Chloroplasts Of Bundle Sheath Cells Research Articles

Single-cell RNA-sequencing provides new insights into the cell-specific expression patterns and transcriptional regulation of photosynthetic genes in bermudagrass leaf blades

As an important warm-season turfgrass species, bermudagrass (Cynodon dactylon L.) flourishes in warm areas around the world due to the existence of the C4 photosynthetic pathway. However, how C4 photosynthesis operates in bermudagrass leaves is still poorly understood. In this study, we performed single-cell RNA-sequencing on 5296 cells from bermudagrass leaf blades. Eight cell clusters corresponding to mesophyll, bundle sheath, epidermis and vascular bundle cells were successfully identified using known cell marker genes. Expression profiling indicated that genes encoding NADP-dependent malic enzymes (NADP-MEs) were highly expressed in bundle sheath cells, whereas NAD-ME genes were weakly expressed in all cell types, suggesting C4 photosynthesis of bermudagrass leaf blades might be NADP-ME type rather than NAD-ME type. The results also indicated that starch synthesis-related genes showed preferential expression in bundle sheath cells, whereas starch degradation-related genes were highly expressed in mesophyll cells, which agrees with the observed accumulation of starch-filled chloroplasts in bundle sheath cells. Gene co-expression analysis further revealed that different families of transcription factors were co-expressed with multiple C4 photosynthesis-related genes, suggesting a complex transcription regulatory network of C4 photosynthesis might exist in bermudagrass leaf blades. These findings collectively provided new insights into the cell-specific expression patterns and transcriptional regulation of photosynthetic genes in bermudagrass.

An iron-deficiency tolerant genotype of sorghum effectively localizes iron to the thylakoid membranes of the bundle sheath cells

ABSTRACT Although sorghum (Sorghum bicolor L.), a C4 grass plant, requires a higher iron (Fe) concentration in its leaves for sufficient growth compared to C3 grass plants, there are differences in Fe-deficiency tolerance among sorghum genotypes. C4 plant sorghum localizes Fe in bundle sheath (BS) cells as a major site for CO2 assimilation; however, the response of chloroplasts in BS cells to Fe deficiency remains unknown. We compared the allocation of Fe and proteins involved in the photosystems between Fe-deficiency-tolerant and Fe-deficiency-susceptible sorghum genotypes. The distribution patterns of the Fe-requiring core proteins of photosystems I and II were the same in both genotypes, whereas the tolerant genotype, line D100, allocated more Fe to the BS thylakoid membranes than the susceptible genotype, regardless of Fe nutritional status and leaf Fe content. These results suggest that the tolerant D100 sorghum line has mechanisms to supply sufficient Fe to the BS thylakoid membranes.

Stress-induced higher vein density in the C3-C4 intermediate Moricandia suffruticosa under drought and heat stress.

The evolution of C4 photosynthesis involved multiple anatomical and physiological modifications, yet our knowledge of the genetic regulation involved remains elusive. In this study, systematic analyses were conducted comparing the C3-C4 intermediate Moricandia suffruticosa and its C3 relative Brassica napus (rapeseed). We found that the leaves of M. suffruticosa had significantly higher vein density than those of B. napus, and the vein density was further increased in M. suffruticosa under drought and heat stress. Moreover, the bundle sheath distance, as the mean distance from the outer wall of one bundle sheath to the outer wall of an adjacent one, decreased and the number of centripetal chloroplasts in bundle sheath cells was found to be altered in M. suffruticosa leaves under drought and heat treatments. These results suggest that abiotic stress can induce a change in an intermediate C3-C4 anatomy towards a C4-like anatomy in land plants. By integrating drought and heat factors, co-expression network and comparative transcriptome analyses between M. suffruticosa and B. napus revealed that inducible auxin signaling regulated vascular development, and autophagy-related vesicle trafficking processes were associated with this stress-induced anatomical change. Overexpressing three candidate genes, MsERF02, MsSCL01, and MsDOF01, increased leaf vein density and/or enhanced photosynthetic assimilation and drought adaptability in the transgenic lines. The findings of this study may improve our understanding of the genetic regulation and evolution of C4 anatomy.

