A MORPHOLOGICAL AND HISTOLOGICAL STUDY OF THE TOMATO MUTANT ‘CURL’

The dominant tomato mutant ‘Curl’ differs from normal plants in several striking respects including the following: misshapen laminar structures such as leaves, sepals, and petals; stunted petiole and rachis; and persistent growth of blade and stem tissue from the adaxial surface of the rachis. These tissues as well as others which appear morphologically normal show gross histological abnormalities. Also evident in sections of mutant tissue is the appearance of areas containing numerous crystalline inclusions and a lack of bodies showing a stainable starch reaction in palisade and mesophyll of leaves and in endodermis and pith cells of the stem.     

Kernel abortion in maize : I. Carbohydrate concentration patterns and Acid invertase activity of maize kernels induced to abort in vitro

The distribution of the glycolytic enzymes, phosphofructokinase, aldolase, triosephosphate isomerase, phosphoglycerate kinase, pyruvate kinase, and the oxidative pentose phosphate pathway enzymes, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, was determined in the leaf tissues of two C₃-plants, pea and leek, and two C₄-plants, maize and sorghum. All enzymes examined were found in epidermal tissue. In pea, maize, and sorghum leaves, the specific activities of these enzymes were usually higher in the nonphotosynthetic epidermal tissue than in the photosynthetic tissues of the leaves. In leek leaves, which were etiolated, specific activities were similar in both epidermal and mesophyll tissue. The distribution of the rate limiting enzymes of glycolysis and the oxidative pentose phosphate pathways probably reflects the capacity of each tissue to generate NADH, NADPH, and ATP from the oxidation of glucose. This capacity appears to be greater in leaf tissues unable to generate reducing equivalents and ATP by photosynthesis, that is, in epidermal tissues and etiolated mesophyll tissue.     

Enzymes of starch and sucrose metabolism in Zea mays leaves

Mesophyll and bundle sheath cells of maize leaves were separated and enzymes of starch and sucrose metabolism assayed. The starch content and activities of ADPglucose (ADPG) starch synthetase and phosphorylase expressed both on a chlorophyll and a protein basis were much lower in mesophyll cells compared to bundle sheath preparations. Exposure of the leaves to continuous illumination for 2·5 days caused the starch content of mesophyll cells to rise greatly and led to considerable increases in ADPG starch synthetase and phosphorylase activity. In glasshouse grown leaves the bulk of invertase, sucrose phosphate synthetase, sucrose phosphatase, UDPglucose pyrophosphorylase and amylase was situated in the mesophyll layer. Sucrose synthetase, ADPG starch synthetase and phosphorylase were largely confined to the bundle sheath. No enzyme could be completely assigned to one particular cell layer. Upon continuous illumination both ADPG starch synthetase and phosphorylase increased in the mesophyll bythe same relative amount. The mesophyll is likely to be a major site for sucrose synthesis in maize leaves.     

A comparative analysis of fructose 2,6-bisphosphate levels and photosynthate partitioning in the leaves of some agronomically important crop species

Starch, sucrose, and fructose 2,6-bisphosphate (F2, 6BP) levels were measured in pea (Pisum sativum L.), maize (Zea mays L.), onion (Allium cepa L.) and soybean (Glycine max L.) leaves throughout a light/dark cycle. Leaf starch accumulated in pea, maize, and soybean but not in onion. Sucrose was a major leaf storage reserve in pea, maize, and onion but was only found at low levels in soybean. In all species examined, the most dramatic changes in F2,6BP concentration coincided with light/dark transitions. During the light period F2,6BP levels were about 0.1 nanomole/milligram chlorophyll in soybean source leaves and there was a small increase in effector concentration in the dark. Levels of F2,6BP were also low in pea and maize leaves during the light period but then increased 10- or 20-fold in the dark. Dark onion leaf F2,6BP levels were about 1.1 to 1.3 nanomole/milligram chlorophyll and these values decreased by 20 to 30% in the light. Thus, three different patterns were identified that describe diurnal F2,6BP levels in source leaves. These results support the suggestion that F2,6BP is involved in the regulation of sucrose biosynthesis. However, it was not possible to demonstrate that high levels of F2,6BP are essential for starch synthesis in the chloroplast.     

Starch synthetases from Vitis vinifera and zea mays

ADPglucose: α-1,4-glucan α-4-glucosyltransferases (starch synthetases) from leaves of Vitis vinifera and leaves and kernels of Zea mays were chromatographed on DEAE-cellulose columns. One form of the enzyme was present in grape leaves having activity both in the presence and absence of primer. Two forms were present in both leaves and kernels of maize. The second peak of activity in maize leaves and the first peak in maize kernels synthesized a polyglucan in the absence of primer. A peak of branching enzyme (Q-enzyme) occurred between the two starch synthetase peaks with both tissues. When fractions containing starch synthetase and branching enzyme were added to the first leaf starch synthetase peak, up to 100-fold activation of the unprimed reaction occurred. Branching enzyme did not stimulate the unprimed activity of the first kernel peak and no branching enzyme could be detected in this peak. The unprimed product was a branched polyglucan with mainly α-1,4-links.     

