Localization of nitrite and sulfite reductase in bundle sheath and mesophyll cells of maize leaves

The distribution of nitrite reductase (EC 1.7.7.1) and sulfite reductase (EC 1.8.7.1) between mesophyll ceils and bundle sheath cells of maize ( Zea mays L. cv. Seneca 60) leaves was examined. This examination was complicated by the fact that both of these enzymes can reduce both NO-₂ and SO^2-₃ In crude extracts from whole leaves, nitrite reductase activity was 6 to 10 times higher than sulfite reductase activity. Heat treatment (10 min at 55°C) caused a 55% decrease in salfite reductase activity in extracts from bundle sheath cells and mesophyll cells, whereas the loss in nitrite reductase activity was 58 and 82% in bundle sheath cells and mesophyll cell extracts, respectively. This result was explained, together with results from the literature, by the hypothesis that sulfite reductase is present in both bundle sheath cells and mesophyll cells, and that nitrite reductase is restricted to the mesophyll cells. This hypothesis was tested i) by comparing the distribution of nitrite reductase activity and sulfite reductase activity between bundle sheath and mesophyll cells with the presence of the marker enzymes ribulose-l, 5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoe-nolpyruvate carboxylase (EC 4.1.1.32), ii) by examining the effect of cultivation of maize plants in the dark without a nitrogen source on nitrite reductase activity and sulfite reductase activity in the two types of cells, and iii) by studying the action of S^2-on the two enzyme activities in extracts from bundle sheath and mesophyll cells. The results from these experiments are consistent with the above hypothesis.     

Glycolate oxidase isoforms are distributed between the bundle sheath and mesophyll tissues of maize leaves

Glycolate oxidase (EC 1.1.3.15) activity was detected both in the bundle sheath (79%) and mesophyll (21%) tissues of maize leaves. Three peaks of glycolate oxidase activity were separated from maize leaves by the linear KCl gradient elution from the DEAE-Toyopearl column. The first peak corresponded to the glycolate oxidase isoenzyme located in the bundle sheath cells, the second peak had a dual location and the third peak was related to the mesophyll fraction. The mesophyll isoenzyme showed higher affinity for glycolate (Km 23 micromol x L(-1)) and a higher pH optimum (7.5-7.6) as compared to the bundle sheath isoenzyme (Km 65 micromol x L(-1), pH optimum 7.3). The bundle sheath isoenzyme was strongly activated by isocitrate and by succinate while the mesophyll isoenzyme was activated by isocitrate only slightly and was inhibited by succinate. It is concluded that although the glycolate oxidase activity is mainly attributed to the bundle sheath, conversion of glycolate to glyoxylate occurs also in the mesophyll tissue of C4 plant leaves.     

Distribution of two isoforms of glutamine synthetase in bundle sheath and mesophyll cells of corn leaves

Localization of two isoforms of glutamine synthetase (GS; EC 6.3.1.2) was investigated in different cell types, mesophyll cells and bundle sheath cells, of corn ( Zea mays L. var. W64A × W182E) leaves by using ion exchange chrotnatography. In whole leaf extracts, relative activities of GS1 (cytosolic GS) and GS2 (chloroplastic GS) were almost equal. Purified mesophyll protoplasts and bundle sheath strands also showed similar proportions of GS1 and GS2. Methionine sulfoximine (1 mM ) enhanced the accumulation of ammonia when mesophyll protoplasts were incubated with nitrite or when bundle sheath strands were incubated with glycine. This clearly indicates a spatial separation of metabolism of NH⁺₄ derived from photorespiration and from reduction of NOJ.     

Response of the malate dehydrogenase system of maize mesophyll and bundle sheath to salt stress

In maize (Zea mays L., cv. Voronezhskaya-76) seedlings subjected to salinity, the values of indicators of stress response development (contents of proline and lactate, activity of peroxidase) were higher in the cells of mesophyll than in the bundle sheath. At short-term NaCl (150 mM) action, the main reactions of the total adaptation syndrome were located in the cell of mesophyll. At salinity, substantial rearrangements of the isoenzyme composition of the malate dehydrogenase (MDH) system main enzymes occurred, which determined cell energization, the synthesis of reducing equivalents, maintenance of the osmotic balance, and functioning of the Hatch-Slake cycle. The changes in some intermediate concentrations and MDH-system enzyme functioning occurring under stress conditions permit a suggestion that, in maize tissues subjected to salt stress, an additional metabolic pathway related to aspartate synthesis and transport is induced.     