Two major metabolic factors for an efficient NADP-malic enzymetype C4 photosynthesis.

Compared to the large number of studies focused on the factors controlling C3 photosynthesis efficiency, there are relatively fewer studies of the factors controlling photosynthetic efficiency in C4 leaves. Here, we used a dynamic systems model of C4 photosynthesis based on maize (Zea mays) to identify features associated with high photosynthetic efficiency in NADP-malic enzyme (NADP-ME) type C4 photosynthesis. We found that two additional factors related to coordination between C4 shuttle metabolism and C3 metabolism are required for efficient C4 photosynthesis: (1) accumulating a high concentration of phosphoenolpyruvate through maintaining a large PGA concentration in the mesophyll cell chloroplast and (2) maintaining a suitable oxidized status in bundle sheath cell chloroplasts. These identified mechanisms are in line with the current cellular location of enzymes/proteins involved in the starch synthesis, the Calvin–Benson cycle and photosystem II of NADP-ME type C4 photosynthesis. These findings suggested potential strategies for improving C4 photosynthesis and engineering C4 rice.

Physiological and Proteomic Analyses of Different Ecotypes of Reed (Phragmites communis) in Adaption to Natural Drought and Salinity.

Drought and salinity are the two major abiotic stresses constraining the crop yield worldwide. Both of them trigger cellular dehydration and cause osmotic stress which leads to cytosolic and vacuolar volume reduction. However, whether plants share a similar tolerance mechanism in response to these two stresses under natural conditions has seldom been comparatively reported. There are three different ecotypes of reed within a 5 km2 region in the Badanjilin desert of Northwest China. Taking the typical swamp reed (SR) as a control, we performed a comparative study on the adaption mechanisms of the two terrestrial ecotypes: dune reed (DR) and heavy salt meadow reed (HSMR) by physiological and proteomic approaches coupled with bioinformatic analysis. The results showed that HSMR and DR have evolved C4-like photosynthetic and anatomical characteristics, such as the increased bundle sheath cells (BSCs) and chloroplasts in BSCs, higher density of veins, and lower density and aperture of stomata. In addition, the thylakoid membrane fluidity also plays an important role in their higher drought and salinity tolerance capability. The proteomic results further demonstrated that HSMR and DR facilitated the regulation of proteins associated with photosynthesis and energy metabolism, lipid metabolism, transcription and translation, and stress responses to well-adapt to the drought and salinity conditions. Overall, our results demonstrated that HSMR and DR shaped a similar adaption strategy from the structural and physiological levels to the molecular scale to ensure functionality in a harsh environment.

Engineering chloroplast development in rice through cell-specific control of endogenous genetic circuits.

SummaryThe engineering of C4 photosynthetic activity into the C3 plant rice has the potential to nearly double rice yields. To engineer a two‐cell photosynthetic system in rice, the rice bundle sheath (BS) must be rewired to enhance photosynthetic capacity. Here, we show that BS chloroplast biogenesis is enhanced when the transcriptional activator, Oryza sativa Cytokinin GATA transcription factor 1 (OsCGA1), is driven by a vascular specific promoter. Ectopic expression of OsCGA1 resulted in increased BS chloroplast planar area and increased expression of photosynthesis‐associated nuclear genes (PhANG), required for the biogenesis of photosynthetically active chloroplasts in BS cells of rice. A further refinement using a DNAse dead Cas9 (dCas9) activation module driven by the same cell‐type specific promoter, directed enhanced chloroplast development of the BS cells when gRNA sequences were delivered by the dCas9 module to the promoter of the endogenous OsCGA1 gene. Single gRNA expression was sufficient to mediate the transactivation of both the endogenous gene and a transgenic GUS reporter fused with OsCGA1 promoter. Our results illustrate the potential for tissue‐specific dCas9‐activation and the co‐regulation of genes needed for multistep engineering of C4 rice.

