reaction is the carboxylation of ribulose 1,5-bisphosphate (RuBP), which initiates plant carbohydrate synthesis. The other is the oxygenation of RuBP, which initiates photorespiration ³ . The common assumption is that photorespiration is a futile cycle that dissipates more than 25% of a plant’s energy as waste heat 4–6 , but inhibiting photorespiration decreases shoot protein synthesis 7–11 . Here is evidence for a previously unrecognized photorespiratory cycle in which rubisco converts RuBP into pyruvate, malic enzyme carboxylates pyruvate into malate, and malate dehydrogenase oxidizes malate, generating reductants that convert nitrate into amino acids (Fig. 1). This cycle becomes prominent only when rubisco or malic enzyme are associated with manganese, but prior experiments replaced the manganese bound to these enzymes with magnesium 3,12,13 . The proposed cycle coordinates photorespiration with several other processes including C 3 carbon fixation, pentose phosphate shunt, malate valve, and nitrogen metabolism. It thereby balances plant organic carbon and nitrogen as atmospheric CO 2 fluctuates daily, seasonally, and over millennia ¹⁴ . This carbon:nitrogen homeostasis improves photosynthetic efficiency ³ and explains why C 3 species, plants that photorespire at substantial rates, remain dominant in most habitats.
... Shifting the glycine decarboxylation step exclusively to the bundle sheath cells leads to increased CO 2 release in these cells, creating an elevated CO 2 environment where the oxygenase reaction of Rubisco is considerably reduced. The bundle sheath specific localisation of the Pprotein from the glycine decarboxylase complex has been shown in C3-C4 species from diverse phylogenetic backgrounds by immunolocalization (Khoshravesh et al., 2016;Oono et al., 2022;Rawsthorne et al., 1988;Schlüter & Weber, 2016). The glycine shuttle biochemistry is accompanied by enhanced centripetal organelle accumulation in the bundle sheath cells (for reviews see: Lundgren, 2020;Schlüter and Weber, 2016). ...
Research on C4 and C3-C4 photosynthesis has attracted significant attention because the understanding of the genetic underpinnings of these traits will support the introduction of its characteristics into commercially relevant crop species. We used a panel of 19 taxa of 18 Brassiceae species with different photosynthesis characteristics (C3 and C3-C4) with the following objectives: (i) create draft genome assemblies and annotations, (ii) quantify orthology levels using synteny maps between all pairs of taxa, (iii) describe the phylogenetic relatedness across all the species, and (iv) track the evolution of C3-C4 intermediate photosynthesis in the Brassiceae tribe. Our results indicate that the draft de novo genome assemblies are of high quality and cover at least 90% of the gene space. Therewith we more than doubled the sampling depth of genomes of the Brassiceae tribe that comprises commercially important as well as biologically interesting species. The gene annotation generated high-quality gene models, and for most genes extensive upstream sequences are available for all taxa, yielding potential to explore variants in regulatory sequences. The genome-based phylogenetic tree of the Brassiceae contained two main clades and indicated that the C3-C4 intermediate photosynthesis has evolved five times independently. Furthermore, our study provides the first genomic support of the hypothesis that Diplotaxis muralis is a natural hybrid of D. tenuifolia and D. viminea. Altogether, the de novo genome assemblies and the annotations reported in this study are a valuable resource for research on the evolution of C3-C4 intermediate photosynthesis.
... However, one of the main bottlenecks is an incomplete understanding of how bundle sheath cells become photosynthetically activated in C 4 plants. On average, the bundle sheath chloroplast content of C 4 species is ~ 30% more than in C 3 species [16,18], but how this evolved is not fully understood. The GOLDEN2-LIKE family of transcription factors known to regulate chloroplast development in C 4 species [19][20][21]. ...
... However, the bundle sheath has been challenging to phenotype in C 3 plants. Classical bright-field light microscopy after embedding samples in resin and thin sectioning has been used [18]. Although this is simple and easily available, it only captures two-dimensional (2D) information from a thin section. ...
... It has been challenging to phenotype bundle sheath tissue in C 3 species as these cells are deeper in the leaf because of the many layers of mesophyll cells [27]. Approaches including bright-field light microscopy [18], transmission electron microscopy [23], serial block-face scanning electron microscopy [26] and single-cell isolation methods [22] are slow and so this hinders rapid analysis of transgenic lines harbouring candidate genes that are hypothesized to control chloroplast proliferation in the bundle sheath. ...
