Gene Expression and Accumulation of Rubisco in Bundle Sheath and Mesophyll Cells during Leaf Development and Senescence in Rice,a C3 Plant

Abstract

Gene expression of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (rbcL) and small subunit (rbcS) in bundle sheath and mesophyll cells of rice, a C₃ plant, was examined during leaf development and senescence by in situ hybridization. Localization of Rubisco protein in both cells was also examined by immuno-electron microscopy. Gene expression and accumulation of Rubisco were related with the chlorophyll fluorescence parameters. The chlorophyll fluorescence parameters, such as F_v/F_m and Φ_psii, gradually increased during leaf development with the increase in the accumulation of Rubisco. However, the chlorophyll fluorescence parameters decreased earlier than the Rubisco content during leaf senescence. The expression of rbcS decreased earlier in bundle sheath cells than in mesophyll cells during leaf development, whereas the expression of rbcL in both cells was retained during leaf development and decreased during leaf senescence. On the other hand, Rubisco content of bundle sheath and mesophyll cells increased during leaf development and decreased during leaf senescence. Rubisco was retained even after the disappearance of the expression of rbcS and rbcL detectable by in situ hybridization. The present results suggest that the expression pattern of rbcS in bundle sheath cells was somewhat different from that in mesophyll cells, but this difference was not reflected in Rubisco content.     

Presence of chloroplasts in mestome sheath cells of the C3 Pooid grass Elymus tsukushiensis

ABSTRACT

The successful introduction of the C₄ pathway into C₃ crops would increase photosynthetic rates and crop productivity. However, our poor understanding of how Kranz leaf anatomy develops poses a great obstacle. In particular, the origin, development, and genetics of bundle sheath (BS) cells in C₄ plants are key points to elucidate. Here we report that Elymus tsukushiensis, a common C₃ grass of the subfamily Pooideae, contains chloroplasts in the mestome sheath (MS) cells of the leaf, unlike most MS cells of C₃ grasses. The chloroplasts are smaller than those of mesophyll cells. Immunogold localization showed that the chloroplasts and mitochondria of MS cells, respectively, accumulate ribulose 1,5-bisphosphate carboxylase/oxygenase and a photorespiratory enzyme, glycine decarboxylase, as in mesophyll cells. Thus, we suggest that the MS cells have weak photosynthetic and photorespiratory functions. This finding provides an insight into the development and evolution of C₄-type BS cells in leaves of C₃ grasses.     

Differential Sensitivity of Chloroplasts in Mesophyll and Bundle Sheath Cells in Maize,an NADP-Malic Enzyme-Type C4 Plant,to Salinity Stress

Abstract

The changes in chloroplast ultrastructure and the contents of chlorophyll, Na and K in response to salinity stress were investigated in leaves of maize, an NADP-malic enzyme-type C₄ plant species possessing dimorphic chloroplasts. The seedlings were treated with 0, 1, 2 or 3% NaCl for three or five days under a light or dark condition. In both light and dark conditions, the dry weight of salt-treated plants decreased as NaCl concentration increased. Chlorophyll and K contents of the second leaf blade decreased as NaCl concentration increased under the light condition but not under the dark condition. Na content of the second leaf blade was significantly higher at high NaCl concentrations under both light and dark conditions. However, Na content was much lower under the dark condition than light condition. Higher concentrations (2 and 3%) of NaCl significantly increased the size of plastoglobules, decreased the number and size of starch granules and altered the chloroplast ultrastructure. Under the light condition, mesophyll cell (MC) chloroplasts appeared more sensitive to the damaging effect of salinity than the bundle sheath cell (BSC) chloroplasts. MC chloroplasts became more globular in shape and showed swollen and disorganized thylakoids and reduced thickness of grana by salinity. BSC chloroplasts were less affected by salinity than MC chloroplasts. Although chloroplast size and number and size of starch granules were reduced, there was no structural distortion in the thylakoids of BSC chloroplasts. However, the thickness of grana was increased by salinity. Under the dark condition, the chloroplast structure was less affected by salinity. Though the envelope of BSC chloroplasts was occasionally damaged, the thylakoids in both MC and BSC chloroplasts were preserved under salinity stress. The present study suggests that the chloroplast damage caused by salinity is light-dependent and MC chloroplasts are more sensitive to salinity than BSC chloroplasts.     

