Photosystem II Activity in Agranal Bundle Sheath Chloroplasts from Zea mays

The photochemical activities of chloroplasts isolated from bundle sheath and mesophyll cells of maize (Zea mays var. DS606A) have been measured. Bundle sheath chloroplasts are almost devoid of grana, except in very young leaves, while mesophyll chloroplasts contain grana at all stages of leaf development.     

Comparative Studies of the Thylakoid Proteins of Mesophyll and Bundle Sheath Plastids of Zea mays

The proteins from both grana and stroma lamellae of maize (Zea mays) mesophyll plastids and from maize bundle sheath plastid membranes have been compared by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels using a discontinuous buffer system. Peptide differences between grana and stroma lamellae were essentially quantitative and not qualitative. Bundle sheath plastid membrane peptides more closely resembled those of the ultrastructurally similar stroma lamellae. However, bundle sheath membranes contained several peptides not apparent in the stroma lamellae.     

CO(2) Assimilation and Malate Decarboxylation by Isolated Bundle Sheath Chloroplasts from Zea mays

Conditions for optimal CO₂ fixation and malate decarboxylation by isolated bundle sheath chloroplasts from Zea mays were examined. The relative rates of these processes varied according to the photosynthetic carbon reduction cycle intermediate provided. Highest rates of malate decarboxylation, measured as pyruvate formation, were seen in the presence of 3-phosphoglycerate, while carbon fixation was highest in the presence of dihydroxyacetone phosphate; only low rates were measured with added ribose-5-phosphate. Chloroplasts exhibited a distinct phosphate requirement and this was optimal at a level of 2 millimolar inorganic phosphate in the presence of 2.5 millimolar 3-phosphoglycerate, dihydroxyacetone phosphate, or ribose-5-phosphate. Malate decarboxylation and CO₂ fixation were stimulated by additions of AMP, ADP, or ATP with half-maximal stimulation occurring at external adenylate concentrations of about 0.15 millimolar. High concentrations (>1 millimolar) of AMP were inhibitory. Aspartate included in the incubation medium stimulated malate decarboxylation and CO₂ assimilation. In the presence of aspartate, the apparent Michaelis constant (malate) for malate decarboxylation to pyruvate by chloroplasts decreased from 6 to 0.67 millimolar while the calculated V_max for this process increased from 1.3 to 3.3 micromoles per milligram chlorophyll. Aspartate itself was not metabolized. It was concluded that the processes mediating the transport of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate transport on the one hand, and also of malate might differ from those previously described for chloroplasts from C₃ plants.     

The Involvement of Aspartate and Glutamate in the Decarboxylation of Malate by Isolated Bundle Sheath Chloroplasts from Zea mays

Aspartate or glutamate stimulated the rate of light-dependent malate decarboxylation by isolated Zea mays bundle sheath chloroplasts. Stimulation involved a decrease in the apparent K_m (malate) and an increased maximum velocity of decarboxylation. In the presence of glutamate other dicarboxylates (succinate, fumarate) competitively inhibited malate decarboxylation by intact chloroplasts with respect to malate with an apparent K_i of about 6 millimolar. For comparison the K_i for inhibition of nicotinamide adenine dinucleotide phosphate-malic enzyme from freshly lysed chloroplasts by these dicarboxylates was 15 millimolar. A range of compounds structurally related to aspartate stimulated malate decarboxylation by intact chloroplasts. K_a values for stimulation at 5 millimolar malate were 1.7, 5, and 10 millimolar for l-glutamate, l-aspartate, and β-methyl-dl-aspartate, respectively. Certain compounds, notably cysteic acid, which stimulated malate decarboxylation by intact chloroplasts inhibited malate decarboxylation by nicotinamide adenine dinucleotide phosphate-malic enzyme obtained from lysed chloroplasts and assayed under comparable conditions. It was concluded that aspartate, glutamate, and related compounds affect the transport of malate into the intact chloroplasts and that malate translocation does not take place on the general dicarboxylate translocator previously reported for higher plant chloroplasts.     

