Changes in Protein Content and in the Structure and Number of Chloroplasts during Leaf Senescence in Rice Seedlings

Two types of experiment were carried out to examine whetheror not the inactivation of photosynthesis is related to lossof chloroplasts during foliar senescence of rice seedlings.Levels of both soluble and insoluble leaf proteins decreasedduring senescence, the loss of the soluble proteins being fasterthan that of the insoluble ones. There was a good positive correlationbetween the rate of oxygen evolution and the level of solubleproteins. The inactivation of photosynthesis was also linearlyrelated to the loss of a major fraction of insoluble proteins.Thus, the loss of photosynthetic ability is ascribable to thedegradation of relevant proteins and enzymes during leaf senescence.Electron microscopy revealed that senescence caused the disorientationof the grana and stroma thylakoids, a decrease in the numberof starch granules, and an increase in the size and number ofplastoglobuli. Large grana consisting 20 to 30 thylakoids appearedin aged leaves. In addition to these changes in ultrastructure,there was a significant decrease in the size of chloroplasts.Furthermore, the number of chloroplasts in mesophyll cells wasalso notably reduced during senescence. Thus, the loss of leafproteins and inactivation of photosynthesis are both relatedto the decrease in the total mass of chloroplasts during senescenceof rice seedlings. ³Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan. (Received January 4, 1989; Accepted April 19, 1989)     

Changes in the Number and Size of Chloroplasts during Senescence of Primary Leaves of Wheat Grown under Different Conditions

Changes in the number and size of chloroplasts in mesophyllcells were investigated in primary leaves of wheat from fullexpansion to yellowing under different growth conditions. Thenumber of chloroplasts per cell decreased slowly, although thedecrease was steady and statistically significant, until thelast stage of leaf senescence, when rapid degradation of chloroplaststook place. Rates of leaf senescence, or the decline in thenumber of chloroplasts, varied greatly among plants grown atdifferent seasons of the year, but about 20% of chloroplastsalways disappeared during the phase when steady loss of chloroplastsoccurred. The area of chloroplast disks also decreased graduallybut significantly, with a rapid decrease late in senescence.Thus, the total quantity of chloroplasts per mesophyll celldecreased substantially during leaf senescence. Yellowed leavescontained numerous structures that resemble oil drops but nochloroplasts. Decreases in rates of photosynthesis that occurduring senescence may, therefore, be largely due to decreasesin the quantity of chloroplasts. However, a better correlationwas found between the decrease in the maximum capacity for photosynthesisand the degradation of RuBP carboxylase. When plants had beengrown with a sufficient supply of nutrients, the number of chloroplastsdecreased steadily but at a reduced rate and the reduction inthe area of chloroplast disks was strongly suppressed. Thus,the quantitative decrease in chloroplasts in senescing leavesappears to be regulated by the requirements for nutrients (nitrogen)of other part of the plant. ³Present address: Department of Biology, Faculty of Science,Toho University, Miyama, Funabashi, Chiba, 274 Japan     

A Genetic Analysis of Chloroplast Division and Expansion in Arabidopsis thaliana

A nuclear recessive mutant of Arabidopsis thaliana, arc5, has been isolated in which there is no significant increase in chloroplast number during leaf mesophyll cell expansion and in which there are only 13 chloroplasts per mesophyll cell compared with 121 in wild-type cells. Mature arc5 chloroplasts in fully expanded mesophyll cells are 6-fold larger than in wild-type cells. A large proportion of arc5 chloroplasts also show some degree of central constriction, suggesting that the mutation has prevented the completion of the chloroplast division process. To examine the interaction of arc loci, a double mutant was constructed between arc1, a mutant possessing many small chloroplasts, and arc5. A second double mutant was also constructed between arc3, a previously discovered mutant also possessing few large chloroplasts per cell, and arc1. Analysis of these double mutants shows that chloroplast number per mesophyll cell is greater when arc5 and arc3 mutations are expressed in the arc1 background than when expressed alone. The cell-specific nature of arc mutants was also analyzed. The phenotypic traits characteristic of arc3 and arc5 are a reduction in chloroplast number and an increase in chloroplast size in mesophyll cells: these changes are also observed in reduced form in the epidermal and guard cell chloroplasts of arc3 and arc5 plants. Analysis of parenchyma sheath cell chloroplasts suggests that in leaves of arc1 plants the normal developmental distinction between mesophyll and parenchyma sheath chloroplasts is perturbed. The relevance of these findings to the analysis of the control of chloroplast division in mesophyll cells is discussed.     

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.     