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

Abstract

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

大豆光合生理生态的研究——第14报 大豆不同类型叶细胞中叶绿体的分布及超微结构

大豆叶片棚栏细胞和海绵细胞中叶绿体数量多、体积大,基质较稠密,叶绿体基粒及片层结构较复杂。维管束鞘细胞和平脉叶肉细胞中叶绿体数目较少、体积小、基质较稀疏,叶绿体基粒及片层结构较简单。维管束薄壁细胞中叶绿体数最少、体积最小,结构最简单。叶绿体在不同细胞中的分布和结构的复杂程度与各类细胞的功能及在叶片中的解剖位置有关。     

Structural and physiological responses of the C4 grass Sorghum bicolor to nitrogen limitation

Nitrogen (N) is one of the major nutrients influencing photosynthesis and productivity of C₄ plants as well as C₃ plants. C₄ photosynthesis operates through close coordination between mesophyll (M) and bundle sheath (BS) cells. However, how the development of structural and physiological traits in leaves of C₄ plants is regulated under N limitation remains uncertain. We investigated structural and physiological responses of leaves of the NADP-ME-type C₄ grass Sorghum bicolor to N limitation. Plants were grown under four levels of N supply (.05 to .6 g N per 5-L pot). Decreasing N supply resulted in decreases in net photosynthetic rate, stomatal conductance, leaf N and chlorophyll contents, and the activity ratio of phosphoenolpyruvate carboxylase to ribulose 1,5-bisphosphate carboxylase/oxygenase and increases in δ¹³C values and photosynthetic N use efficiency. Low-N leaves were thinner and had smaller photosynthetic cells, especially in M, resulting in lower M/BS tissue area ratio, and contained smaller and fewer chloroplasts. The BS chloroplasts in the low-N leaves accumulated abundant starch grains. The number of thylakoids per granal stack was reduced in M chloroplasts but not in BS chloroplasts. The low-N leaves had thicker cell walls, especially in the BS cells, which might be associated with less negative δ¹³C values, and fewer plasmodesmata in the BS cells. These data reveal structural and physiological responses of C₄ plants to N limitation, most of which would be related to cellular N allocation, light use, CO₂ diffusion and leakiness, and metabolite transport under N limitation.     

叶肉细胞特异表达焦磷酸化酶基因OsIP1在不同源库类型水稻品种中的效应

 将水稻自身焦磷酸化酶基因OsIP1置于叶肉细胞特异表达启动子下,利用农杆菌介导法将嵌合基因cyFBPase:OsIP1分别导入“源限制型” 水稻品种中超123和“库限制型” 品种农垦57。 根据荧光定量PCR结果和农艺性状,筛选高表达且农艺性状没有明显改变的T2转基因纯合株系,用以检测和分析在水稻叶肉细胞中特异性过表达水稻焦磷酸化酶基因的效应。初步的研究结果显示：1）在营养生长期,转基因株系的最高茎蘖数和穗数比各受体亲本均有不同程度的提高,尤其是在分蘖性较强的品种上;2）在籽粒灌浆结实期,叶片、叶鞘中可溶性总糖、蔗糖和淀粉含量在整个灌浆期的变化趋势与对照相似,且多数转基因植株叶片和叶鞘中的蔗糖含量（除了灌浆高峰期时的叶鞘）均显著高于对照;3）转基因株系单株干物质量较野生型对照有显著提高。单株产量呈现不同程度的增加,其中“源限制型”品种中超123转基因株系单株产量较对照增幅达显著水平。     