Polyamine Content Related to Seed Storage Performance in Zea mays

As part of a search for biochemical indicators of seed withgood survival characteristics during storage, polyamine (PA)levels were measured in embryos for Zea mays seeds. PA levelswere determined in individual embryos of Mean Generation Analysis(MGA) of crosses between lines which show good (C²) and poor(C⁶) storage characteristics. A correlation was found betweenhigher PA contents (putrescine and spermidine) and superiorsurvival characteristics within the populations that were analysedby MGA. Titres of putrescine (PUT) and spermidine (SPD) showedthat three-quarters of the F² resembled the C² parent and aquarter resembled the C⁶ parent. Furthermore, the backcrossC⁶F¹ showed that half of the embryos had PUT levels above theparental C⁶ line. A maternal inheritance effect was not observed.Neither spermine nor cadaverine content was correlated withthe survival inheritance pattern. Given the survival kineticsobtained, it is suggested that both the PA titres and the seedsurvival characteristics are determined by nuclear dominantfactors. Key words: Polyamines, Zea mays, seeds     

Photoreduction and Oxidation of Cytochrome f in Bundle Sheath Cells of Maize

The photo-oxidation of cytochrome f (cytochrome c₅₅₄) in bundle sheath cells isolated from leaves of maize (Zea mays var. DS 606A) has been compared with that in intact maize leaf and in isolated pea leaf cells (Pisum sativum L.). In all cases, illumination with red light caused a negative absorbance change at 554 nm which was attributed to the oxidation of cytochrome f. The extent of this change was greater using monochromatic red light at wavelengths above 700 nm compared with wavelengths below 700 nm. 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea abolished this difference in bundle sheath cells. After illumination for 1 minute or longer in bundle sheath cells, reduction of cytochrome f in the dark was rapid only if the wavelength of the illuminating light was below 700 nm. In the presence of 3-(3,4-dichlorophenyl)-1, 1-dimethlyurea, reduction was slow after illumination at all wavelengths.     

Intracellular Localization of Phosphoenolpyruvate Carboxykinase in Bundle Sheath Cells of C4 Plants

Bundle sheath protoplasts (BSP) were isolated and purified fromfour C₄ species of the phosphoenolpyruvate (PEP) carboxykinasetype (Panicum maximum, P. texanum, Chloris gayana and Eriochloaborumensis), and cell organellses were separated from the BSPextract by differential centrifugation or sucrose density gradientcentrifugation. Separation of the organelles was ascertainedby the distribution of marker enzymes for chloroplasts, mitochondria,peroxisomes and cytoplasm. Contrary to the previous report [Rathnamand Edwards (1975) Arch. Biochem. Biophys. 171: 214], the distributionof PEP carboxykinase in BSP of P. maximum was the same as thatof UDP-glucose pyrophosphorylase, a marker for cytoplasm, andPEP carboxykinase activity was not recovered in the intact chloroplasts.The same results were obtained with P. texanum, C. gayana andE. borumensis. Therefore, we conclude that PEP carboxykinase is exclusivelylocalized in the cytoplasm of bundle sheath cells of C₄ plants. (Received July 23, 1983; Accepted October 17, 1983)     

玉米纹枯病抗性相关miRNA的鉴定与功能分析

MicroRNA (miRNA)是一类内源性非编码小分子RNA,通过指导剪切或者抑制翻译等方式调节植物基因的表达,参与调控植物的生长发育,并在多种非生物与生物胁迫响应中发挥重要作用. 但目前关于玉米纹枯病抗性相关miRNA表达调节与功能尚不十分清楚. 本研究结合直接克隆法与生物信息学分析,鉴定玉米纹枯病抗性相关9个新的玉米miRNA和已知的zma-miR168a、zma-miR168a*;WMD 3软件进行靶基因预测显示,共获得靶基因总数34个,靶基因功能主要涉及玉米的抗氧化胁迫机制、自身反馈调节、转录调控途径、抗病相关代谢途径以及毒物转运外排等调控过程;实时定量PCR检测miRNA显示,耐感纹枯病材料R15和Ye478叶片和叶鞘中共有9个miRNA受纹枯病感染诱导发生特异性差异表达. 本研究结果提示,玉米纹枯病抗性相关 miRNA介导的玉米对纹枯病诱导产生可能的抗病途径构成了玉米抗纹枯病侵染复杂的防御机制.     

玉米根尖细胞中液泡发生过程的电镜观察

关于液泡的起源与发育目前存在多种看法,Mesquita(1969)认为是由粗糙型内质网膨大形成液泡前体,然后发育成液泡,Bowes(1965)则认为根的分生区细胞液泡来源于内质网,而非分生区细胞的液泡则来源于线粒体或质体的降解;Sitte(1961)认为液泡由部分细胞质溶解后形成液泡膜     

Storage protein characteristics of proline-requiring mutants of Zea mays (L.)