Effects of artificial warming on the structural, physiological, and biochemical changes of maize (Zea mays L.) leaves in northern China

We examined the effects of artificial warming on physiological, biochemical, and structural changes in leaves of maize plants (Zea mays L.) with a field warming experiment in the North China Plain. Stomatal characters, leaf anatomy and ultrastructure, gas exchange, and carbohydrate and mineral nutrition concentrations were examined using light microscopy, electron microscopy, portable photosynthesis system (Licor-6400), and inductively coupled plasma atomic emission spectroscopy. We found that artificial warming (about 2 °C) increased both the stomatal index and stomatal size, and thus increased net photosynthesis rate (A), stomatal conductance (g _s), and transpiration rate (E). Artificial warming also significantly increased the profile area of chloroplast and mitochondria, but decreased leaf width and thickness, mesophyll thickness, and mesophyll cell size (mainly palisade cell size). In addition, artificial warming also significantly increased the foliar C:N ratio and soluble sugar contents (glucose, fructose, and sucrose), but not the mineral nutrients and starch contents. Our findings suggest that future global warming may affect the maize growth and production in northern China due to the direct warming effects on the structures (anatomy and ultrastructure), biochemical properties and gas exchanges of the maize leaves.     

冠盘二胞Marssonina coronaria侵染不同抗性苹果叶片的组织病理学研究

利用光学和电子显微镜,从组织细胞学水平系统研究了冠盘二胞Marssonina coronaria在苹果抗、感病品种叶片上的侵染过程及侵染后寄主细胞的超微结构特征。结果表明：冠盘二胞的侵入和定殖过程可以分为6个阶段：孢子萌发与芽管形成、附着胞形成、侵入细胞角质层、在叶肉细胞内产生吸器、菌丝在叶肉细胞间和细胞内扩展、分生孢子盘形成。随着菌丝扩展,受侵寄主细胞出现细胞壁加厚,细胞壁降解,质壁分离,叶绿体内淀粉粒、嗜饿颗粒积累,叶绿体基粒片层瓦解,线粒体空泡化等现象。在不同抗性的苹果品种上,分生孢子萌发率差别不明     

Tissue Distribution of beta-Cyanoalanine Synthase in Leaves

β-Cyanoalanine synthase, which catalyzes the reaction between cysteine and HCN to form β-cyanoalanine and H₂S, was assayed in leaf tissues from cyanogenic (Sorghum bicolor × Sorghum sudanense [sorghum]) and noncyanogenic (Pisum sativum [pea], Zea mays [maize], and Allium porrum [leek]) plants. The activity in whole leaf extracts ranged from 33 nanomoles per gram fresh weight per minute in leeks, to 1940 nanomoles per gram fresh weight per minute in sorghum. The specific activities of β-cyanoalanine synthase in epidermal protoplasts from maize and sorghum and in epidermal tissues from peas were in each case greater than the corresponding values for mesophyll protoplasts or tissues, or for strands of bundle sheath cells.

The tissue distributions for this enzyme were determined for pea, leek, and sorghum: the mesophyll protoplasts and tissues in these three plants contained 65% to 78% of the enzyme, while epidermal protoplasts and tissues contained 10% to 35% of the total leaf activity. In sorghum, the bundle sheath strands contained 13% of the leaf activity. The presence of β-cyanoalanine synthase in all tissues and species studied suggests a fundamental role for this enzyme in plant metabolism.

     

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells,Growth, Photosynthetic Capacity,and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.     

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.     

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.     

玉米叶肉细胞和维管束鞘细胞中光合产物的分析

玉米苗照光后,叶肉细胞和维管束鞘细胞大量积累淀粉和可溶性糖(包括蔗糖),其中淀粉95％以上在维管束鞘细胞中。阻断光合产物输出时,两类细胞中蔗糖和淀粉积累都显著增加。离体维管束鞘细胞也能合成蔗糖。离体玉米叶内原生质体饲喂NaH~(14)CO_3并照光后,通常90％以上的~(14)C参入到有机酸和氨基酸中,3～10％参入糖和淀粉中。玉米叶肉原生质体具有直接利用CO_2合成碳水化合物的能力。     

The distribution of carbonic anhydrase and ribulose diphosphate carboxylase in maize leaves

