Bundle Sheath Strands Research Articles

Compartmentation of photosynthesis gene expression in C4 maize depends on time of day.

Compared with the ancestral C3 state, C4 photosynthesis occurs at higher rates with improved water and nitrogen use efficiencies. In both C3 and C4 plants, rates of photosynthesis increase with light intensity and are maximal around midday. We determined that in the absence of light or temperature fluctuations, photosynthesis in maize (Zea mays) peaks in the middle of the subjective photoperiod. To investigate the molecular processes associated with these temporal changes, we performed RNA sequencing of maize mesophyll and bundle sheath strands over a 24-h time course. Preferential expression of C4 cycle genes in these cell types was strongest between 6 and 10 h after dawn when rates of photosynthesis were highest. For the bundle sheath, DNA motif enrichment and gene coexpression analyses suggested members of the DNA binding with one finger (DOF) and MADS (MINICHROMOSOME MAINTENANCE FACTOR 1/AGAMOUS/DEFICIENS/Serum Response Factor)-domain transcription factor families mediate diurnal fluctuations in C4 gene expression, while trans-activation assays in planta confirmed their ability to activate promoter fragments from bundle sheath expressed genes. The work thus identifies transcriptional regulators and peaks in cell-specific C4 gene expression coincident with maximum rates of photosynthesis in the maize leaf at midday.

Dark respiration rates are not determined by differences in mitochondrial capacity, abundance and ultrastructure in C4 leaves

Our understanding of the regulation of respiration in C4 plants, where mitochondria play different roles in the different types of C4 photosynthetic pathway, remains limited. We examined how leaf dark respiration rates (Rdark ), in the presence and absence of added malate, vary in monocots representing the three classical biochemical types of C4 photosynthesis (NADP-ME, NAD-ME and PCK) using intact leaves and extracted bundle sheath strands. In particular, we explored to what extent rates of Rdark are associated with mitochondrial number, volume and ultrastructure. Based on examination of a single species per C4 type, we found that the respiratory response of NAD-ME and PCK type bundle sheath strands to added malate was associated with differences in mitochondrial number, volume, and/or ultrastructure, while NADP-ME type bundle sheath strands did not respond to malate addition. In general, mitochondrial traits reflected the contributions mitochondria make to photosynthesis in the three C4 types. However, despite the obvious differences in mitochondrial traits, no clear correlation was observed between these traits and Rdark . We suggest that Rdark is primarily driven by cellular maintenance demands and not mitochondrial composition per se, in a manner that is somewhat independent of mitochondrial organic acid cycling in the light.

Photosynthesis-independent production of reactive oxygen species in the rice bundle sheath during high light is mediated by NADPH oxidase

When exposed to high light, plants produce reactive oxygen species (ROS). In Arabidopsis thaliana, local stress such as excess heat or light initiates a systemic ROS wave in phloem and xylem cells dependent on NADPH oxidase/respiratory burst oxidase homolog (RBOH) proteins. In the case of excess light, although the initial local accumulation of ROS preferentially takes place in bundle-sheath strands, little is known about how this response takes place. Using rice and the ROS probes diaminobenzidine and 2',7'-dichlorodihydrofluorescein diacetate, we found that, after exposure to high light, ROS were produced more rapidly in bundle-sheath strands than mesophyll cells. This response was not affected either by CO2 supply or photorespiration. Consistent with these findings, deep sequencing of messenger RNA (mRNA) isolated from mesophyll or bundle-sheath strands indicated balanced accumulation of transcripts encoding all major components of the photosynthetic apparatus. However, transcripts encoding several isoforms of the superoxide/H2O2-producing enzyme NADPH oxidase were more abundant in bundle-sheath strands than mesophyll cells. ROS production in bundle-sheath strands was decreased in mutant alleles of the bundle-sheath strand preferential isoform of OsRBOHA and increased when it was overexpressed. Despite the plethora of pathways able to generate ROS in response to excess light, NADPH oxidase-mediated accumulation of ROS in the rice bundle-sheath strand was detected in etiolated leaves lacking chlorophyll. We conclude that photosynthesis is not necessary for the local ROS response to high light but is in part mediated by NADPH oxidase activity.

