Chilling-induced ultrastructural changes to mesophyll cells of Arabidopsis grown under short days are almost completely reversible by plant re-warming

Exposure of plants to chilling (low temperatures above freezing) limits growth and development in all environments outside the lowest latitudes. Cell ultrastructure and morphometric studies may allow associations to be made between chilling-induced changes at the ultrastructural level, molecular events and their physiological consequences. We examined changes in the shape, size and membrane organization of the organelles of mesophyll cells in Arabidopsis thaliana (Col 0), a cold-resistant species, after subjecting 6-week-old plants grown at normal growth temperatures to chilling (2.5–4°C; 14-h dark/10-h light cycle) for 6, 24 and 72 h and after a re-warming period of 50 h. No ultrastructural differences were seen in the first 6 h of chilling but after 24 h we observed swollen and rounded chloroplasts with larger starch grains and dilated thylakoids compared to control plants. By 72 h, chilling had resulted in a large accumulation of starch in chloroplasts, an apparent crowding of the cytosol and a lower abundance of peripheral reticulum than in the controls. The average area per chloroplast in cell sections increased after 72-h chilling while the number of chloroplasts remained the same. Ring-shaped and other morphologically aberrant mitochondria were present in significantly higher abundance in plants given 72 h chilling than in the controls. Plant re-warming for 50 h reduced chloroplast size to those of the controls and returned mitochondria to standard morphology, but peripheral reticulum remained less abundant than in plants never given a cold treatment. The near full return to normal ultrastructure upon plant re-warming indicates that the morphological changes may be part of acclimation to cold.     

Effects of Temperature Acclimation Pretreatment on the Ultrastructure of Mesophyll Cells in Young Grape Plants （Vitis vinifera L. cv. Jingxiu） Under Cross-Temperature Stresses

Leaves from annual young grape plants （Vitis vinifera L. cv. Jingxiu） were used as experimental materials. The ultrastructural characteristics of mesophyll cells in chilling-treated plants after heat acclimation （HA） and in heat-treated plants after cold acclimation （CA） were observed and compared using transmission electron microscopy. The results showed that slight injury appeared in the ultrastructure of mesophyll cells after either HA （38℃ for 10 h） or CA （8℃ for 2.5 d）, but the tolerance to subsequent extreme temperature stress was remarkably improved by HA or CA pretreatment. The increases in membrane permeability and malondialdehyde concentration under chilling （0℃） or heat （45℃） stress were markedly inhibited by HA or CA pretreatment. The mesophyll cells of plants not pretreated with HA were markedly damaged following chilling stress. The chloroplasts appeared irregular in shape, the arrangement of the stroma lamellae was disordered, and no starch granules were present. The cristae of the mitochondria were disrupted and became empty. The nucleus became irregular in shape and the nuclear membrane was digested. In contrast, the mesophyll cells of HA-pretreated plants maintained an intact ultrastructure under chilling stress. The mesophyll cells of control plants were also severely damaged under heat stress. The chloroplast became round in shape, the stroma lamellae became swollen, and the contents of vacuoles formed clumps. In the case of mitochondria of control plants subjected to heat stress, the outer envelope was digested and the cristae were disrupted and became many small vesicles. Compared with cellular organelles in control plants, those in CA plant cells always maintained an integrated state during whole heat stress, except for the chloroplasts, which became round in shape after 10 h heat stress. From these data, we suggest that the stability of mesophyll cells under chilling stress can be increased by HA pretreatment. Similarly, CA pretreatment can protect chloroplasts, mitochondria, and the nucleus against subsequent heat stress; thus, the thermoresistance of grape seedlings was improved. The results obtained in the present study are the first, to our knowledge, to offered cytological evidence of cross-adaptation to temperature stresses in grape plants.     

A comparative study of low-temperature-induced ultrastructural alterations of three species with differing chilling sensitivities

Abstract. The effects of light and water stress upon chilling injury of chloroplasts have been assessed by electron microscopy in seedlings of three species known to differ in their chilling susceptibility. Chilling injury to chloroplasts was first manifested by distortion and swelling of thylakoids, reduction in starch granule size, and the formation of small vesicles of the envelope, called the peripheral reticulum. More prolonged treatment produced accumulations of lipid droplets, increased staining of the stroma, disintegration of the envelope, and mixing with cytoplasmic contents. Cotton, a notably chilling-sensitive plant, and bush bean, a somewhat less sensitive plant, showed damage within 6 h when exposed to both light and water stress at chilling temperatures (5°C). Even collard, a chilling-resistant species, exhibited signs of chilling injury to chloroplasts after 6 h when exposed to both light and water stress but the plastids remained intact throughout the 48 h of treatment. Comparable chilling injury does not occur in cotton until around 72 h if the plants are exposed to water stress or light separately. Bush bean was affected less by separate treatments of light and water stress. The least chilling injury occurred in all three species when they were kept in the dark at a high humidity.     