The C4 cycle and beyond: diverse metabolic adaptations accompany dual-cell photosynthetic functions in Setaria

C4 photosynthesis is typically characterized by the spatial compartmentalization of the photosynthetic reactions into mesophyll (M) and bundle sheath (BS) cells. Initial carbon fixation within M cells gives rise to C4 acids, which are transported to the BS cells. There, C4 acids are decarboxylated so that the resulting CO2 is incorporated into the Calvin cycle. This work is focused on the study of Setaria viridis, a C4 model plant, closely related to several major feed and bioenergy grasses. First, we performed the heterologous expression and biochemical characterization of Setaria isoforms for chloroplastic NADP-malic enzyme (NADP-ME) and mitochondrial NAD-malic enzyme (NAD-ME). The kinetic parameters obtained agree with a major role for NADP-ME in the decarboxylation of the C4 acid malate in the chloroplasts of BS cells. In addition, mitochondria-located NAD-ME showed regulatory properties that could be important in the context of the operation of the C4 carbon shuttle. Secondly, we compared the proteomes of M and BS compartments and found 825 differentially accumulated proteins that could support different metabolic scenarios. Most interestingly, we found evidence of metabolic strategies to insulate the C4 core avoiding the leakage of intermediates by either up-regulation or down-regulation of chloroplastic, mitochondrial, and peroxisomal proteins. Overall, the results presented in this work provide novel data concerning the complexity of C4 metabolism, uncovering future lines of research that will undoubtedly contribute to the expansion of knowledge on this topic.

Активность антиоксидантных ферментов в хлоропластах мезофилла и обкладки сосудистых пучков кукурузы (Zea mays L.), под воздействием солевого стресса

Antioxidant defense systems have been studied in the mesophyll (MC) and bundle sheath cell (BSC) chloroplasts of maize (Zea mays L.) leaves cultivated in an artificial climate chamber under various concentrations (0%, 1%, 2%, 3%) of NaCl. The amounts of some of the main products of lipid peroxidation malondialdehyde (MDA) and reactive oxygen species hydrogen peroxide (H2O2) as well as activities of superoxide dismutase (SOD) and ascorbate peroxidase (APO) were determined in MC and BSC chloroplasts. BSC chloroplasts were found to be more tolerant to salt stress compared with MC chloroplasts. The MDA amount increased in both chloroplasts. H2O2 was found to be localized mainly in MC chloroplasts at various NaCl concentrations. The SOD and APO activities increased in both chloroplasts of the plants exposed to salt stress.

The effect of salt (NaCl) stress on the ultrastructure of mesophyll and bundle sheath cell chloroplasts and the activity of superoxide dismutase in maize plants (Zea mays L.)

The effect of salt (NaCl) stress on the ultrastructure of mesophyll and bundle sheath cell chloroplasts and the activity of superoxide dismutase in maize plants (Zea mays L.)

Characteristics of photosynthesis in maize leaves (С4 plants) upon changes in the level of illuminance and nitrate nutrition