A bipartite transcription factor module controlling expression in the bundle sheath of Arabidopsis thaliana.

C4 photosynthesis evolved repeatedly from the ancestral C3 state, improving photosynthetic efficiency by ~50%. In most C4 lineages, photosynthesis is compartmented between mesophyll and bundle sheath cells, but how gene expression is restricted to these cell types is poorly understood. Using the C3 model Arabidopsis thaliana, we identified cis-elements and transcription factors driving expression in bundle sheath strands. Upstream of the bundle sheath preferentially expressed MYB76 gene, we identified a region necessary and sufficient for expression containing two cis-elements associated with the MYC and MYB families of transcription factors. MYB76 expression is reduced in mutant alleles for these transcription factors. Moreover, downregulated genes shared by both mutants are preferentially expressed in the bundle sheath. Our findings are broadly relevant for understanding the spatial patterning of gene expression, provide specific insights into mechanisms associated with the evolution of C4 photosynthesis and identify a short tuneable sequence for manipulating gene expression in the bundle sheath.

Genome-Wide Transcription Factor Binding in Leaves from C3 and C4 Grasses.

The majority of plants use C3 photosynthesis, but over 60 independent lineages of angiosperms have evolved the C4 pathway. In most C4 species, photosynthesis gene expression is compartmented between mesophyll and bundle-sheath cells. We performed DNaseI sequencing to identify genome-wide profiles of transcription factor binding in leaves of the C4 grasses Zea mays, Sorghum bicolor, and Setaria italica as well as C3 Brachypodium distachyon In C4 species, while bundle-sheath strands and whole leaves shared similarity in the broad regions of DNA accessible to transcription factors, the short sequences bound varied. Transcription factor binding was prevalent in gene bodies as well as promoters, and many of these sites could represent duons that influence gene regulation in addition to amino acid sequence. Although globally there was little correlation between any individual DNaseI footprint and cell-specific gene expression, within individual species transcription factor binding to the same motifs in multiple genes provided evidence for shared mechanisms governing C4 photosynthesis gene expression. Furthermore, interspecific comparisons identified a small number of highly conserved transcription factor binding sites associated with leaves from species that diverged around 60 million years ago. These data therefore provide insight into the architecture associated with C4 photosynthesis gene expression in particular and characteristics of transcription factor binding in cereal crops in general.

An optimized protocol for isolation of high-quality RNA through laser capture microdissection of leaf material.

Laser Capture Microdissection is a powerful tool that allows thin slices of specific cell types to be separated from one another. However, the most commonly used protocol, which involves embedding tissue in paraffin wax, results in severely degraded RNA. Yields from low abundance cell types of leaves are particularly compromised. We reasoned that the relatively high temperature used for sample embedding, and aqueous conditions associated with sample preparation prior to microdissection contribute to RNA degradation. Here, we describe an optimized procedure to limit RNA degradation that is based on the use of low‐melting‐point wax as well as modifications to sample preparation prior to dissection, and isolation of paradermal, rather than transverse sections. Using this approach, high‐quality RNA suitable for down‐stream applications such as quantitative reverse transcriptase–polymerase chain reactions or RNA‐sequencing is recovered from microdissected bundle sheath strands and mesophyll cells of leaf tissue.