Cell size and chloroplast size in relation to chloroplast replication in light-grown wheat leaves

As part of an investigation into the control of chloroplast replication the number and size of chloroplasts in mesophyll cells was examined in relation to the size of the cells. In first leaves of Triticum aestivum L. and T. monococcum L. the number of chloroplasts in fully expanded mesophyll cells is positively correlated with the plan area of the cells. The linear relationship between chloroplast number per cell and cell plan area is also consistent over a fivefold range of cell size in isogenic diploid and tetraploid T. monococcum. In T. aestivum the chloroplast number per unit cell plan area varies among cells in relation to the size of the chloroplasts. Those cells containing chloroplasts with a relatively small face area have a correspondingly higher density of chloroplasts, and consequently, the total chloroplast area per unit cell plan area is very similar in all the cells. The results indicate that the proportion of the cell surface area covered by chloroplasts is precisely regulated, and that this is achieved during cell development by growth and replication of the chloroplasts.     

Responses of Chlorophyll Fluorescence and Carotenoids Biosynthesis to High Light Stress in Rice Seedling Leaves at Different Leaf Position

In the present study, we investigated the changes of photosynthesis, chlorophyll fluorescence and the content of carotenoid pigments in rice ( Oryza sativa L.) seedling leaves and their responses to high light. The results showed that the rate of photosynthesis, the contents of individual and total carotenoids and the pool size of xanthophyll cycle decreased with age increasing of the leaf. When the leaves were exposed to high light for 2 h, the qN of mature leaf (5th leaf) increased more significantly than that of younger (6th leaf) and older leaves (3rd and 4th leaf). Comparing with the leaves before exposure to high light, the excitation pressure on PSⅡ (1-qP) increased by 44%, 57%, 19% and 45% in the 3rd, 4th, 5th and 6th leaf under high light, respectively. The highest content of carotenoids and the greatest conversion of violaxanthin to zeaxanthin were found in the 5th leaf, and it was consistent with the 5th leaf exhibiting the strongest resistance to high light. Our results suggested that the ability of rice leaf to resist photoinhibition is related to the level of carotenoids and the ability of carotenoids biosynthesis.      

Relationship between Photosynthesis and Chlorophyll Content during Leaf Senescence of Rice Seedlings

Photosynthetic oxygen evolution, chlorophyll contents and chlorophylla /b ratios of 3rd to 6th leaves of rice seedlings were measuredto examine whether or not inactivation of photosynthesis duringsenescence is related to loss of chlorophyll. Photosyntheticactivity decreased more rapidly than chlorophyll content duringleaf senescence; as a result, the lower the leaf position, thelower was the rate of oxygen evolution determined on the basisof chlorophyll. Chlorophyll ab ratio also decreased with advancingsenescence. Electrophoretic analysis revealed that the declinein chlorophyll ab ratio is due to more rapid degradation ofthe reaction center complexes than light-harvesting chlorophyllab proteins of photosystem II and that the photosystem I reactioncenter disappears in parallel with the inactivation of photosynthesis.A simple method was developed to estimate the amounts of chlorophylla associated with the reaction center complexes of the two photosystemsfrom the total chlorophyll contents and chlorophyll ab ratiosof leaves. Rates of oxygen evolution, determined on the basisof chlorophyll a bound to the reaction center complexes, remainedconstant throughout the course of senescence. Thus, inactivationof photosynthesis is closely related with loss of the reactioncenter complexes during leaf senescence of rice seedlings. Theresults suggest that loss of photosynthesis is mainly causedby loss of a functional unit of photosynthesis or by a decreasein the number of whole chloroplasts. (Received April 22, 1987; Accepted August 13, 1987)     

Costs and benefits of photosynthetic light acclimation by tree seedlings in response to gap formation

Some shade leaves increase their photosynthetic capacity (P _max) when exposed to a higher irradiance. The increase in P _max is associated with an increase in chloroplast size or number. To accommodate those chloroplasts, plants need to make thick leaves in advance. We studied the cost and benefit of photosynthetic acclimation in mature leaves of a tree species, Kalopanax pictus Nakai, in a cool-temperate deciduous forest. Costs were evaluated as the additional investment in biomass required to make thick leaves, while the benefit was evaluated as an increase in photosynthetic carbon gain. We created gaps by felling canopy trees and examined the photosynthetic responses of mature leaves of the understorey seedlings. In the shade, leaves of K. pictus had vacant spaces that were not filled by chloroplasts in the mesophyll cells facing the intercellular space. When those leaves were exposed to higher irradiance after gap formation, the area of the mesophyll surface covered by chloroplasts increased by 17% and P _max by 27%. This increase in P _max led to an 11% increase in daily carbon gain, which was greater than the amount of biomass additionally invested to construct thicker leaves. We conclude that the capacity of a plant to acclimate to light (photosynthetic acclimation) would contribute to rapid growth in response to gap formation.     