We studied assimilation of 14СО2 and distribution of 14С among the products of 3-min-long photosynthesis of maize (Zea mays L.) leaves. The day before the experiment, half of the plants were fertilized with Ca(NO3)2 (1 g/L of water) at a rate of 6 L/m2. Five days before the experiment, some plants were shaded for adaptation (illuminance was reduced by 50%). On the day of the experiment (before the application of 14СО2), several shaded plants were exposed to direct sunlight for 3 min, and some plants grown at full light (light plants) were shaded for 3 min (illuminance of 50%). Unfertilized plants adapted for 5 days to shading showed photosynthesis of 75.9% of control level (full light). If light plants were transferred to shading for 3 min, their photosynthesis decreased to 42.1%. In plants shaded for 5 days and then transferred to full light, photosynthesis in 3 min was 96.3% of control level. At full light, fertilization with nitrate boosted photosynthesis to 132.6% as compared with control material, but photosynthesis decreased to 43.5 and 65.4% of control level in plants shaded for 5 days and those shaded for 3 min, respectively. At the same time, the plants shaded for 5 days and then exposed for 3 min to full light restored photosynthesis to almost control level (95.5%). Analysis of 14С distribution among the products of 3-min-long photosynthesis showed that, the same as in C3 plants, a decrease in illuminance (especially a sudden one) in maize reduced the ratio between labeled sucrose and hexoses and elevates incorporation of 14С into malate, which indicated that its consumption in bundle sheath cells was suppressed. A decrease in the ratio between labeled sucrose and hexoses became more pronounced under the influence of nitrates with this effect also occurring in transport products of photosynthesis (20 cm below 14С-providing leaf area). In plants fertilized with nitrates, radioactivity of sucrose (% of radioactivity of soluble compounds) decreased in all the types of illumination. When illuminance was suddenly reduced for 3 min, incorporation of 14С into sucrose was 21.5 against 51.2% in light plants, and radioactivity of aspartate and malate sharply rose to 13.7 and 26.1% (against 2.1 and 8.9% in control material). Incorporation of 14С into compounds of glycolate pathway was low (less than 2.5%), but it was somewhat greater in nitrate plants. We concluded that the same mechanism of interaction between stomatal apparatus of leaf epidermis, invertase of mesophyll apoplast, and photosynthetic metabolism of carbon with electron flux via electron transport chain in chloroplasts of bundle sheath cells, which governs the rate of photosynthesis and assimilate export from the leaf but is triggered by the extent of consumption in the bundle sheath cells of C4 acids produced in the mesophyll operates in C4 plants (the same as in C3 plants).

Salinity induces membrane structure and lipid changes in maize mesophyll and bundle sheath chloroplasts.

The membranes of Zea mays (maize) mesophyll cell (MC) chloroplasts are more vulnerable to salinity stress than are those of bundle sheath cell (BSC) chloroplasts. To clarify the mechanism underlying this difference in salt sensitivity, we monitored changes in the glycerolipid and fatty acid compositions of both types of chloroplast upon exposure to salinity stress. The monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) contents were higher in MC chloroplasts than in BSC chloroplasts, in both the presence and absence of salt treatment. Under salt conditions, the MGDG level in MC chloroplasts was significantly lower than under normal conditions, while it was unchanged in BSC chloroplasts. In both types of chloroplast, the contents of DGDG, phosphatidylglycerol and phosphatidylinositol remained at the same levels in control and salt-treated plants, whereas sulfoquinovosyldiacylglycerol and phosphatidylcholine were significantly lower and higher, respectively, upon salt treatment. In addition, the fatty acid composition and double bond index of individual lipid classes were changed by salt treatment in both BSC and MC chloroplasts, although these factors had no effect on glycerolipid content. These findings suggest that the difference in salt sensitivity of MC and BSC chloroplast membranes is related to differences in MGDG responses to salinity. Thus, we propose that the low MGDG content and the low sensitivity of MGDG to salinity in BSC chloroplasts render them more tolerant than MC chloroplasts to salinity stress.