Background:
It has been proposed that engineering the C4 photosynthetic pathway into C3 crops could significantly increase yield. This goal requires an increase in the chloroplast compartment of bundle sheath cells in C3 species. To facilitate large-scale testing of candidate regulators of chloroplast development in the rice bundle sheath, a simple and robust method to phenotype this tissue in C3 species is required.
Results:
We established a leaf ablation method to accelerate phenotyping of rice bundle sheath cells. The bundle sheath cells and chloroplasts were visualized using light and confocal laser microscopy. Bundle sheath cell dimensions, chloroplast area and chloroplast number per cell were measured from the images obtained by confocal laser microscopy. Bundle sheath cell dimensions of maize were also measured and compared with rice. Our data show that bundle sheath width but not length significantly differed between C3 rice and C4 maize. Comparison of paradermal versus transverse bundle sheath cell width indicated that bundle sheath cells were intact after leaf ablation. Moreover, comparisons of planar chloroplast areas and chloroplast numbers per bundle sheath cell between wild-type and transgenic rice lines expressing the maize GOLDEN-2 (ZmG2) showed that the leaf ablation method allowed differences in chloroplast parameters to be detected.
Conclusions:
Leaf ablation is a simple approach to accessing bundle sheath cell files in C3 species. We show that this method is suitable for obtaining parameters associated with bundle sheath cell size, chloroplast area and chloroplast number per cell.
... Shifting the glycine decarboxylation step exclusively to the bundle sheath cells leads to increased CO2 release in these cells, creating an elevated CO 2 environment around the bundle sheath Rubisco and considerably reducing the oxygenase reaction in this compartment. The bundle sheath specific localisation of the Pprotein from the glycine decarboxylase complex has been shown in C3-C4 species from diverse phylogenetic backgrounds by immunolocalization (Rawsthorne et al., 1988, Schlüter and Weber, 2016, Khoshravesh et al., 2016, Oono et al., 2022. The glycine shuttle biochemistry is accompanied by enhanced centripetal organelle accumulation in the bundle sheath cells (for reviews see: Weber, 2016, Lundgren, 2021). ...
Research on C4 and C3-C4 photosynthesis has attracted significant attention because the understanding of the genetic underpinnings of this trait will support the introduction of its characteristics into commercially relevant crop species. We used a panel of 19 taxa of 18 Brassiceae species with different photosynthesis characteristics (C3 and C3-C4) with the following objectives: (i) create draft genome assemblies and annotations, (ii) quantify the level of orthology using synteny maps between all pairs of taxa, (iii) describe the phylogenetic relatedness across all the species, and (iv) track the evolution of C3-C4 intermediate photosynthesis in the Brassiceae tribe.
Our results indicate that the draft de novo genome assemblies are of high quality and cover at least 90%
of the gene space. Therewith we more than doubled the sampling depth of genomes of the Brassiceae tribe that comprises commercially important as well as biologically interesting species. The gene annotation generated high-quality gene models, and for most genes extensive upstream sequences are available for all taxa, yielding potential to explore variants in regulatory sequences. The genome-based phylogenetic tree of the Brassiceae contained two main clades and indicated that the C3-C4 intermediate photosynthesis has evolved five times independently. Furthermore, our study provides the first genomic support of the hypothesis that Diplotaxis muralis is a natural hybrid of D. tenuifolia and D. viminea. Altogether, the de novo genome assemblies and the annotations reported in this study are a valuable resource for research on the evolution of C3-C4 intermediate photosynthesis.
... In this, we recognize that approaches to minimize rubisco oxygenation contain great promise but are outside of the purview of this chapter focused on the reactions downstream of rubisco oxygenation. We also do not consider the unique adaptation of C 2 photosynthesis and efforts to engineer this modification to photorespiration, where CO 2 release is localized into the bundle sheath for enhanced re-fixation (Lundgren, 2020, Khoshravesh et al., 2016. In the next sections, we will further break down the biochemistry of photorespiration to illustrate where the inefficiencies are and how they might be exploited to engineer higher rates of net photosynthesis, as well as discuss the future of photorespiration with climate change. ...