Photosynthesis research protocols

This book is number 684 in Springer's popular ‘Methods in Molecular Biology’ series, which is well known for its targeted protocol chapters, each with detailed recipes, photographs, and a set of notes for additional practical guidance. As its name says, Photosynthesis research protocols focuses on various aspects of photosynthesis research and its chapters reflect the whole complexity of this research field. Its editor Robert Carpentier is a very active member of the photosynthesis research community and is involved in three contributions to this book. The first part of the book, with 12 chapters, consists of biochemical protocols. Detailed descriptions of how to purify entire thylakoid multi-protein complexes or parts of them, as well as protocols of their crystallisation, are provided. Moreover, protocols on how to isolate the luminal proteins plastocyanin and cytochrome c6, as well as chloroplast lipids, are provided in this section. The second part of the book with seven chapters is less homogenous and includes protocols on in vitro import of proteins, proteomics, detection of free radicals and reactive oxygen species, photoinhibition assays, reconstitution of PSII into liposomes and a protocol on how to immobilize photosynthetic material. The third part of the book, with four chapters, deals with genetic approaches, focusing on Arabidopsis mutants, cyanobacterial genetics and chloroplast transformation in Chlamydomonas. A small fourth part with only two chapters, the content of which is related to the first biochemical part of this book, presents protocols on chloroplast isolation and the isolation of bundle-sheath cell strands and thylakoids from leaves of C4 grasses. The fifth and final part of the book, with five chapters, describes recipes centred around Rubiso and Rubisco activase, including its isolation, quantitation and activity measurements. All chapters are of high quality and written by leading scientists in their fields. Illustrations are provided when needed and each protocol is sufficiently detailed also to allow access to beginners in the field. The book occasionally gives the impression that the authors were undecided as to whether to focus on photosynthesis or to cover chloroplast biology in general. Hence, this collection of protocols is too diverse to expect that all of it will appeal to every reader. On the other hand, this same diversity ensures that every photosynthesis researcher will surely find several chapters of interest to them within this book.

Analysis of donors of electrons to photosystem I and cyclic electron flow by redox kinetics of P700 in chloroplasts of isolated bundle sheath strands of maize

Bundle sheath chloroplasts of NADP-malic enzyme (NADP-ME) type C4 species have a high demand for ATP, while being deficient in linear electron flow and oxidation of water by photosystem II (PSII). To evaluate electron donors to photosystem I (PSI) and possible pathways of cyclic electron flow (CEF1) in isolated bundle sheath strands of maize (Zea mays L.), an NADP-ME species, light-induced redox kinetics of the reaction center chlorophyll of PSI (P700) were followed under aerobic conditions. Donors of electrons to CEF1 are needed to compensate for electrons lost from the cycle. When stromal electron donors to CEF1 are generated during pre-illumination with actinic light (AL), they retard the subsequent rate of oxidation of P700 by far-red light. Ascorbate was more effective than malate in generating stromal electron donors by AL. The generation of stromal donors by ascorbate was inhibited by DCMU, showing ascorbate donates electrons to the oxidizing side of PSII. The inhibitors of NADPH dehydrogenase (NDH), amytal and rotenone, accelerated the oxidation rate of P700 by far-red light after AL, indicating donation of electrons to the intersystem from stromal donors via NDH. These inhibitors, however, did not affect the steady-state level of P700+ under AL, which represents a balance of input and output of electrons in P700. In contrast, antimycin A, the inhibitor of the ferredoxin-plastoquinone reductase-dependent CEF1, substantially lowered the level of P700+ under AL. Thus, the primary pathway of ATP generation by CEF1 may be through ferredoxin-plastoquinone, while function of CEF1 via NDH may be restricted by low levels of ferredoxin-NADP reductase. NDH may contribute to redox poising of CEF1, or function to generate ATP in linear electron flow to O2 via PSI, utilizing NADPH generated from malate by chloroplastic NADP-ME.

Characterization of photosynthetic electron transport in bundle sheath cells of maize. I. Ascorbate effectively stimulates cyclic electron flow around PSI.