Extraction of maize (Zea mays) leaves by progressive grinding under suitably protective conditions yields total carbonic anhydrase activities (4800 units per milligram chlorophyll) comparable to the activity in spinach (Spinacia oleracea) leaves. The total ribulose diphosphate carboxylase activity was also equal to or greater than the best literature values for maize. Of the total leaf carbonic anhydrase, 72.5% on a chlorophyll basis was present in the mesophyll cells and 14.2% in the bundle-sheath cells. The distribution of the total leaf ribulose diphosphate carboxylase between the mesophyll and bundle-sheath cells was 42.0 and 48.7% respectively. There was three times as much total chlorophyll in extracts of the mesophyll cells compared with the bundle-sheath cells of maize. Similar results for the above distribution of the two enzymes were found using a differential grinding technique. The possible function of carbonic anhydrase in photosynthesis is discussed. The equal distribution of ribulose diphosphate carboxylase activity between the mesophyll and bundle-sheath cells casts doubt upon the hypothesis that a rigid biochemical compartmentation exists between these cell types in maize.     

Tissue specific variation of C-glycosylflavone patterns in oat leaves as influenced by the environment

A comparison is made between the flavone patterns accumulating in epidermal tissues and in the mesophyll of oat primary leaves grown in a phytotron and under field conditions. In developing leaves cultivated under standard conditions, varying patterns of two vitexin-derived O-rhamnosides and of one isovitexin O-arabinoside are produced in the basal region as the result of basal meristem activity. These patterns are tissue specific and differ quantitatively in the epidermis and the mesophyll. During the course of subsequent growth and differentiation, this pattern is constant as the compounds are moved upwards due to basipetal leaf growth. In comparison, the flavone patterns generated in the basal section of leaves grown in the field do not vary significantly. There is the additional accumulation of isoorientin O-arabinoside. Again flavone patterns are tissue specific, but in contrast to standard growth they are modified characteristically in those leaf tissues which are already morphologically differentiated. It is possible that the isovitexin moiety of the O-arabinoside is oxidized to the corresponding isoorientin derivative in the mesophyll. Moreover, field-grown leaves show a two-fold increase in flavone content in each leaf epidermis and a six-fold increase in the mesophyll when compared to the corresponding tissues of phytotron-grown leaves.     

Chalcone synthase and flavonoid products in primary-leaf tissues of rye and maize

During cell and tissue differentiation of developing rye (Secale cereale L.) and maize (Zea mays L.) primary leaves, various flavonoids are synthesized and accumulate in both epidermal and mesophyll tissues. In order to prove either the biosynthetic autonomy of each tissue type and- or intercellular transport of flavonoids, the tissue distributions of chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the pathway, and of flavonoids have been comparatively investigated. Monoclonal antibodies raised against CHS from rye were used to relate enzyme activity in a particular tissue extract to the corresponding amount of CHS protein. A close correlation was found between CHS activities and amounts of CHS protein during leaf development and in the various tissues. The simultaneous occurrence of CHS in both epidermal layers as well as in the mesophyll correlated with the accumulation of flavonoid products in these tissues, indicating tissue autonomy of flavonoid biosynthesis. These data are in contrast to previous reports (Knogge and Weissenböck, 1986, Planta 167, 196–205) on primary leaves of oat (Avena sativa) where CHS and several subsequent enzymes were located mainly in the mesophyll whereas major flavonoid products accumulated predominantly in both epidermal cell layers, indicating that intertissue transport of flavonoids might occur.     

Chloroplast fine structure in the japonica-2 maize mutant exposed to continuous illumination. 1. The green tissues.

Exposure to continuous illumination causes the appearance of numerous plastoglobuli in the stroma of both the mesophyll and bundle sheath chloroplasts of the green tissues of the leaves of the japonica-2 mutant of maize. In the pale green tissues the thylakoids have markedly swollen membranes. Another feature of the plastids exposed to continuous illumination is the heavy accumulation of starch. The japonica-2 chloroplasts show a different sensitivity to light, the chloroplasts of the pale green tissues being affected more markedly than the ones of the dark green tissues, and the bundle sheath chloroplasts more than those of the mesophyll. The effects of continuous illumination may be interpreted as an acceleration of chloroplast ontogenesis.     

In situ starch degradation of different feeds in the rumen

The in situ starch degradation of 5 feeds (barley, maize, pea, oats and wheat bran) has been measured (trial 1), and the influence of particle size on starch degradation investigated with 3 feeds (barley, maize, pea) (trial 2). The starch degradability of barley, oats and wheat bran was found to be higher than that of pea, and higher again than that of maize: 98, 97, 96, 90 and 58% respectively. For barley, oats and wheat bran, starch was degraded more rapidly than the other dry matter (pm) components. Maize and pea starches were degraded at the same rate as non-starchy components. The particle size variations between feeds ground on the same screen may partly explain variations in starch degradability. When the particle size increased from 0.8 to 6.0 mm screen grinding, in situ starch degradability decreased; the decrease was higher for maize (13.8 points) than for barley (7.4 points) or pea (10.4 points).