Changes in the Number and Composition of Chloroplasts during Senescence of Mesophyll Cells of Attached and Detached Primary Leaves of Wheat (Triticum aestivum L.)

Changes in the number and composition of chloroplasts of mesophyll cells were followed during senescence of the primary leaf of wheat (Triticum aestivum L.). Senescence was due to the natural pattern of leaf ontogeny or was either induced by leaf detachment and incubation in darkness, or incubation of attached leaves in the dark. In each case discrete sections (1 centimeter) of the leaf, representing mesophyll cells of the basal, middle, and tip regions, were examined. For all treatments, senescence was characterized by a loss of chlorophyll and the protein ribulose 1,5-bisphosphate carboxylase (RuBPCase). Chloroplast number per mesophyll cell remained essentially constant during senescence. It was not until more than 80% of the plastid chlorophyll and RuBPCase was degraded that some reduction (22%) in chloroplast number per mesophyll cell was recorded and this was invariably in the mesophyll cells of the leaf tip. We conclude that these data are consistent with the idea that degradation occurs within the chloroplast and that all chloroplasts in a mesophyll cell senesce with a high degree of synchrony rather than each chloroplast senescing sequentially.     

Variation in Mesophyll Cell Number and Size in Wheat Leaves

The numbers of mesophyll cells in wheat leaves were determinedin a variety of wheat species differing in ploidy level andin leaves from different positions on the wheat plant. Leafsize and mesophyll cell number are linearly related in bothcases but differences were observed in mesophyll cell numberper unit leaf area with changing leaf size. Where changes incell size are caused either by nuclear ploidy or leaf position,differences in mesophyll cell number per unit leaf are negativelycorrelated with mesophyll cell plan area. The decrease in cellsize with increasing leaf position also results in a greaternumber of chloroplasts per unit leaf area. These results arediscussed in relation to anatomical variation of the wheat leaf. Mesophyll cell, cell numbers, leaf size, Triticum     

Characteristics of leaf structure and photosynthetic apparatus within the crown of systematically shadedQuercus petraea andNothofagus procera seedlings

Four-year-old seedlings ofQuercus petraea (Matt.) Liebl. andNothofagus procera (Poepp. et Endl.) Querst were grown outdoors in pots while subjected to full, medium and low irradiances. Shading and decrease in height of leaf attachment generally increased specific leaf area, the diameters of chloroplasts and of palisade and spongy mesophyll cells, but decreased leaf thickness, number of palisade cell layers, length of palisade and spongy mesophyll cells, number of chloroplasts per mesophyll cell and epidermal cell and cuticle thickness, stomata and hair densities per unit leaf area, hair length, maximum hair breath and cell wall thickness in the two species. However, inN. procera grown under full irradiance, leaves at the upper and middle positions had hairs on both upper and lower epidermes, whereas those in other treatments and all leaves in all treatments inQ. petraea, had theirs only on the upper epidermis.     

Accumulation of pathogenesis-related proteins in barley leaf intercellular spaces during leaf senescence

Accumulation of the pathogenesis-related (PR) proteins localised in intercellular spaces of barley primary leaves, chlorophyll content, structure of chloroplasts, and photosynthesis were examined during natural and in vitro induced leaf senescence (cultivation of whole plants in the dark or detached leaves under nutrient deficiency). Some of PR proteins accumulated during natural senescence, but their accumulation pattern was different from those of pathogen-induced as well as during in vitro-induced senescence, which indicate different molecular bases of these processes. Photosynthetic rate and chlorophyll content indicate that natural senescence of barley primary leaves began from 15th day after sowing. In 35-d-old first leaves, the chloroplasts showed typical characteristics of senescence as significant decrease of size, greater grana, and prominent plastoglobuli. The chloroplasts contained more grana under in vitro induced senescence and they had reduced length in the dark. Correspondingly, accumulation of PR proteins was detectable on about the 15th day but the content of some PR proteins increased in later stages of senescence. This revised version was published online in July 2006 with corrections to the Cover Date.     