Plants under Climatic Stress: II. Low Temperature, High Light Effects on Chloroplast Ultrastructure

Mesophyll chloroplasts of the C₄-pathway grasses Sorghum and Paspalum and of the C₃-pathway legume soybean undergo ultrastructural changes under moderate light intensities (170 w·m⁻², 400-700 nanometers) at a tme when photosynthesis is much reduced by low temperature (10 C). The pattern of ultrastructural change was similar in these species, despite some differences in the initial sites of low temperature action on photosynthesis and differences in their mechanisms of CO₂ fixation. Starch grains in the chloroplasts rapidly reduce in size when chilling stress is applied. At or before the time starch grains completely disappear the membranes of the individual stromal thylakoids close together, reducing the intraspace between them while the chloroplast as a whole begins to swell. Extensive granal stacking appears to hold the thylakoids in position for some time, causing initial swelling to occur in the zone of the peripheral reticulum, when present. At more advanced stages of swelling the thylakoid system unravels while the thylakoid intraspaces dilate markedly. Initial thylakoid intraspace contraction is tentatively ascribed to an increase in the transmembrane hydrogen ion gradient causing movement of cations and undissociated organic acids from the thylakoid intraspace to the stroma. Chloroplast swelling may be caused by a hold-up of some osmotically active photosynthetic product in the chloroplast stroma. After granal unraveling and redilation of the thylakoid intraspaces, chloroplasts appear similar to those isolated in low salt hypotonic media. At the initial stages of stress-induced ultrastructural change, a marked gradient in degree of chloroplast swelling is seen within and between cells, being most pronounced near the surface of the leaf directly exposed to light.     

Low-temperature induced changes in the ultrastructure of maize mesophyll chloroplasts strongly depend on the chilling pattern/intensity and considerably differ among inbred and hybrid genotypes

The ultrastructure and dimensions of chloroplasts in leaf mesophyll cells were quantitatively examined in three parental inbred lines of maize (Zea mays L.) and their four hybrids subjected to two types of four-week low-temperature (LT) treatment: the abrupt onset of chilling temperatures (“severe chilling”, SC) and the gradual, more moderate one (“moderate chilling”, MC). The relationship between the response of individual genotypes to one or the other type of chilling was analyzed as well as the possibility to predict the behaviour of chloroplasts in hybrids from that of their parents. Although selected parameters of chloroplast ultrastructure (e.g. volume densities of granal and intergranal thylakoids, plastoglobuli, and peripheral reticulum) and dimensions changed due to the exposure of maize plants to LT, no general pattern of such changes was found for this species due to the observed intraspecific variability. The response of some genotype to SC could not be predicted from its behaviour under MC (and vice versa) and no clear rules could be applied for the inheritance of chloroplast response to chilling in the general sense. Thus, great caution should be always taken when interpreting the results of studies aimed at the dissection of chloroplast ultrastructure as affected by LT, particularly in case such studies are made with one genotype or under one type of chilling only.     

Ultrastructural features of chilling injury: injured cells and the early events during chilling of suspension-cultured mung bean cells

The ultrastructure of cells of mung bean (Vigna radiata L. var. Wilczek) in suspension culture was studied during chilling. During such treatment, three kinds of injured cells were observed: swollen cells, cells with broken vacuolar membranes, and cells with shrunken plasma membranes. Swelling was observed from the early stages of chilling, and in most cells during chilling. The other two types of cells were observed at the late stages of chilling. At the early stage of chilling, whorls of rough endoplasmic reticulum that surrounded clear regions of cytoplasm were observed. At the same time, markedly rough vacuolar membranes, plastids and mitochondria with vacuoles, enlargement of Golgi vesicles, and dilation of the ER were seen. These changes preceded the swelling of cells. These ultrastructural features of chilling injury are discussed in terms of biochemical observations. The disruption of the vacuolar membrane and the shrinking of the plasma membrane are discussed in terms of destruction of the cytoskeleton.     