Summary Protein and amino acid composition of mature karnels from three allelic proline-requiring mutants in maize, pro _1-1, pro _1-2, and pro _1-3 were analyzed and compared to kernels of the stock A 188 containing the wild type allele. The amount of free proline was specifically reduced in the embryos of all three mutants, while in the endosperm such a reduction was only found for pro _1-2 and pro _1-3 Accumulation of the proline-rich zeins was strongly reduced in the mutants, but in contrast to opaque-2 the reduction affected all major zein polypeptides to the same extent, possibly as a consequence of the defective proline metabolism. Albumins and globulins as well as free amino acids were more abundant in the endosperms of the mutants than in the wild type. Analysis of the albumins and globulins by SDS-PAGE revealed specific increases as well as reductions of certain polypeptides in the endosperms and embryos of the mutants.     

Structural Associations Between Mitochondria and Chloroplasts in the Bundle Sheath Cells of Portulaca oleracea

Mitochondria and chloroplasts are structurally associated inthe bundle sheath cells of Portulaca oleracea L., an NAD malicenzyme type C₄ plant. These associations occur in some restrictedregions where the mitochondrial cristae extend inwards. Exposureof plants to 1.0 µ11^–1 SO₂ for 3 h induced shrinkageof mitochondria of the bundle sheath cells, which further visualizedthe structural association between the organelles. C₄ plant, chloroplast, mitochondrion, Portulaca oleracea, sulphur dioxide     

Site-specific recombination in Zea mays

The elimination of marker genes after selection is recommended for the commercial use of genetically modified plants. We compared the applicability of the two site-specific recombination systems Cre/lox and Flp/FRT for marker gene elimination in maize plants. The selection marker gene pat surrounded by two identically directed lox or FRT sites was introduced into maize. Sexual crossing with plants harboring the corresponding constitutively expressed recombinase led to the precise and complete excision of the lox-flanked marker gene in the F₁ progeny, whereas Flp-mediated recombination of FRT sequences occurred rarely. Further examination of site-specific integration was done by biolistic bombardment of immature embryos harboring only one lox site with a lox.uidA sequence with results indicating directed integration.     

Isolation of Bundle Sheath Cell Chloroplasts from the NADP-ME Type C(4) Plant Zea mays: Capacities for CO(2) Assimilation and Malate Decarboxylation

Bundle sheath chloroplasts have been isolated from Zea mays leaves by a procedure involving enzymic digestion of mechanically prepared strands of bundle sheath cells followed by gentle breakage and filtration. The resulting crude chloroplast preparation was enriched by Percoll density layer centrifugation to yield intact chloroplasts (about 20 micrograms chlorophyll per 10-gram leaf tissue) with high metabolic activities. Based on activities of marker enzymes in the chloroplast and bundle sheath cell extracts, the chloroplasts were essentially free of contamination by other organelles and cytoplasmic material, and were generally about 70% intact. Chlorophyll a/b ratios were high (about 10). With appropriate substrates these chloroplasts displayed high rates of malate decarboxylation, measured as pyruvate formation, and CO₂ assimilation (maximum rates approximately 5 and 3 micromoles per minute per milligram chlorophyll, respectively). These activities were light dependent, linear for at least 20 minutes at 30°C, and displayed highest rates at pH 8.0. High metabolic rates were dependent on addition of an exogenous source of carbon to the photosynthetic carbon reduction cycle (3-phosphoglycerate or dihydroxyacetone phosphate) and a nucleotide (ATP, ADP, or AMP), as well as aspartate. Generally, neither malate decarboxylation nor CO₂ assimilation occurred substantially in the absence of the other activity indicating a close relationship between these processes. Presumably, NADPH required for the photosynthetic carbon reduction cycle is largely supplied during the decarboxylation of malate by NADP-malic enzyme. The results are discussed in relation to the role of bundle sheath chloroplasts in C₄ photosynthesis by species of the NADP-malic enzyme type.     