Redox changes of the reaction-center chlorophyll of photosystem I (P700) and chlorophyll fluorescence yield were measured in bundle sheath strands (BSS) isolated from maize (Zea mays L.) leaves. Oxidation of P700 in BSS by actinic light was suppressed by nigericin, indicating the generation of a proton gradient across the thylakoid membranes of BSS chloroplasts. Methyl viologen, which transfers electrons from photosystem I (PSI) to O2, caused a considerable decrease in the reduction rate of P700+ in BSS after turning off actinic light, showing that electron flow from the acceptor side of PSI to stromal components is critical for this reduction. Ascorbate (Asc), and to a lesser extent malate (Mal), caused a lower level of P700+ in BSS under aerobic conditions in far-red light, implying electron donation from these substances to the intersystem carriers. When Asc or Mal was added to BSS during pre-illumination under anaerobic conditions in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), the far-red-induced level of P700+ was lowered. The results suggest Asc and Mal can cause reduction of stromal donors, which in turn establishes conditions for rapid PSI-driven P700+ reduction. Addition of these metabolites also strongly stimulated the development of a proton gradient in thylakoids under aerobic conditions in the absence of DCMU, i.e. under conditions analogous to those in vivo. Ascorbate was a much more effective electron donor than Mal, suggesting it has a physiological role in activation of cyclic electron flow around PSI.

Isolation and characterization of cDNAs for differentially accumulated transcripts between mesophyll cells and bundle sheath strands of maize leaves.

To characterize novel genes functioning specifically in mesophyll cells (MCs) or bundle sheath cells (BSCs) of C4 plants, differential screening of a maize cDNA library was conducted using 32P-labeled single-strand cDNAs prepared from MCs and bundle sheath strands (BSS) as probes. Ten genes encoding thylakoid membrane proteins in chloroplasts were identified as MC-abundant genes. These included genes for chlorophyll a/b binding proteins, plastocyanin, PsaD, PsbT, PsbR, PsbO, PsaK, PsaG, PsaN and ferredoxin. Seven genes identified as BSS-abundant genes encoded PEP carboxykinase, salt-inducible SalT homolog, heavy metal-inducible metallothionein-like protein, ABA- and drought-inducible glycine-rich protein, and three proteins of unknown function (one of which was named Bss1). In situ hybridization analyses for several selected genes revealed that mRNAs for the metallothionein-like protein and Bss1 were accumulated specifically in BSCs, and that mRNA for the SalT homolog was accumulated in vascular cells around phloem cells. Results suggest that the functional differentiation of MC chloroplasts accompany preferential expression of these small proteins in photosystem complexes and that BSCs are the major site of stress responses.

Phosphoenolpyruvate carboxykinase is involved in the decarboxylation of aspartate in the bundle sheath of maize

We recently showed that maize (Zea mays L.) leaves contain appreciable amounts of phosphoenolpyruvate carboxykinase (PEPCK; R.P. Walker, R.M. Acheson, L.I. Tecsi, R.C. Leegood [1997] Aust J Plant Physiol 24: 459-468). In the present study, we investigated the role of PEPCK in C4 photosynthesis in maize. PEPCK activity and protein were enriched in extracts from bundle-sheath (BS) strands compared with whole-leaf extracts. Decarboxylation of [4-14C]aspartate (Asp) by BS strands was dependent on the presence of 2-oxoglutarate and Mn2+, was stimulated by ATP, was inhibited by the PEPCK-specific inhibitor 3-mercaptopicolinic acid, and was independent of illumination. The principal product of Asp metabolism was phosphoenolpyruvate, whereas pyruvate was a minor product. Decarboxylation of [4-14C]malate was stimulated severalfold by Asp and 3-phosphoglycerate, was only slightly reduced in the absence of Mn2+ or in the presence of 3-mercaptopicolinic acid, and was light dependent. Our data show that decarboxylation of Asp and malate in BS cells of maize occurs via two different pathways: Whereas malate is mainly decarboxylated by NADP-malic enzyme, decarboxylation of Asp is dependent on the activity of PEPCK.