Variations in internal water distribution and leaf anatomical structure in maize under persistently reduced soil water content and growth recovery after re-watering

Episodes of regional drought are increasing and are frequently associated with increased duration and intensity. However, relatively little is known about effects of long-lasting drought on leaf microscopic structure and physiological metabolism of plants. In this study, we investigated internal water re-distribution and leaf anatomical structure of maize (Zea mays L.) grown under persistently reduced soil water content. Meanwhile, the threshold of soil water content at which maize cannot recover growth vitality after re-watering was determined. Our results showed that during persistent reductions in the field water capacity from 75 to 25 %, plant growth declined, while the water content in maize decreased following the order from the lower to upper leaves and their leaf sheathes to the stem and roots. At 20 % of field water capacity, the volume of bulliform cells declined, accompanied by an inward shrinkage of cell walls. Under field water capacity below 20 %, the number of chloroplasts in bundle-sheath cells decreased, chloroplasts in mesophyll cells deformed from oval to round, concomitant of a near to zero net photosynthetic rate. It was demonstrated that the growth vitality of maize plants could be recovered by re-watering even if field water capacity reduced to 15 %, but not to 10 %.

Silicon alleviates cadmium toxicity by enhanced photosynthetic rate and modified bundle sheath's cell chloroplasts ultrastructure in maize

Silicon was shown to alleviate the negative effects of various biotic and abiotic stresses on plant growth. Although the positive role of Si on toxic and heavy metal Cd has been already described, the mechanisms have been explained only partially and still remain unclear. In the present study we investigated the effect of Si on photosynthetic-related processes in maize exposed to two different levels of Cd via measurements of net photosynthetic rate (AN), chlorophyll a fluorescence and pigment analysis, as well as studies of leaf tissue anatomy and cell ultrastructure using bright-field and transmission electron microscopy. We found that Si actively alleviated the toxic syndromes of Cd by increasing AN, effective photochemical quantum yield of photosystem II (ϕPSII) and content of assimilation pigments, although did not decrease the concentration of Cd in leaf tissues. Cadmium did not affect the leaf anatomy and ultrastructure of leaf mesophyll’s cell chloroplasts; however, Cd negatively affected thylakoid formation in chloroplasts of bundle sheath cells, and this was alleviated by Si. Improved thylakoid formation in bundle sheath’s cell chloroplasts may contribute to Si-induced enhancement of photosynthesis and related increase in biomass production in C4 plant maize.

Abscisic acid signalling determines susceptibility of bundle sheath cells to photoinhibition in high light-exposed Arabidopsis leaves

The rapid induction of the bundle sheath cell (BSC)-specific expression of ASCORBATE PEROXIDASE2 (APX2) in high light (HL)-exposed leaves of Arabidopsis thaliana is, in part, regulated by the hormone abscisic acid (ABA) produced by vascular parenchyma cells. In this study, we provide more details of the ABA signalling that regulates APX2 expression and consider its importance in the photosynthetic responses of BSCs and whole leaves. This was done using a combination of analyses of gene expression and chlorophyll a fluorescence of both leaves and individual BSCs and mesophyll cells. The regulation of APX2 expression occurs by the combination of the protein kinase SnRK2.6 (OST1):protein phosphatase 2C ABI2 and a Gα (GPA1)-regulated signalling pathway. The use of an ost1-1/gpa1-4 mutant established that these signalling pathways are distinct but interact to regulate APX2. In HL-exposed leaves, BSC chloroplasts were more susceptible to photoinhibition than those of mesophyll cells. The activity of the ABA-signalling network determined the degree of susceptibility of BSCs to photoinhibition by influencing non-photochemical quenching. By contrast, in HL-exposed whole leaves, ABA signalling did not have any major influence on their transcriptomes nor on their susceptibility to photoinhibition, except where guard cell responses were observed.