Gene Expression of Enzymes for Starch and Sucrose Metabolism and Transport in Leaf Sheaths of Rice (Oryza sativa L.) during the Heading Period in Relation to the Sink to Source Transition

Abstract

Leaf sheaths of rice plants are known to temporarily accumulate starch prior to heading, which is subsequently remobilized and transported into the panicle after heading. We investigated the time course for both carbohydrate content and steady state mRNA levels of enzymes related to starch and sucrose metabolism in the rice leaf sheath (Oryza sativa L. cv. Nipponbare). Leaf sheaths from the second leaf below the flag leaf accumulated high levels of starch before heading but they rapidly decreased after heading. In contrast, the flag leaf sheath did not accumulate as much starch. In the second leaf sheath, the mRNA levels of enzymes involved in starch synthesis, ADP glucose pyrophosphorylase (EC 2. 7. 7. 27), soluble starch synthase (EC 2. 4. 1.21) and branching enzyme (EC 2. 4. 1. 18) were high before heading, which coincided with rapid accumulation of starch. The mRNA levels of sucrose synthesis enzymes, cytosolic FBPase (EC 3. 1. 3. 11) and sucrose phosphate synthase (EC 2. 4. 1. 14), and the sucrose transporter (OsSUTI) increased at the time of heading, which was largely coincident with a decrease in the mRNA levels of starch synthesis enzymes. In the flag leaf sheaths, changes in mRNA levels of starch synthesis enzymes were not pronounced, however mRNA levels of sucrose synthesis enzymes and the sucrose transporter showed a clear increase throughout the heading period. The different characteristics observed between the two leaf sheaths will be discussed in relation to the sink to source transition.     

Differences in lamina joint anatomy cause cultivar differences in leaf inclination angle of rice

ABSTRACT

Leaf erectness is an important agronomic trait for improving canopy photosynthesis in rice. It is well known that leaf inclination angle (LIA) decreases after expansion during ripening. However, the high-yielding indica cultivar ‘Takanari’ retains a greater LIA during ripening than the high-quality japonica cultivar ‘Koshihikari’. To clarify the cause of the cultivar difference in LIA, we investigated anatomical characteristics of the lamina joint of a flag leaf. We found a close linear correlation between LIA at the centre and at the base of the leaf blade in both cultivars during ripening. The length of the lamina joint increased significantly more on the adaxial side of a leaf (the margin of the collar) than on the abaxial side (the abaxial side of the central part of the collar) in ‘Koshihikari’ after leaf expansion, but there was no clear difference in ‘Takanari’. We found a close linear correlation between the ratio of lamina joint length on the adaxial to abaxial sides and LIA in ‘Koshihikari’ and ‘Takanari’ during ripening. In ‘Koshihikari’, the average length of cells on the adaxial side increased significantly after leaf expansion, with no significant increase in that on the abaxial side and no significant change in cell number on either side. In ‘Takanari’, cell length and cell number showed no significant changes on either side of the lamina joint. We conclude that the cultivar difference in LIA during ripening is caused mainly by cell elongation on the adaxial side of the lamina joint.

List of Abbreviations: k: light extinction coefficient; LIA: leaf inclination angle; QTL: quantitative trait locus     

The Effect of the Amount of Nitrogen Fertilizer on Starch Metabolism in Leaf Sheath of Japonica and Indica Rice Varieties during the Heading Period