The isolation and characterisation of a mutant of the C 4 plant Amaranthus edulis deficient in NAD-malic enzyme activity

A mutant of Amaranthus edulis (Speg.) lacking activity of the C4 leaf form of NAD-malic enzyme (ME; EC 1.1.1.39) has been isolated. Homozygous mutant (5% wild-type ME activity) and heterozygous (50% wild-type ME activity) F2 plants were shown to contain both the α and β NAD-ME subunits in similar amounts to those detected in the wild-type leaves. The rate of photosynthetic CO2 assimilation was reduced in the homozygous mutant to 5% of that observed for the wild-type leaves. Other C4 enzymes were not down-regulated in the mutant plants. There was little difference in photosynthetic rate of the heterozygous plants compared to the wild-type, suggesting that NAD-ME exerts little control over the rate of C4 photosynthesis, and that in the wild-type the enzyme has a very low control coefficient. The activity loss in the heterozygote may therefore be compensated by regulatory mechanisms that increase the activity of the enzyme in vivo. Data for bundle-sheath strands indicated that although the homozygous mutants were able to oxidise malate via the Krebs cycle, they were unable to convert malate to pyruvate and alanine via NAD-ME.

Cyst(e)ine is the transport metabolite of assimilated sulfur from bundle-sheath to mesophyll cells in maize leaves

The intercellular distribution of the enzymes and metabolites of assimilatory sulfate reduction and glutathione synthesis was analyzed in maize (Zea mays L. cv LG 9) leaves. Mesophyll cells and strands of bundle-sheath cells from second leaves of 11-d-old maize seedlings were obtained by two different mechanical-isolation methods. Cross-contamination of cell preparations was determined using ribulose bisphosphate carboxylase (EC 4.1.1.39) and nitrate reductase (EC 1.6.6.1) as marker enzymes for bundle-sheath and mesophyll cells, respectively. ATP sulfurylase (EC 2.7.7.4) and adenosine 5'-phosphosulfate sulfotransferase activities were detected almost exclusively in the bundle-sheath cells, whereas GSH synthetase (EC 6.3.2.3) and cyst(e)ine, gamma-glutamylcysteine, and glutathione were located predominantly in the mesophyll cells. Feeding experiments using [35S]sulfate with intact leaves indicated that cyst(e)ine was the transport metabolite of reduced sulfur from bundle-sheath to mesophyll cells. This result was corroborated by tracer experiments, which showed that isolated bundle-sheath strands fed with [35S]sulfate secreted radioactive cyst(e)ine as the sole thiol into the resuspending medium. The results presented in this paper show that assimilatory sulfate reduction is restricted to the bundle-sheath cells, whereas the formation of glutathione takes place predominantly in the mesophyll cells, with cyst(e)ine functioning as a transport metabolite between the two cell types.

Purification and characterization of non-chloroplastic α-1,4-glucan phosphorylases from leaves of Digitaria eriantha Stent

Summary We have previously identified four major α-glucan Phosphorylase (GP) enzymes in crude leaf extracts from pangolagrass ( Digitaria eriantha Stent.). One co-isolates with the chloroplasts (cGP) while the other three are non-chloroplastic (nGP). In this report we present further characterization and leaf cell-type localization of the non-chloroplastic enzymes. In contrast to observations in maize, all of the pangolagrass GP enzymes were present in both isolated bundle sheath strands and mesophyll cells. Ion exchange column chromatography separated the leaf GPs into three peaks: A, B and C. Peak A was the most active and contained three non-chloroplastic GP (nGP), active bands separable by native Polyacrylamide gel electrophoresis (NPGE). Peak B GP enzymes migrated identically to peak A enzymes during NPGE. Peak C contained a single chloroplastic GP (cGP). The two major nGPs in the peak A fraction co-purified and migrated as a single band during SDS-PAGE, but they could be separated by IEF-column chromatography. Kinetic properties of these peak A nGP enzymes were similar to those of other plant nGP enzymes, with the exception that the pangolagrass nGP is not inhibited by dinucleotide sugars. The co-localization of the leaf GP enzymes in both bundle sheath and mesophyll cells, and the separation of the non-chloroplastic GPs into two pools (peaks A and B) during ion exchange chromatography are unique characteristics not previously described for plant leaf GPs.