Lipid biosynthesis by intact mesophyll and bundle sheath chloroplasts from maize

The two dimorphic forms of chloroplast isolated from maize leaves utilized acetate for fatty acid biosynthesis and had similar requirements for cofactors. The oleate:palmitate ratio of the fatty acid products was lower for bundle sheath chloroplasts as was acetate incorporation into total fatty acids. Galactose from UDP-galactose was incorporated into galactolipids by both morphological forms to give monogalactosyl diacylglycerol and digalactosyl diacylglycerol in the ratio of 4:1.     

Comparative proteomic analysis of amaranth mesophyll and bundle sheath chloroplasts and their adaptation to salt stress

The effect of salt stress was analyzed in chloroplasts of Amaranthus cruentus var. Amaranteca, a plant NAD-malic enzyme (NAD-ME) type. Morphology of chloroplasts from bundle sheath (BSC) and mesophyll (MC) was observed by transmission electron microscopy (TEM). BSC and MC from control plants showed similar morphology, however under stress, changes in BSC were observed. The presence of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) was confirmed by immunohistochemical staining in both types of chloroplasts. Proteomic profiles of thylakoid protein complexes from BSC and MC, and their changes induced by salt stress were analyzed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (2-D BN/SDS-PAGE). Differentially accumulated protein spots were analyzed by LC–MS/MS. Although A. cruentus photosynthetic tissue showed the Kranz anatomy, the thylakoid proteins showed some differences at photosystem structure level. Our results suggest that A. cruentus var. Amaranteca could be better classified as a C₃–C₄ photosynthetic plant.     

Glutamine synthetase from mesophyll and bundle sheath maize cells: isoenzyme complements and different sensitivities to phosphinothricin

 Anion-exchange FPLC has been used to resolve the isoforms of glutamine synthetase (GS, EC 6.3.1.2) from Zea mays mesophyll (MC) and bundle sheath cells (BSC). Two different isoforms were detected in both types of photosynthetic cells. The predominantly active isoform was GS1 (61%) in MC and GS2 (67%) in BSC. The relative contribution of GS1 and GS2 to the overall GS activity in BSC in maize here reported resembles the proportion described for most C3 plants. Differences among these isoforms in terms of their susceptibility to phosphinothricin (PPT), an analogue of glutamate and known inhibitor of GS, were found. The GS1 isoenzyme from MC was the most sensitive form, being inhibited by 50% at approximately 2.0 μM DL-PPT, whereas the GS2 from BSC presented the highest tolerance to the inhibitor (I₅₀=30 μM). The transferase-to-semibiosynthetic activity ratio for the MC isoforms, which was higher than the ratio for the BSC isoforms, and the differences shown by the isoforms in susceptibility to PPT predict important differences in the biochemical properties and regulation of GS isoenzymes. In this regard, the cytoplasmic isoenzymes, and especially the one in MC, due to its relatively high contribution to mesophyll cell GS activity, could play a vital role in nitrogen metabolism in maize. Received: 1 December 1999 / Revised: 7 February 2000 / Accepted: 23 February 2000     

盐胁迫下芦苇叶肉细胞超微结构的研究

对青藏高原柴达木盆地柯柯盐湖边盐碱地上生长的芦苇叶肉细胞的超微结构进行了研究,并以西宁地区非盐碱地上生长的芦苇作对照。结果表明：西宁地区的芦苇叶肉细胞的叶绿体呈椭圆形,其膜系统完整,基粒片层和基质片层发育良好。在盐碱地上生长的芦苇叶肉细胞的叶绿体呈圆形,叶绿体内出现较大的淀粉粒,并发现有线粒体嵌入叶绿体的现象。叶绿体的类囊体膨大,线粒体的嵴也有膨大的现象。在盐湖水中生长的芦苇叶肉细胞,叶绿体的类囊体排列紊乱、扭曲、松散。类囊体膜局部被破坏,部分类囊体膜解体,空泡化,甚至消失,一些溶解了的类囊体流进细胞质中。综上所述,芦苇叶肉细胞超微结构的变化是该植物适应柯柯盐湖地区盐渍、低温、低气压、强辐射等环境因子的结果。     

Effects of moderate drought on ascorbate peroxidase and glutathione reductase activities in mesophyll and bundle sheath cells of maize