Evolutionary transition from C3 to C4 photosynthesis and the route to C4 rice

AbstractCompared with C3 plants, C4 plants possess a mechanism to concentrate CO2 around the ribulose-1,5-bisphosphate carboxylase/oxygenase in chloroplasts of bundle sheath cells so that the carboxylation reaction work at a much more efficient rate, thereby substantially eliminate the oxygenation reaction and the resulting photorespiration. It is observed that C4 photosynthesis is more efficient than C3 photosynthesis under conditions of low atmospheric CO2, heat, drought and salinity, suggesting that these factors are the important drivers to promote C4 evolution. Although C4 evolution took over 66 times independently, it is hypothesized that it shared the following evolutionary trajectory: 1) gene duplication followed by neofunctionalization; 2) anatomical and ultrastructral changes of leaf architecture to improve the hydraulic systems; 3) establishment of two-celled photorespiratory pump; 4) addition of transport system; 5) co-option of the duplicated genes into C4 pathway and adaptive changes of C4 enzymes. Based on our current understanding on C4 evolution, several strategies for engineering C4 rice have been proposed to increase both photosynthetic efficiency and yield significantly in order to avoid international food crisis in the future, especially in the developing countries. Here we summarize the latest progresses on the studies of C4 evolution and discuss the strategies to introduce two-celled C4 pathway into rice.

Localization of reactive oxygen species and change of antioxidant capacities in mesophyll and bundle sheath chloroplasts of maize under salinity

In maize, the structure of bundle sheath cell (BSC) chloroplasts is less subject to salinity stress than that of mesophyll cell (MC) chloroplasts. To elucidate the difference in sensitivity to salinity, antioxidant capacities and localization of reactive oxygen species were investigated in both chloroplasts. Transmission electron microscopic observation showed that O2 (-) localization was found in both chloroplasts under salinity, but the accumulation was much greater in MC chloroplasts. H2 O2 localization was observed only in MC chloroplasts of salt-treated plants. In isolated chloroplasts, the activities of superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) were increased by salinity. While the enhancement of SOD activity was similar in both chloroplasts, the increase of APX and DHAR activities were more pronounced in BSC chloroplasts than in MC chloroplasts. Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) and glutathione reductase (GR, EC 1.6.4.2) were undetectable in BSC chloroplasts, while they increased in MC chloroplasts under salinity. Although ascorbate content increased by salinity only in BSC chloroplasts, glutathione content increased significantly in both chloroplasts, and was higher in MC chloroplasts than in BSC chloroplasts. The content of thiobarbituric acid-reactive substances, which is an indicator of lipid peroxidation, was significantly increased by salinity in both chloroplasts. These results suggested O2 (-) -scavenging capacity was comparable between both chloroplasts, whereas H2 O2 -scavenging capacity was lower in MC chloroplasts than in BSC chloroplasts. Moreover, the increased lipid peroxidation under salinity was associated with the structural alteration in MC chloroplasts, while it had less impact on the structure of BSC chloroplasts.

Effects of Salinity Stress on the Structure of Bundle Sheath and Mesophyll Chloroplasts in NAD-Malic Enzyme and PCK Type C4 Plants

The effect of NaCl stress on the structure of leaf chloroplasts was investigated in several NAD-Malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK) type C4 plant species. Seedlings of the monocot species, except Zoysia japonica, grown in 300 mL pots were subjected to salt stress by adding 50 mL of 3% NaCl solution per day to the soil for 5 d after the fourth leaf blades were fully developed. Z. japonica and the dicot species, Amaranthus tricolor, were also treated with 3% NaCl in a similar manner from 5 wk after germination. Salt stress negatively affected the growth, chlorophyll content and chloroplast structure in all the species. At the ultrastructure level, swelling of thylakoids and disruption of envelopes were more or less observed in mesophyll cell (MC) chloroplasts after salt treatment. The structure of bundle sheath cell (BSC) chloroplasts, on the other hand, was hardly damaged under salt condition although stromal and starch areas were considerably decreased. Furthermore, salinity induced granal development in BSC chloroplasts in most species; the number of thylakoids per granum, granal indices and appressed thylakoid density in salt-treated plants were generally higher than those in control. Since the similar responses have also been reported in all NADP-ME type C4 species investigated in our previous study, the high sensitivity to salt stress in MC chloroplasts and the granal development in BSC chloroplasts by salinity were considered to be common phenomena in all three C4 subtypes.