Abstract

The effects of the amount of nitrogen fertilizer on the starch metabolism of rice leaf sheath during the heading period in the japonica rice variety, cv. Nipponbare were compared with those in the indica varieties, cv. Tetep and Johna. The rice plants were grown under a low- (similar to the standard nitrogen level in paddy field) or high-nitrogen condition, and the starch content of the second leaf sheaths below the flag leaf was analyzed from the second leaf stage (growth stage 1) until 21 days after the heading (growth stage 7). The starch content of the plants grown under the high-nitrogen condition at the heading stage (growth stage 4) was lower than that under a low-nitrogen condition in all the varieties. The decrease in the activity of starch branching enzyme (SBE) was considered to be important for the repression of starch accumulation under a high-nitrogen condition. Under the high-nitrogen condition, Nipponbare accumulated more starch in the second leaf sheath than indica varieties at the heading stage. However, the phenomenon could not be accounted for by the activities of AGPase and SBE. Semi-quantitative RT-PCR analysis suggested that the lower activities of SBE in the second leaf sheath under the high-nitrogen condition may be due to, at least in part, the decrease in the expression level of RBE4.     

Intracellular position of mitochondria and chloroplasts in bundle sheath and mesophyll cells of C3 grasses in relation to photorespiratory CO2 loss

In C₃ plants, photosynthetic efficiency is reduced by photorespiration. A part of CO₂ fixed during photosynthesis in chloroplasts is lost from mitochondria during photorespiration by decarboxylation of glycine by glycine decarboxylase (GDC). Thus, the intracellular position of mitochondria in photosynthetic cells is critical to the rate of photorespiratory CO₂ loss. We investigated the intracellular position of mitochondria in parenchyma sheath (PS) and mesophyll cells of 10 C₃ grasses from 3 subfamilies (Ehrhartoideae, Panicoideae, and Pooideae) by immunostaining for GDC and light and electron microscopic observation. Immunostaining suggested that many mitochondria were located in the inner half of PS cells and on the vacuole side of chloroplasts in mesophyll cells. Organelle quantification showed that 62–75% of PS mitochondria were located in the inner half of cells, and 62–78% of PS chloroplasts were in the outer half. In mesophyll cells, 61–92% of mitochondria were positioned on the vacuole side of chloroplasts and stromules. In PS cells, such location would reduce the loss of photorespiratory CO₂ by lengthening the path of CO₂ diffusion and allow more efficient fixation of CO₂ from intercellular spaces. In mesophyll cells, it would facilitate scavenging by chloroplasts of photorespiratory CO₂ released from mitochondria. Our data suggest that the PS cells of C₃ grasses have already acquired an initial structure leading to proto-Kranz and further C₃–C₄ intermediate anatomy. We also found that in the Pooideae, organelle positioning in PS cells on the phloem side resembles that in mesophyll cells.     

不同播期下优质稻桂华占、八桂香花后碳氮物质流转与籽粒生长相关性

以桂华占、八桂香为材料,不同播期调控下,研究不同播期下优质稻花后植株碳氮流转与籽粒生长及品质的相关性.结果表明:(1)花后茎鞘、叶片干物质运转速度和运转率都与籽粒起始灌浆势呈正相关.籽粒活跃灌浆期、持续灌浆时间与花后茎鞘、叶片干物质运转速度和运转率呈极显著正相关.(2)播种期推迟不利于茎鞘碳同化物向穗部流转,茎鞘碳同化物转运对籽粒的产量和淀粉产量的贡献率表现为淀粉＞可溶性糖＞蔗糖,茎鞘碳同化物对籽粒产量及淀粉产量的贡献率远高于叶片.可溶性糖转运对籽粒产量和淀粉产量贡献率表现为SD1＞SD2＞SD3;蔗糖、淀粉对籽粒的产量贡献率表现为SD1＞SD2＞SD3.茎叶可溶性糖积累量的减少与籽粒直链淀粉含量和积累量增加是同步的,并且,茎叶可溶性糖积累量快速递减期(花后3～12 d)与直链淀粉含量和积累量快速递增期(花后6～12d)同步.(3)播种期推迟减少茎鞘和叶片总氮的积累,籽粒氮收获指数降低,但是播种期的推迟却增加茎鞘和叶片器官蛋白氮积累,有利于籽粒蛋白质含量提高.     