Regulation of Starch Synthesis in the Bundle Sheath and Mesophyll of Zea mays L. : Intercellular Compartmentalization of Enzymes of Starch Metabolism and the Properties of the ADPglucose Pyrophosphorylases

The intercellular localization of enzymes involved in starch metabolism and the kinetic properties of ADPglucose pyrophosphorylase were studied in mesophyll protoplasts and bundle sheath strands separated by cellulase digestion of Zea mays L. leaves. Activities of starch synthase, branching enzyme, and ADPglucose pyrophosphorylase were higher in the bundle sheath, whereas the degradative enzymes, starch phosphorylase, and amylase were more evenly distributed and slightly higher in the mesophyll. ADPglucose pyrophosphorylase partially purified from the mesophyll and bundle sheath showed similar apparent affinities for Mg²⁺, ATP, and glucose-1-phosphate. The pH optimum of the bundle sheath enzyme (7.0-7.8) was lower than that of the mesophyll enzyme (7.8-8.2). The bundle sheath enzyme showed greater activation by 3-phosphoglycerate than did the mesophyll enzyme, and also showed somewhat higher apparent affinity for 3-phosphoglycerate and lower apparent affinity for the inhibitor, orthophosphate. The observed activities of starch metabolism pathway enzymes and the allosteric properties of the ADPglucose pyrophosphorylases appear to favor the synthesis of starch in the bundle sheath while restricting it in the mesophyll.     

Occurrence of sorbitol in Zea mays

The identity of sorbitol (d-glucitol) from maize seeds was confirmed by GC/MS of the TMSi-ether and by co-chromatography with authentic sorbitol. Sorbitol was found in seeds and silks but not in pollen or leaves. Both endosperm and embryo contained sorbitol, but endosperm accounted for most of the sorbitol recovered from intact seeds.     

Purification and Properties of Mesophyll and Bundle Sheath Cell alpha-Glucan Phosphorylases from Zea mays L. : Equivalence of the Enzymes with the Cytosol and Plastid Phosphorylases from Spinach

Two major α-glucan phosphorylases (I and II) from leaves of the C₄ plant corn (Zea mays L.) were previously shown to be compartmented in mesophyll and bundle sheath cells, respectively (C Mateyka, C Schnarrenberger 1984 Plant Sci Lett 36: 119-123). The two enzymes were separated by chromatography on DEAE-cellulose and purified to homogeneity by affinity chromatography on immobilized starch, according to published procedures, as developed for the cytosol and chloroplast phosphorylase from the C₃ plant spinach. The two α-glucan phosphorylases have their pH optimum at pH 7. The specificity for polyglucans was similar for soluble starch and amylopectin, however, differed for glycogen (K_m = 16 micrograms per milliliter for the mesophyll cell and 250 micrograms per milliliter for the bundle sheath cell phosphorylase). Maltose, maltotriose, and maltotetraose were not cleaved by either phosphorylase. If maltopentaose was used as substrate, the rate was about twice as high with the bundle sheath cell phosphorylase, than with the mesophyll cell phosphorylase. The phosphorylase I showed a molecular mass of 174 kilodaltons and the phosphorylase II of 195 kilodaltons for the native enzyme and of 87 and of 53 kilodaltons for the SDS-treated proteins, respectively. Specific antisera raised against mesophyll cell phosphorylase from corn leaves and against chloroplast phosphorylase from spinach leaves implied high similarity for the cytosol phosphorylase of the C₃ plant spinach with mesophyll cell phosphorylase of the C₄ plant corn and of chloroplast phosphorylase of spinach with the bundle sheath cell phosphorylase of corn.     

The Proportion of Cells that Divide in Root Meristems of Zea mays L.

The proportion of cells that divide in four regions of the rootmeristem of Zea mays has been determined by an analysis of itscells pulse-labelled with tritiated thymidine. In the quiescent centre less than half of the cells divide andthe fastest of these (less than half of them) have a mitoticcycle duration of about 40 h at 23 °C compared with a cell-doublingtime of 230 h for the region. In the cap initials 80–90per cent of the cells divide and about 80 per cent of thesedivide once in 10 h. In the stele about 80 per cent of cellsdivide near the quiescent centre and all divide at 200 µmfrom the quesecent centre. The fast cells divide every 14 hin both regions, but the cell-doubling time increases from 18to 25 h near the quiescent centre. The root cap is completely replaced by its initials every dayand 10 000 cells are sloughed off. The rest of the meristemadds about 170 000 cells to the root every day. These figures are discussed in relation to the role of the quiescentcentre and the control of cell division.