Mesophyll and bundle sheath cells of maize leaves ( Zea mays L.) both contain the enzymes ascorbate peroxidase (AP; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) which are involved in hydrogen peroxide detoxification. Since bundle sheath cells of maize are deficient in photosystem II and have high CO₂ levels, oxidative stress may be less severe in these cells than in mesophyll cells. The present study was conducted to determine if AP and GR activity levels preferentially increase in mesophyll cells relative to bundle sheath cells when plants are subjected to moderate drought. Although drought inhibited the growth of greenhouse-grown plants, it did not affect the levels of protein, chlorophyll or AP. GR was unaffected by drought in whole leaf tissue and mesophyll cells, but did increase slightly in bundle sheath cells. This slight increase is of questionable biological importance. AP and GR activity levels were similar in mesophyll cells, bundle sheath cells and in whole leaf tissue. The data suggest that moderate drought has little effect on enzymes of the hydrogen peroxide scavenging system and that mesophyll and bundle sheath cells may be exposed to similar levels of hydrogen peroxide.     

Ultrastructural alterations in mesophyll and bundle sheath chloroplasts of two maize cultivars in response to chilling at high irradiance

Maize (Zea mays L.) seedlings of two cultivars (cv. Bastion adapted to W. Europe, and cv. Batan 8686 adapted to the highlands of Mexico), raised in a glasshouse (19–25 °C), were transferred to 4.5 or 9 °C at photon flux density (PPFD) of 950 μmol m⁻² s⁻¹ with 10-h photoperiod for 58 h and then allowed to recover at 22 °C for 16 h (14 h dark and 2 h at PPFD of 180 μmol m⁻² s⁻¹). The ultrastructural responses after 4 h or 26 h at 4.5 °C were the disappearance of starch grains in the bundle sheath chloroplasts and the contraction of intrathylakoid spaces in stromal thylakoids of the mesophyll chloroplasts. At this time, bundle sheath chloroplasts of cv. Batan 8686 formed peripheral reticulum. Prolonged stress at 4.5 °C (50 h) caused plastid swelling and the dilation of intrathylakoid spaces, mainly in mesophyll chloroplasts. Bundle sheath chloroplasts of cv. Batan 8686 seedlings appeared well preserved in shape and structure. Batan 8686 had also higher net photosynthetic rates during chilling and recovery than Bastion. Extended leaf photobleaching developed during the recovery period after chilling at 4.5 °C. This was associated with collapsed chloroplast envelopes, disintegrated chloroplasts and very poor staining.     

The chloroplast nucleoids of the bundle sheath and mesophyll cells of Zea mays

Dimorphic chloroplasts of Zea mays L. cv. GH5004 from bundle sheath and mesophyll cells contained similar amounts of DNA, while bundle sheath chloroplasts contained twice the number of nucleoids compared to mesophyll chloroplasts. On average bundle sheath nucleoids were half the size of mesophyll nucleoids and contained half as much DNA. Electron microscope autoradiography of the chloroplasts showed that the nucleoid DNA is associated with the thylakoids and in the case of mesophyll chloroplasts preferentially with the grana. These observations suggest that the differences in nucleoid distribution may be due to differences in membrane morphology, with the small nucleoids of agranal bundle sheath chloroplasts being widely dispersed.     

Differentiation of bundle sheath,mesophyll, and distinctive cells in the C4 grass Arundinella hirta (Poaceae)

The C₄ grass Arundinella hirta is characterized by unusual leaf blade anatomy: veins are widely spaced and files of bundle-sheath-like cells, the distinctive cells, form longitudinal strands that are not associated with vascular tissue. While distinctive cells (DCs) appear to function like bundle sheath cells (BSCs), they differ developmentally in two ways: they are derived from ground meristem rather than procambium and they are formed 1–2 plastochrons later. This study describes ultrastructural features of differentiating of BSCs, DCs, and associated mesophyll cells (MCs) during leaf development. BSCs and DCs differ from adjacent MCs by undergoing earlier cell enlargement, greater rates of chloroplast enlargement, reduction of chloroplast thylakoids at late stages of differentiation, more extensive starch formation, greater wall thickening, and deposition of a suberin lamella. The precocious delimitation of the bundle sheath layer is reflected in earlier BSC enlargement and vacuole growth. Derivation of DCs from ground meristem is correlated with late developmental changes in chloroplast size, wall thickness, and plasmodesmatal density. Despite these differences in timing of events, particularly at early stages, the development of the specialized structural features of BSCs and DCs is essentially similar. Thus, proximity to vascular tissue appears to be nonessential for the coordination and regulation of BSC- and MC-specific developmental events.     