CHLOROPLAST SPECIALIZATION IN DICOTYLEDONS POSSESSING THE C4-DICARBOXYLIC ACID PATHWAY OF PHOTOSYNTHETIC CO2 FIXATION

The C₄-dicarboxylic-acid pathway of photosynthetic CO₂ fixation found in tropical grasses has recently been demonstrated in members of the Amaranthaceae and Chenopodiaceae. In the tropical grasses this CO₂-fixation pathway is correlated with specialized leaf anatomy and chloroplast structure. This investigation was undertaken to determine if leaf cells of some representatives of these other families had structural features similar to those of tropical grasses. The leaf anatomy of Amaranthus edulis and a variety of Atriplex species is very similar and it resembles that of grasses such as sugar cane. Prominent bundle sheaths are surrounded by a layer of palisade cells. Bundle-sheath cells of Am. edulis have large chloroplasts containing much starch, but the chloroplasts have grana. The palisade cells have much smaller chloroplasts containing very little starch. The bundle-sheath cell chloroplasts of At. lentiformis have grana, their profiles tend to be ovoid, and they contain abundant starch grains. The palisade cell chloroplasts contain little starch and their profiles are discoid. The bundle-sheath cells of both species contain mitochondria which are much larger than those in the palisade cells. The chloroplasts in both types of cells in both species have a highly developed peripheral reticulum. This reticulum is composed of anastomosing tubules which are contiguous with the inner plastid membrane. The leaf anatomy and cell ultrastructure of these dicots are similar to those of the tropical grasses possessing this new photosynthetic carbon-fixation pathway. These morphological features are interpreted as adaptations for the rapid transport of precursors and end products of photosynthesis. A hypothesis is presented stating that the unique morphological and biochemical characters of these plants represent adaptations for efficient and rapid carbon fixation in environments where water stress frequently limits photosynthesis.     

Na2CO3胁迫对星星草叶肉细胞超微结构的影响

利用透射电镜技术对Na₂CO₃胁迫下星星草叶肉细胞超微结构进行了观察。结果表明：未胁迫的叶肉细胞排列疏松,各种细胞器结构完整,叶绿体含少量淀粉粒和脂质球。轻度盐胁迫（2g/L,4g/L Na₂CO₃）对叶肉细胞超微结构影响较小。中度盐胁迫（6g/L,8g/L Na₂CO₃）引起叶肉细胞超微结构的变化,叶绿体类囊体肿胀,基粒紊乱,不含淀粉粒,脂质球数量增加,叶绿体由原来的梭形或椭球形变成圆球状;部分线粒体嵴消失,出现晶体结构;中央大液泡破裂;核逐渐降解。高度盐胁迫（10g/L,12g/L Na₂CO₃）下,叶绿体片层结构消失,脂质球数量增加,体积变大,被大量的膜片层所包围,叶绿体内、外膜消失,叶肉细胞中看不到叶绿体的存在;膜片层包围线粒体;叶肉细胞中可见大量的泡状结构和膜片层,叶肉细胞死亡。上述结果表明,细胞器特别是叶绿体膜结构的破坏与盐胁迫叶肉细胞最终死亡密切相关     

MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox‐balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states. We demonstrate that MSL1 localises to the inner mitochondrial membrane. When expressed in Escherichia coli, MSL1 forms a stretch‐activated ion channel with a slight preference for anions and provides protection against hypo‐osmotic shock. In contrast, loss of MSL1 in Arabidopsis did not prevent swelling of isolated mitochondria in hypo‐osmotic conditions. Instead, our data suggest that ion transport by MSL1 leads to dissipation of mitochondrial membrane potential when it becomes too high. The importance of MSL1 function was demonstrated by the observation of a higher oxidation state of the mitochondrial glutathione pool in msl1‐1 mutants under moderate heat‐ and heavy‐metal‐stress. Furthermore, we show that MSL1 function is not directly implicated in mitochondrial membrane potential pulsing, but is complementary and appears to be important under similar conditions.     

Na2CO3胁迫对星星草叶肉细胞超微结构的影响

利用透射电镜技术对Na2CO3胁迫下星星草叶肉细胞超微结构进行了观察。结果表明：未胁迫的叶肉细胞排列疏松,各种细胞器结构完整,叶绿体含少量淀粉粒和脂质球。轻度盐胁迫（2g/L,4g／LNa2CO3）对叶肉细胞超微结构影响较小。中度盐胁迫（6g／L,8g／L Na2CO3）引起叶肉细胞超微结构的变化,叶绿体类囊体肿胀,基粒紊乱,不含淀粉粒,脂质球数量增加,叶绿体由原来的梭形或椭球形变成圆球状;部分线粒体嵴消失,出现晶体结构;中央大液泡破裂;核逐渐降解。高度盐胁迫（10g／L,12g／LNa2CO3）下,叶绿体片层结构消失,脂质球数量增加,体积变大,被大量的膜片层所包围,叶绿体内、外膜消失,叶肉细胞中看不到叶绿体的存在;膜片层包围线粒体;叶肉细胞中可见大量的泡状结构和膜片层,叶肉细胞死亡。上述结果表明,细胞器特别是叶绿体膜结构的破坏与盐胁迫叶肉细胞最终死亡密切相关。