In vitro digestion of bundle sheath cells in rumen fluid and its relation to the suberized lamella and C4 photosynthetic type in Panicum species

Differences in ease of digestion of bundle sheath cell walls of leaf blades of Panicum spp. are shown to be associated with differences in bundle sheath cell wall structure, including the presence or absence of a suberized lamella. These structural differences are correlated with photo-synthetic type (viz. C₃, intermediate C₃/C₄, and the C₄ types, PCK, NADP-ME and NAD-ME), as are mesophyll: bundle sheath area ratios.     

Temporal and Spatial Variations of Carbohydrate Content in Rice Leaf Sheath and Their Varietal Differences

Abstract

In rice plant, carbohydrates accumulated in leaf sheaths before heading are translocated to grain and affect yield formation greatly. To clarify the intrinsic mechanism of carbohydrate metabolism in the leaf sheath, we investigated the temporal and spatial variations of carbohydrate metabolism in the third leaf sheath counted from the top and their varietal differences. The results revealed that the amount of carbohydrate decreased from the base to the tip of the leaf sheath, irrespective of variety and developmental stage. However, the proportion of starch content in the basal one-fifth of the leaf sheath to that in the whole sheath varied from 35% to 60% with the variety. Comparing the activities of enzymes related to starch metabolism at the base, middle and tip of the leaf sheath in IR65598-112-2 (New plant type) with those in Nipponbare, the activities of ADP-glucose pyrophosphorylase, branching enzyme and granule-bound starch synthase (GBSS) showed varietal differences. Particularly, the activity of GBSS may play an important role in the varietal difference in spatial variation of starch content in the leaf sheath. In IR65598-112-2, the sucrose content in the leaf sheath was extremely high, suggesting that sucrose may be one of the carbohydrate reserves in this line.     

Two β-amylase genes,OsBAM2 and OsBAM3, are involved in starch remobilization in rice leaf sheaths

To identify mechanisms of starch degradation in rice leaf sheaths at the post-heading stage, we investigated the function of OsBAM2 and OsBAM3, which encode plastid-targeted active β-amylase isoforms, in starch remobilization in leaf sheaths. The starch content in the second leaf sheaths below the flag leaf (the third leaf sheaths) peaked at the flag leaf emergence stage and gradually decreased until 15 days after heading. The mRNA levels of OsBAM2 and OsBAM3 in the third leaf sheaths increased from the flag leaf emergence stage to the heading stage when the starch content began to decrease. However, these mRNA levels did not always remain high during post-heading. Overexpression of OsBAM2 or OsBAM3 markedly repressed starch accumulation in the third leaf sheaths, showing that OsBAM2 and OsBAM3 function in starch degradation in rice leaf sheaths. In contrast, no significant differences in starch content in the third leaf sheaths were detected between knockdown plants of OsBAM2 or OsBAM3 and non-transgenic wild-type plants. Our results suggest that reduced expression of the individual genes, OsBAM2 or OsBAM3, does not result in excess accumulation of starch in the leaf sheaths, probably because of the complementary function of another gene or the action of other genes encoding starch-degrading .     

Effect of various drought stresses and subsequent recovery on proline,total soluble sugar and starch metabolisms in Rice (Oryza sativa L.) varieties

ABSTRACT

Rice (Oryza sativa L.) is one of the most important staple foods in the world, however most improved rice varieties are susceptible to drought stress. A two-year study was conducted to explore the effects of various drought stresses and subsequent recovery on the accumulation and degradation of proline, total soluble sugar and starch in different rice varieties at vegetative stage. The results showed that relative water content in the leaves and sheaths of rice varieties significantly decreased under drought stresses, but not at the same rate. Under control and drought conditions, the water content in sheaths was higher than that in leaves. Interestingly, under severe drought stress in 2015, the leaf water content was higher than the sheath water content. The water distribution between leaves and sheaths might be a response of plants to protect leaf system from devastation by drought. Proline was highly accumulated under drought stress but rapidly decreased after re-watering. The drought tolerant variety DA8 expressed higher ability in accumulation of proline than susceptible varieties. In general, total soluble sugar and starch contents in leaves and sheaths of varieties decreased under drought stress conditions. Total soluble sugar and starch content of DA8 were less affected than other varieties under drought conditions. Our study indicated that metabolisms of total soluble sugar and starch in rice were affected by both environmental conditions and characteristics of varieties. Proline accumulation ability of varieties can be used as a useful indicator for drought tolerant potential in rice breeding for water-limited environments.     