ULTRASTRUCTURAL CHANGES OF CHLOROPLASTS IN ATTACHED AND DETACHED,AGING PRIMARY WHEAT LEAVES

Degradation of chloroplasts is shown in mesophyll cells of primary leaves of wheat. The sequence of ultrastructural changes in chloroplasts of naturally senescing leaves is compared with that of detached, aging leaves. In chloroplasts of naturally senescing leaves, the first indications of aging are the appearance of osmiophilic globuli and reorientation of the thylakoidal system. The membranes of the grana and intergrana lamellae then become distended and later dissociate into distinct vesicles. Concurrent with these membrane changes, osmiophilic globuli increase in size and number, and the stroma breaks down. Finally, the chloroplast envelope ruptures and plastid contents disperse throughout the cell's interior. In chloroplasts of mesophyll cells in detached, aging leaves, initial changes also include appearance of osmiophilic globuli, but later stages of chloroplast degradation are different. The chloroplast envelope ruptures before the lamellae break down. Swelling of grana and intergrana lamellae is not pronounced and, additionally, the thylakoidal system degenerates without forming vesicles or numerous osmiophilic globuli. These differences in the sequence of chloroplast degradation indicate that naturally senescing leaves rather than detached, aging leaves should be used in studies of chloroplast senescence.     

Response of senescing rice leaves to flooding stress

Flooding stress (FS) induced changes in pigment and protein contents and in photochemical efficiency of thylakoid membranes of chloroplasts were investigated during senescence of primary leaves of rice seedlings. Leaf senescence was accompanied by loss in 2,6-dichlorophenolindophenol (DCPIP) photoreduction, rate of oxygen evolution, quantum yield of photosystem 2 with an increase in MDA accumulation, and non-photochemical quenching (NPQ) of chlorophyll fluorescence. These changes were further aggravated when the leaves during this period experienced FS. The increase in NPQ value under stress may indicate photosynthetic adaptation to FS.     

Cellular compartmentation of ammonium assimilation in rice and barley

This review describes immunolocalization studies of the tissue and cellular location of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (Fd GOGAT; EC 1.4.7.1 and NADH-GOGAT; EC 1.4.1.14) proteins in roots and leaves of rice (Oryza sativa L.) and barley (Hordeum vulgare L.). In rice, cytosolic GS (GS1) protein was distributed homogeneously through all cells of the root. NADH GOGAT protein was strongly induced and its cellular location altered by ammonium treatment, becoming concentrated within the epidermal and exodermal cells. Fd GOGAT protein location changed with root development, from a widespread distribution in young cells to becoming concentrated within the central cylinder as cells matured. Plastid GS protein was barely detectable in rice roots, but was the major isoform in leaves, being present in the mesophyll and parenchyma sheath cells. GS1 was specific to the vascular bundle, as was NADH GOGAT, whereas Fd GOGAT was primarily found in mesophyll cells. In barley roots, GS1 protein was found in the cortical and vascular parenchyma and its concentration was highest in N-deficient seedlings. Plastid GS protein was detected in both cortical and vascular cells, where different plastid forms, containing different concentrations of GS protein, were identified. In barley leaves, GS2 protein was detected in the mesophyll chloroplasts and GS1 was found in the mesophyll and vascular cells. N nutrition strongly influenced this distribution, with a marked increase in GS1 concentration in the vascular cells in response to nitrate and ammonium, and an increase in mesophyll GS2 concentration in nitrate-grown seedlings. Fd GOGAT protein was found in both the mesophyll and vascular plastids. These localization studies show that the GS/GOGAT cycle is highly compartmentalized at both the subcellular and cellular levels. Reasons for this compartmentation, and the roles of each isoform, are discussed.     

Dismantling of Arabidopsis thaliana mesophyll cell chloroplasts during natural leaf senescence

One of the earliest events in the process of leaf senescence is dismantling of chloroplasts. Mesophyll cell chloroplasts from rosette leaves were studied in Arabidopsis thaliana undergoing natural senescence. The number of chloroplasts decreased by only 17% in fully yellow leaves, and chloroplasts were found to undergo progressive photosynthetic and ultrastructural changes as senescence proceeded. In ultrastructural studies, an intact tonoplast could not be visualized, thus, a 35S-GFP::δ-TIP line with a GFP-labeled tonoplast was used to demonstrate that chloroplasts remain outside of the tonoplast even at late stages of senescence. Chloroplast DNA was measured by real-time PCR at four different chloroplast loci, and a fourfold decrease in chloroplast DNA per chloroplast was noted in yellow senescent leaves when compared to green leaves from plants of the same age. Although chloroplast DNA did decrease, the chloroplast/nuclear gene copy ratio was still 31:1 in yellow leaves. Interestingly, mRNA levels for the four loci differed: psbA and ndhB mRNAs remained abundant late into senescence, while rpoC1 and rbcL mRNAs decreased in parallel to chloroplast DNA. Together, these data demonstrate that, during senescence, chloroplasts remain outside of the vacuole as distinct organelles while the thylakoid membranes are dismantled internally. As thylakoids were dismantled, Rubisco large subunit, Lhcb1, and chloroplast DNA levels declined, but variable levels of mRNA persisted.     