Light-Induced Chloroplast [alpha]-Amylase in Pearl Millet (Pennisetum americanum)

In pearl millet (Pennisetum americanum) seedlings light induces the appearance of a leaf [alpha]-amylase isozyme. The leaf [alpha]-amylase isozyme was present in enriched amounts in isolated chloroplast but it could not be detected in isolated etioplasts. The chloroplast [alpha]-amylase was present in both mesophyll and bundle-sheath chloroplasts. Preliminary characterization indicated that molecular properties of chloroplast [alpha]-amylase were like those of a typical [alpha]-amylase. The plastidic [alpha]-amylase had a molecular mass of 46 kD, pH optimum of 6.2, required Ca2+ for activity and thermostability, but lost activity in the presence of ethylenediaminetetracetate. Plastidic [alpha]-amylase activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis could be renatured in situ by Triton X-100. Western blot analysis demonstrated that this protein was antigenically similar to a maize seed [alpha]-amylase. In vivo [35S]methionine labeling of bundle-sheath strands isolated from light-grown leaves followed by immunoprecipitation revealed that bundlesheath strands synthesized plastidic [alpha]-amylase de novo.

Chapter 21 Methods for Mesophyll and Bundle Sheath Cell Separation

Publisher Summary This chapter describes the methods for the separation of mesophyll and bundle sheath cells from maize seedlings. The resultant cells, which can be easily manipulated, are physiologically active. The overall purity and viability are usually greater than 95%. Physical destruction method for bundle sheath cell isolation is used to purify intact bundle sheath strands from which bundle sheath protoplasts, nuclei, and chloroplasts can be isolated if desired. Mesophyll cells are broken during the grinding process. Although the suspension of the first grinding is enriched for mesophyll chloroplasts and nuclei, it is heavily contaminated with bundle sheath chloroplasts and nuclei. The grinding is usually performed with a Polytron homogenizer for its efficiency and flexibility. For large batch isolation, a Cuisinart food processor can be used instead of manual cutting to first chop the leaves into small pieces. Enzymatic digestion method allows the simultaneous isolation of mesophyll protoplasts and bundle sheath strands. To obtain pure bundle sheath strands by grinding, it is important to remove the thick midvein and cut leaves to 1- to 3-mm strips for efficient grinding to eliminate the contamination of mesophyll cells.

Subcellular and immunocytochemical localization of the enzymes involved in ammonia assimilation in mesophyll and bundle-sheath cells of maize leaves

The cellular localization of the enzymes involved in primary nitrogen assimilation was investigated following separation of mesophyll protoplasts and bundle-sheath cells of maize (Zea mays L.) leaves. Determination of the enzymatic activities in the two types of cell revealed that nitrate and nitrite reductase are principally located in the mesophyll cells whereas glutamine synthetase (GS) and ferredoxin-dependent glutamate synthase (Fd-GOGAT) are present in both tissues with a preferential location in the bundle-sheath strands. In order to confirm the results obtained by this conventional biochemical method we have used an in-situ immunofluorescence technique to unambiguously localize GS and Fd-GOGAT at the cellular level. Thin-sectioned maize leaves treated with specific GS and Fd-GOGAT antisera followed by conjugation with fluorescein-isothiocyanate-labelled sheep anti-rabbit immunoglobulins clearly show that GS is equally distributed within the leaf whereas Fd-GOGAT is mostly present in the chloroplasts of the bundle-sheath cells. The cellular localization of nitrate reductase, nitrite reductase, GS-2 and Fd-GOGAT in maize leaf cell types strongly indicates that primary nitrogen assimilation functions in the mesophyll cells while photorespiratory nitrogen recycling is restricted to the bundle-sheath cells.