玉米C4光合叶不同部位“花环”结构及叶绿素含量的变化

以玉米进行C4光合的全展第5位叶片为材料,分析从叶基部到顶部的解剖结构和叶绿素含量变化,研究玉米C4光合叶片"花环"结构随叶片发育的变化规律。结果表明,玉米第5位叶从基部到顶部都具有完整的典型"花环"结构,维管束鞘细胞(BSC)和叶肉细胞(MC)的体积在叶片发育过程中具有渐变性,从叶基部到顶部BSC和MC均呈先增大后变小的趋势,而且叶绿素a、b和a+b含量呈相同的变化趋势,说明BSC和MC细胞体积与叶绿素含量的变化具有相关性。叶绿素a/b总体呈上升趋势,说明玉米第5叶基部到顶部的光合途径存在C3向C4转变的过程。玉米第5叶不同部位C4光合途径发育的渐变性比前3叶更为明显。     

Monoclonal Antibody Production and Immunolocalization of a Salinity Stress-Related Protein in Rice (Oryza sativa)

Among various physiological responses to salt stress, the synthesis of a lectin-related protein of 14.5 kDa was observed in rice plants (Oryza sativa L.) under the treatment of 170 mmol/L NaCl. In order to better understand the role of the SALT protein in the physiological processes involving salinity, it was immunolocalized in mesophilic cells of leaf sheath and blade of a rice variety IAC-4440 following monoclonal antibodies produced by hybridome culture technique. This variety turned out to be an excellent model for that purpose, since it accumulates SALT protein even in absence of salt treatment and it has been classified as moderately sensitive to salinity and a superior grain producer. This feature was relevant for this work since it allowed the use of plants without the deleterious effects caused by salinity. Immunocytochemistry assays revealed that the SALT protein is located in the stroma of chloroplasts under non-stressing condition. Since the chloroplast is the main target affected by salinity and considering that the SALT protein does not present any apparent signal peptide for organelle localization, its lectin-like activity seems to play an important role in the establishment of stable complexes, either to other proteins or to oligosaccharides that are translocated to the chloroplast.     

盐胁迫下海马齿叶肉细胞超微结构观察

通过透射电镜,在超显微结构水平上对淡水和海水栽培的海马齿(Sesuvium portulacastrumL.)植物叶肉细胞结构进行了比较。结果显示:海水栽培的海马齿叶肉细胞质膜明显向内折叠,出现大量大小、形状各异的质膜突起,以及质膜片层;而淡水栽培的海马齿叶肉细胞质膜向内折叠不明显,质膜突起少见。相对于淡水栽培,海水栽培的海马齿植物叶肉细胞叶绿体变小、数量增多;形状变短,由肾形、梭形或弓形变成椭圆形或一端膨大的不规则形状;叶绿体基粒片层结构清晰完整,垛叠程度增加,叶绿体没有受到明显伤害;叶绿体中淀粉粒数量增多,体积变大,淀粉粒表面出现皱褶,形状由长椭圆型变成短椭圆形或不规则形状,电子密度变低;叶绿体上脂质体增多且体积变大。线粒体数量增加,但体积变小;形状由圆球状或棒状变成椭圆体状;线粒体内膜向内折叠所形成的嵴清晰,但海水栽培的海马齿叶肉细胞线粒体外膜模糊,受到轻微伤害。