Observation of Apparently Unchanging Mesophyll Cell Diameters Throughout Leaf Ontogeny in Woody Species

The expansion of plant leaves usually lasts 3–6 weeks and it is widely believed that most cell types (epidermal and mesophyll) continue to expand in unison over a similar time period. The evidence supporting this account was derived from studies of herb leaves. We observed in woody species, however, that the diameter of mesophyll cells (spongy and palisade) changed little during leaf expansion from about 5 to 100 % maximum size. To keep pace with epidermal cell enlargement and leaf area expansion, mesophyll cells divided but palisade cell length expanded as leaves grew thicker. The prolonged division of mesophyll and apparently unchanging mesophyll cell diameters constitute a novel pattern of leaf cell development, different from that previously described for herbs. Possible mechanisms that attribute the varied expansion direction and speed to the different cellulose distributions in woody and herbaceous species are suggested. This finding could contribute to an enhanced understanding of the overall mechanism of leaf development.     

A Method for Calculating the Volume and Surface Area in Rice Mesophyll Cells

A method was developed for determining the surface area and volume of rice mesophyll cells of elaborate configuration. The method was employed to calculate these indices in several types of rice mesophyll cells found in seventy samples (53 species) of diverse origin coming from Japan, China, Korea, India, Nepal, Australia, France, Italy, Uzbekistan, Afghanistan, and Krasnodar and Primorskii regions. The cultivars of diverse geographic origin varied in cell shape and size due to the number, size, and arrangement of chloroplasts. When the volumes and surface areas of leaf mesophyll cells were compared using the method reported herein and a simple empirical model of the cell as a single ellipsoid, the two methods produced relatively similar data for cell volume; however, the surface area calculated by the former method was about two times larger than in the latter case. The method described in this paper allows for accurate calculations of the volume and surface area of rice mesophyll cells when data are available on the cell shape and linear dimensions and the number of chloroplasts per cell.     

Structure of the Photosynthetic Apparatus in Leaves of Freshwater Hydrophytes: 2. Quantitative Characterization of Leaf Mesophyll and the Functional Activity of Leaves with Different Degrees of Submersion

The structure of leaf photosynthetic elements was investigated on 42 boreal plant species characterized by different degrees of submergence (helophytes, neustophytes, and hydatophytes). Six main types of mesophyll structures were identified. Quantitative characteristics for the mesostructure of the photosynthetic apparatus in these groups were determined, such as the size and abundance of cells and chloroplasts in the mesophyll and epidermis, the number of plastids per cell in each tissue, the total surface area of the mesophyll cells, epidermal cells, and chloroplasts per unit leaf area. Analysis showed that quantitative characteristics of the photosynthetic apparatus in hydrophytes are determined by two factors: (a) the degree of leaf submergence and (b) the type of mesophyll structure. With an increasing degree of immersion in water, the mesophyll types change in a sequence isopalisade dorsoventral homogeneous. The leaves become thinner, their weight per unit area diminishes, cells and chloroplasts become less numerous (on a per unit leaf area basis), but their dimensions become larger. Adaptation to aquatic medium is also manifested in the increasing contribution of the epidermal tissue to the overall photosynthesis: in submerged leaves, the epidermis accounts for more than 50% of the photosynthetic activity. The occurrence of six structural types of leaves contrasting in their characteristics was confirmed by discriminatory analysis according to the qualitative parameters of mesophyll.     

Structure of the photosynthetic apparatus of the bean Vicia faba L. when treated with 6-benzylaminopurine

The level of endogenous cytokinins changed with growth and development of faba bean (Vicia faba L.) leaves. Typical of juvenile leaves, amounting to 25% of the final leaf size (Smax) was a low content of these plant hormones. The level of cytokinins increased in growing leaves (50% of Smax) and decreased in the leaves that stopped growing. The content of cytokinins in senescent leaves dropped considerably. Exogenous treatment with 6-benzylaminopurine (BAP) had no effect on the structure of terminal phloem; however, it stimulated elongation of mesophyll cells; increases in the area and thickness of leaf blade, amount of photosynthetic pigments, and assimilation potential; and delayed senescence of leaves and defoliation, thereby increasing biomass of the aboveground plant part. It was inferred that BAP had a potential for induction of photosynthetic apparatus development and increase in the yield of faba bean green mass.