Lincomycin-induced alteration in the contents of chlorophyll-protein complexes of dimorphic maize chloroplasts and its effect on the temperature-induced spectral changes

The difference spectroscopy technique has been utilized to investigate the temperature-induced spectral changes in mesophyll and bundle sheath chloroplasts of maize ( Zea mays L. cv. Ganga-5) in order to assess the role of different pigment-protein complexes in the manifestation of temperature effect on the chloroplast membranes. Cooling and heating of both mesophyll and bundle sheath chloroplasts resulted in absorbance difference (AA) bands at similar wavelengths but the degree of absorb-ance changes were significantly higher in bundle sheath chloroplasts. For example, upon cooling to 7-8°C, positive AA bands were observed at 440, 490 and 680 nm in mesophyll chloroplasts and at 440, 495–500 and 680 nm in bundle sheath chloroplasts but the absorbance change at 680 nm was ca 2% in mesophyll chloroplasts, whereas it was ca 5% in bundle sheath chloroplasts, which have a lower content of light-harvesting pigment-protein complex. The role of chlorophyll-protein complexes was further investigated by monitoring the temperature-induced spectral changes of mesophyll and bundle sheath chloroplasts isolated from lincomycin-treated maize plants where lincomycin selectively inhibits the biosynthesis of specific chlorophyll-protein complexes. Results indicated that depletion of certain pigment-protein complexes in mesophyll chloroplasts made them more susceptible (a ca 4% vs ca 2% absorbance change upon cooling and a ca 6% vs ca 4% absorbance change upon heating) and less tolerant to temperature variation (a 76% vs 39% reversibility during ambient→Cooling→ambient temperature cycle). The data indicate that pigment-protein complexes play a significant role in protecting the chloroplast membranes against temperature variation.     

Different pH-dependences of K+ channel activity in bundle sheath and mesophyll cells of maize leaves

The isolation of bundle sheath protoplasts from leaves of Zea mays L. for patch clamp whole-cell experiments presents special problems caused by the suberin layer surrounding these cells. These problems were overcome by the isolation technique described here. Two different types of whole-cell response were found: a small response caused by MB-1 (maize bundle sheath conductance type 1) which was instantaneously activated, and another caused by MB-2 (maize bundle sheath conductance type 2) consisting of an instantaneous response (maize bundle sheath K⁺ instantaneous current type 2; MB-KI2) similar to but stronger than the current through MB-1 plus a small time-dependent outward rectifying component (maize bundle sheath activated outward rectifying current; MB-AOR) with voltage-dependent delayed activation. The occurrence of MB-AOR was often accompanied by a smaller contribution from an inward rectifying channel at negative potentials. Activation of MB-2 required ATP. It is suggested that MB-1 and MB-2 are related to bundle sheath cells with and without direct contact with the xylem vessels. In mesophyll cells, only one type of response caused by MM-2 (maize mesophyll conductance type 2) was found with an instantaneous (maize mesophyll K⁺ instantaneous current type 2, MM-KI2) and a voltage-dependent delayed component (maize mesophyll activated outward rectifying current, MM-AOR). The most striking difference between bundle sheath and mesophyll cells was the pH dependence of K⁺ uptake. At pH 7.2, uptake of K⁺ by MB-2 was identical to that by MM-2 over the whole voltage range. However, acidification stimulated K⁺ conductance in bundle sheath cells, whereas a decrease was found for MM-2. At pH 6.15, the bundle sheath channel MB-2 had more than a 10-fold higher K⁺ uptake at positive and negative potentials than MM-2. The channel MB-1, too, was stimulated by low pH. This seems to indicate a putative role for MB-1 and MB-2 in charge balance during uptake of nutrients via cotransport from the xylem into the symplasm. Received: 23 April 1999 / Accepted: 19 July 1999     

Rice plastidial NAD‐dependent malate dehydrogenase 1 negatively regulates salt stress response by reducing the vitamin B6 content