Selective Excitation of Photosystems in Chloroplasts Inside Plant Leaves Observed by Near-Infrared Laser-Based Fluorescence Spectral Microscopy

In this study, we produced selective images of photosystems in plant chloroplasts in situ. We used a spectroimaging microscope, equipped with a near-infrared (NIR) laser that provided light at wavelengths mainly between 800 and 830 nm, to analyze chlorophyll autofluorescence spectra and images from chloroplasts in leaves of Zea mays at room temperature. Femtosecond laser excitation of chloroplasts in mesophyll cells revealed a spectral shape that was attributable to PSII and its antenna in the centers of grana spots. We found that a continuous wave emitted by the NIR laser at a wavelength as long as 820 nm induced chlorophyll autofluorescence with a high contribution from PSI through a one-photon absorption mechanism. A spectral shape attributable to PSI and its antenna was thus obtained using continuous wave laser excitation of chloroplasts in bundle sheath cells. These highly pure spectra of photosystems were utilized for spectral decomposition at every intrachloroplast space to show distributions of PSI and PSII and their associated antenna. A new methodology using an NIR laser to detect the PSI/PSII ratio in single chloroplasts in leaves at room temperature is described.

Can negligible levels of Rubisco activase be the reason for decreased rubisco activity levels in guard cells

Rubisco in guard cell chloroplasts has been a subject of controversy for decades. Several studies have unequivocally confirmed not only the presence of Rubisco in guard cells but also found it to be catalytically active. Based on a cell basis, the amount of Rubisco in guard cells is suggested to be in the range of 0.6 to 1.4 % whereas its activity is reported to be in the range of 0.35 to 0.5%, of a mesophyll cell. This low activity may be due a) to a low Rubisco content, and/or, b) to either the absence or an inactive functional activation mechanism in guard cells. Rubisco activase, enables rubisco to function under physiological conditions. Rubisco activase has been detected immunologically in cyanobacteria, unicellular green algal species and higher plant species. In this study, evidence have been provided for the presence and/or absence of both rubisco and rubisco activase, in guard cells and mesophyll cells of C3 and C4 plants, using immunoblot and immunogold electron microscopy techniques. Results suggest that: 1.Mesophyll cell chloroplasts in C3 plants and bundle sheath cell chloroplasts of a C4 plant have both rubisco and rubisco activase; 2. In bundle sheath cells, rubisco activase appears to be a low abundant protein when compared to C3 mesophyll cell chroloplasts; 4.Guard cells of both C3 and C4 plants have rubisco, and, 5.rubiso activase, if present, is present in negligible amounts in both C3 and C4 guard cells. Reported inadequate levels of rubisco activity in guard cells are consistent with inadequate level of rubisco activity in Arabidopsis mutants lacking rubisco activase.

The high light response in Arabidopsis involves ABA signaling between vascular and bundle sheath cells.

Previously, it has been shown that Arabidopsis thaliana leaves exposed to high light accumulate hydrogen peroxide (H2O2) in bundle sheath cell (BSC) chloroplasts as part of a retrograde signaling network that induces ASCORBATE PEROXIDASE2 (APX2). Abscisic acid (ABA) signaling has been postulated to be involved in this network. To investigate the proposed role of ABA, a combination of physiological, pharmacological, bioinformatic, and molecular genetic approaches was used. ABA biosynthesis is initiated in vascular parenchyma and activates a signaling network in neighboring BSCs. This signaling network includes the Galpha subunit of the heterotrimeric G protein complex, the OPEN STOMATA1 protein kinase, and extracellular H2O2, which together coordinate with a redox-retrograde signal from BSC chloroplasts to activate APX2 expression. High light-responsive genes expressed in other leaf tissues are subject to a coordination of chloroplast retrograde signaling and transcellular signaling activated by ABA synthesized in vascular cells. ABA is necessary for the successful adjustment of the leaf to repeated episodes of high light. This process involves maintenance of photochemical quenching, which is required for dissipation of excess excitation energy.