Corn phosphoglycolate phosphatase: Immunodetection in kranz cells

Summary Specific polyclonal antibodies raised against corn phosphoglycolate phosphatase were used for immunodetection of this protein in the two chlorophyllous cells, mesophyll protoplasts and bundle sheath strands, isolated enzymatically from expanded corn leaves. Western blot analysis clearly showed an immunoreactive protein band with the bundle sheath cells only; its molecular mass of 31.5 kilodaltons corresponds to the phosphoglycolate phosphatase subunit. This result is totally in accordance with the cellular localization of this specific phosphatase activity, previously shown in bundle sheath cells only, and means that phosphoglycolate phosphatase gene expression was different in the two photosynthetic cell types of maize leaves, a C 4 plant.

Carotenoid distribution and deepoxidation in thylakoid pigment-protein complexes from cotton leaves and bundle-sheath cells of maize

In response to excess light, the xanthophyll violaxanthin (V) is deepoxidized to zeaxanthin (Z) via antheraxanthin (A) and the degree of this deepoxidation is strongly correlated with dissipation of excess energy and photoprotection in PS II. However, little is known about the site of V deepoxidation and the localization of Z within the thylakoid membranes. To gain insight into this problem, thylakoids were isolated from cotton leaves and bundle-sheath strands of maize, the pigment protein-complexes separated on Deriphat gels, electroeluted, and the pigments analyzed by HPLC. In cotton thylakoids, 30% of the xanthophyll cycle pigments were associated with the PS I holocomplex, including the PS I light-harvesting complexes and PS I core complex proteins (CC I), and about 50% with the PS II light-harvesting complexes (LHC II). The Chl was evenly distributed between PS I and PS II. Less than 2% of the neoxanthin, about 18% of the lutein, and as much as 76% of the β-carotene of the thylakoids were associated with PS I. Exposure of pre-darkened cotton leaves to a high photon flux density for 20 min prior to thylakoid isolation caused about one-half of the V to be converted to Z. The distribution of Z among the pigment-protein complexes was found to be similar to that of V. The distribution of the other carotenoids was unaffected by the light treatment. Similarly, in field-grown maize leaves and in the bundle-sheath strands isolated from them, about 40% of the V present at dawn had been converted to Z at solar noon. Light treatment of isolated bundle-sheath strands which initially contained little Z caused a similar degree of conversion of V to Z. As in cotton thylakoids, about 30% the V+A+Z pool in bundle-sheath thylakoids from maize was associated with the PS I holocomplex and the CC I bands and 46% with the LHC II bands, regardless of the extent of deepoxidation. These results demonstrate that Z is present in PS I as well as in PS II and that deepoxidation evidently takes place within the pigment-protein complexes of both photosystems.

Alanine Synthesis by Bundle Sheath Cells of Maize

Because in the phloem sap of maize (Zea mays L.) leaves a quarter of the total amino nitrogen can be found as alanine, the capacity of a de novo synthesis of alanine from 3-phosphoglycerate (3-PGA) was studied with isolated bundle sheath (BS) strands of maize. Inasmuch as these cells have retained their plasmodesmatic openings, it was possible to study the formation of alanine from 3-PGA when glutamate and ADP were being added. Alanine synthesis required the existence of the intact cell structure. From the formation of the intermediates, partially released to the medium, the activities of the enzymes of the reaction chain from 3-PGA to alanine could be measured in the intact cells. The results show that in the BS cells the rate of alanine production from pyruvate (0.5 micromole/minute per milligram BS chlorophyll) is more than sufficient to produce one-fourth of the assimilated nitrogen as alanine. As the activity of pyruvate kinase in intact bundle sheath cells in the light was found to be only 0.2 micromole/minute per milligram BS chlorophyll, it is concluded that in the light part of the conversion of 3-PGA to pyruvate may not occur via pyruvate kinase reaction, but via phosphoeno/pyruvate carboxylase, NADP-malate dehydrogenase, and NADP-malic enzyme in the mesophyll and BS cells.