Salinity is an important environmental factor that adversely impacts crop growth and productivity. Malate dehydrogenases (MDHs) catalyse the reversible interconversion of malate and oxaloacetate using NAD(H)/NADP(H) as a cofactor and regulate plant development and abiotic stress tolerance. Vitamin B6 functions as an essential cofactor in enzymatic reactions involved in numerous cellular processes. However, the role of plastidial MDH in rice (Oryza sativa) in salt stress response by altering vitamin B6 content remains unknown. In this study, we identified a new loss‐of‐function osmdh1 mutant displaying salt stress‐tolerant phenotype. The OsMDH1 was expressed in different tissues of rice plants including leaf, leaf sheath, panicle, glume, bud, root and stem and was induced in the presence of NaCl. Transient expression of OsMDH1‐GFP in rice protoplasts showed that OsMDH1 localizes to chloroplast. Transgenic rice plants overexpressing OsMDH1 (OsMDH1OX) displayed a salt stress‐sensitive phenotype. Liquid chromatography–mass spectrometry (LC‐MS) metabolic profiling revealed that the amount of pyridoxine was significantly reduced in OsMDH1OX lines compared with the NIP plants. Moreover, the pyridoxine content was higher in the osmdh1 mutant and lower in OsMDH1OX plants than in the NIP plants under the salt stress, indicating that OsMDH1 negatively regulates salt stress‐induced pyridoxine accumulation. Furthermore, genome‐wide RNA‐sequencing (RNA‐seq) analysis indicated that ectopic expression of OsMDH1 altered the expression level of genes encoding key enzymes of the vitamin B6 biosynthesis pathway, possibly reducing the level of pyridoxine. Together, our results establish a novel, negative regulatory role of OsMDH1 in salt stress tolerance by affecting vitamin B6 content of rice tissues.     

Bundle sheath chloroplasts of rice are more sensitive to drought stress than mesophyll chloroplasts

We investigated the effects of drought stress on the ultrastructure of chloroplasts in rice plants. After the seedlings were grown in a glasshouse for 1 month, they were treated for drought stress using two methods. One drought treatment was imposed by reducing the water supply to the plants for 1 month. The other was imposed by withholding water for 2 weeks to examine the withering process of leaves by drought stress. The ultrastructural changes of chloroplasts in bundle sheath cells were more prominent than those in mesophyll cells under both drought stress treatments. Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) content in bundle sheath chloroplasts reduced more dramatically than in mesophyll chloroplasts by drought stress. Although a slight swelling of thylakoids was sometimes observed in bundle sheath chloroplasts in moderate stress for 1 month, the thylakoids were less affected by drought stress than chloroplast envelope. These results suggest that chloroplasts in bundle sheath cells were more sensitive to drought stress than those in mesophyll cells and the thylakoids were less damaged by drought stress compared with chloroplast envelope.     

Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies

Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and “malic enzyme” (EC 1.1.1.40) in bundle sheath cells. NADP-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations.     

Vascular bundle sheath and mesophyll cells modulate leaf water balance in response to chitin

Plants can detect pathogen invasion by sensing microbe‐associated molecular patterns (MAMPs). This sensing process leads to the induction of defense responses. Numerous MAMP mechanisms of action have been described in and outside the guard cells. Here, we describe the effects of chitin, a MAMP found in fungal cell walls and insects, on the cellular osmotic water permeability (P_f) of the leaf vascular bundle‐sheath (BS) and mesophyll cells (MCs), and its subsequent effect on leaf hydraulic conductance (K_leaf). BS is a parenchymatic tissue that tightly encases the vascular system. BS cells (BSCs) have been shown to influence K_leaf through changes in their P_f, for example, after sensing the abiotic stress response‐regulating hormone abscisic acid. It was recently reported that, in Arabidopsis, the chitin receptors‐like kinases, chitin elicitor receptor kinase 1 (CERK1) and LYSINE MOTIF RECEPTOR KINASE 5 (LYK5) are highly expressed in the BS as well as the neighboring mesophyll. Therefore, we studied the possible impact of chitin on these cells. Our results revealed that BSCs and MCs exhibit a sharp decrease in P_f in response to chitin treatment. In addition, xylem‐fed chitin decreased K_leaf and led to stomatal closure. However, Atlyk5 mutant showed none of these responses. Complementing AtLYK5 in the BSCs (using the SCARECROW promoter) resulted in the response to chitin that was similar to that observed in the wild‐type. These results suggest that BS play a role in the perception of apoplastic chitin and in initiating chitin‐triggered immunity.