The initial reaction of CO2 fixation has a shared specificity with oxygen, resulting in the formation of 2-phosphoglycolate, an inhibitory sugar. 2-phosphoglycolate is detoxified by photorespiration, but this recycling requires energy and releases carbon, substantially reducing rates of net CO2 fixation in many crop plants. Many strategies are in development to modify native photorespiration and more efficiently detoxify intermediates of photorespiration to boost subsequent net carbon assimilation and crop productivity. Current approaches include increasing the activities of enzymes present in native photorespiration to minimize the pool sizes of inhibitory intermediates or replacing native photorespiration with novel pathways that process 2-phosphoglycolate with improved carbon-conserving, or even fixing, series of reactions. There are also developments in improving photorespiration under elevated temperatures, which occur under typical growing conditions but are outside the scope of many lab-based studies. We finally discuss the potential for improving photorespiration under non-steady-state conditions, which are a hallmark of field crop production systems.
... Arthropogoninae (Khoshravesh et al., 2016) and Chenopodiaceae (Yorimitsu et al., 2019) 824 have shown a close arrangement of mitochondria and chloroplasts. Thus, BS ultrastructure 825 seems to play a major role in prevention of photorespiratory CO2 and NH3 loss and in 826 improvement of leaf carbon and possibly also nitrogen economy. ...
Carbon concentrating mechanisms enhance the carboxylase efficiency of the central photosynthetic enzyme rubisco by providing supra-atmospheric concentrations of CO 2 in its surrounding. In the C 4 photosynthesis pathway, this is achieved by combinatory changes to leaf biochemistry and anatomy. Carbon concentration by the photorespiratory glycine shuttle requires fewer and less complex modifications. It could represent an early step during evolution from C 3 to C 4 photosynthesis and an inspiration for engineering approaches. Plants displaying CO 2 compensation points between 10 to 40 ppm are therefore often termed ‘C 3 –C 4 intermediates’. In the present study, we perform a physiological, biochemical and anatomical survey of a large number of Brassicaceae species to better understand the C 3 -C 4 intermediate phenotype. Our phylogenetic analysis suggested that C 3 -C 4 metabolism evolved up to five times independently in the Brassicaceae. The efficiency of the pathways showed considerable variation between the species but also within species. Centripetal accumulation of organelles in the bundle sheath was consistently observed in all C 3 -C 4 classified accessions indicating a crucial role of anatomical features for CO 2 concentrating pathways. Leaf metabolite patterns were strongly influenced by the individual plant accessions, but accumulation of photorespiratory shuttle metabolites glycine and serine was generally observed. Analysis of PEPC activities suggests that C 4 -like shuttles have not evolve in the investigated Brassicaceae.
Highlight
Our physiological, biochemical and anatomical survey of Brassicaceae revels multiple evolution of C 3 -C 4 intermediacy connected to variation in photorespiratory carbon recapturing efficiency and a distinct C 3 -C 4 bundle sheath anatomy.
... Some genera such as Flaveria and Steinchisma contain both C3 and C4 species or some C3-C4 evolutionary intermediates. Recently, many more C3-C4 intermediates have been found (Brown et al., 2005;Marshall et al., 2007;Keerberg et al., 2014;Khoshravesh et al., 2016). It can be seen that some C3-C4 groups led by Flaveria are important resources for studying the evolutionary mechanism of C4 plants, which provides a valuable biological basis for early attempts to C3 to C4 engineering and for studying the evolutionary mechanism of Kranz anatomical structure (Cui, 2021). ...
How to improve the yield of crops has always been the focus of breeding research. Due to the population growth and global climate change, the demand for food has increased sharply, which has brought great challenges to agricultural production. In order to make up for the limitation of global cultivated land area, it is necessary to further improve the output of crops. Photosynthesis is the main source of plant assimilate accumulation, which has a profound impact on the formation of its yield. This review focuses on the cultivation of high light efficiency plants, introduces the main technical means and research progress in improving the photosynthetic efficiency of plants, and discusses the main problems and difficulties faced by the cultivation of high light efficiency plants. At the same time, in view of the frequent occurrence of high-temperature disasters caused by global warming, which seriously threatened plant normal production, we reviewed the response mechanism of plants to heat stress, introduced the methods and strategies of how to cultivate heat tolerant crops, especially rice, and briefly reviewed the progress of heat tolerant research at present. Given big progress in these area, the era of cultivating smart rice with high light efficiency and heat tolerance has come of age.
