Chloroplast Shape Research Articles

Knockdown of Adenosine 5'-Triphosphate-Dependent Caseinolytic Protease Proteolytic Subunit 6 Enhances Aluminum Tolerance in Peanut Plants (Arachis hypogea L.).

Aluminum (Al3+) toxicity in acidic soils reduces root growth and can lead to a considerable reduction in peanut plants (Arachis hypogea L.). The caseinolytic protease (Clp) system plays the key role in abiotic stress response. However, it is still unknown whether it is involved in peanut response to Al3+ stress. The results from this study showed that Adenosine 5'-triphosphate (ATP)-dependent caseinolytic protease proteolytic subunit 6 (AhClpP6) in peanut plants was involved in the Al3 stress response through its effects on leaf photosynthesis. The AhClpP6 expression levels in the leaf and stem significantly increased with the Al3+ treatment times. Knockdown AhClpP6 peanut lines accumulated significantly more Al3+ when exposed to Al3+ stress, which reduced leaf photosynthesis. Furthermore, in response to Al3+ treatment, knockdown of AhClpP6 resulted in a flattened shape of chloroplasts, disordered and flattened thylakoid, and accumulating more starch grains than those of the wild-type (WT) peanut lines. Taken together, our results suggest that AhClpP6 regulates Al3+ tolerance by maintaining chloroplast integrity and enhancing photosynthesis.

Chloroplasts with clefts and holes: a reassessment of the chloroplast shape using 3D FE-SEM cellular reconstruction of two species of Chlamydomonas.

Chloroplasts are usually considered spheroid organelles, but this is not the only shape of chloroplasts. The chloroplast of Chlamydomonas has been typically described as cup-shaped. However, in old studies, it was also modeled as a complex shape with "perforations" or windows. Here, we reconstructed the cellular architecture of Chlamydomonas reinhardtii and C. applanata using an array tomography system installed on a field emission scanning electron microscope. C. reinhardtii chloroplasts resembled a baseball glove or a cup without a side, featuring numerous large and small holes that may facilitate the transport of metabolites and proteins produced in the Golgi apparatus fitted in the holes. In a lipid-accumulating, high-light condition, the chloroplast volume increased by filling the side cleft with an entire wall. Many accumulated large lipid droplets were accommodated within the chloroplast holes, which could have been considered as "chloroplast lipid droplets." Mitochondrial meshworks surrounded the chloroplast. C. applanata chloroplasts appeared like a folded starfish or a cup with many side clefts and a few holes. There was a single mitochondrion or two that branched in a complex form. Tight contacts of various organelles were also found in C. applanata. These reconstructions illustrate the complexity of chloroplast shape, which necessitates a revised understanding of the localization of lipid droplets and the evolution of chloroplasts: The prevailing image of the spheroid chloroplasts that reminds us of the similarity between chloroplasts and cyanobacteria is no longer tenable.

Are Toxic Substances Always Toxic? Case Studies of Different Organismal Responses Based on Brackish-Water Microphytobenthic Communities from the Baltic Sea

In this manuscript, we present the results of ecotoxicological tests conducted on the Baltic microphytobenthos community consisting largely of cosmopolitan species showing the responses at the community (taxonomic composition and structure), population (abundance), and cellular (chloroplast shape) levels. The tests were carried out for three chemicals with different modes of action on photosynthetic organisms, i.e., copper (II) chloride (at concentrations of 2·10−5 g·dm3 and 2·10−3 g·dm−3), glyphosate in the form of Roundup® (concentrations ranging from 4.2·10−2 to 8.5 g·dm−3), and the ionic liquid [BMIM]Cl (at concentrations of 1.13·10−3 g·dm−3 and 1.75·10−2 g·dm−3). The results of the study indicate that the responses of microphytobenthos at each level analysed are highly variable and difficult to predict a priori based on experiments performed on single strains. In addition, it was shown that microphytobenthic communities are relatively resistant to the substances tested, which is due to taxonomic richness and the resulting potential for substitution of sensitive taxa by more resistant taxa. The results obtained in the tests also indicate that the number of cells of individual taxa can remain at a similar level or increase over short periods of time despite the deformation of chloroplasts in a numerous group of cells.

Ante- and post-mortem cellular injury dynamics in hybrid poplar foliage as a function of phytotoxic O3 dose.

After reaching phytotoxic levels during the last century, tropospheric ozone (O3) pollution is likely to remain a major concern in the coming decades. Despite similar injury processes, there is astounding interspecific-and sometimes intraspecific-foliar symptom variability, which may be related to spatial and temporal variation in injury dynamics. After characterizing the dynamics of physiological responses and O3 injury in the foliage of hybrid poplar in an earlier study, here we investigated the dynamics of changes in the cell structure occurring in the mesophyll as a function of O3 treatment, time, phytotoxic O3 dose (POD0), leaf developmental stage, and mesophyll layer. While the number of Hypersensitive Response-like (HR-like) lesions increased with higher O3 concentrations and POD0, especially in older leaves, most structural HR-like markers developed after cell death, independent of the experimental factors. The pace of degenerative Accelerated Cell Senescence (ACS) responses depended closely on the O3 concentration and POD0, in interaction with leaf age. Changes in total chlorophyll content, plastoglobuli and chloroplast shape pointed to thylakoid membranes in chloroplasts as being especially sensitive to O3 stress. Hence, our study demonstrates that early HR-like markers can provide reasonably specific, sensitive and reliable quantitative structural estimates of O3 stress for e.g. risk assessment studies, especially if they are associated with degenerative and thylakoid-related injury in chloroplasts from mesophyll.

Description of two new species Chattonella tenuiplastida sp. nov. and Chattonella malayana sp. nov. (Raphidophyceae) from South China Sea, with a report of wild fish mortality

Fisheries damage caused by Chattonella red tide has been recorded in Southeast Asia. Molecular studies have clarified the presence of two species, Chattonella marina complex and Chattonella subsalsa in the region, unlike East Asia that had only C. marina complex. To elucidate the phylogeography of Chattonella in Asia, further phylogenetic and morphological examinations were carried out with 33 additional culture strains, including the strains isolated during a bloom of Chattonella sp. (up to 142 cells mL−1) that was associated with a wild fish mortality along the northeastern coast of Peninsular Malaysia in 2016, and those from Yellow Sea, where the Chattonella genotypes have not been determined. LSU rDNA and ITS2 trees showed five intrageneric clades in the genus Chattonella, which were clades I and II (C. subsalsa), clade III (C. marina complex) and two new clades, namely clade IV from Thailand and Malaysia, and clade V from Peninsular Malaysia. The positions of the two new clades were different in LSU rDNA and ITS2 trees. LSU rDNA divergences of clades IV and V from the other clades were ≥ 4.01% and ≥ 5.70%, while their ITS2 divergences were ≥ 7.44% and ≥ 16.43%, respectively. Three and five compensatory base changes (CBCs) were observed in the clades IV and V, respectively, when compared to each of their closest clade. Cells from clades IV and V showed similar morphology to C. marina complex and C. subsalsa clade II, including the presence of button-like granules on cell surface and oboe-shaped mucocysts. However, cell size, the number and shape of chloroplasts in Chattonella clades IV and V, and the non-stacked thylakoids penetrated the pyrenoid in C. subsalsa clade II, were distinctive. Based on the diagnostic chloroplast shape, we proposed the designation of clades IV and V to two new species, Chattonella tenuiplastida sp. nov. and Chattonella malayana sp. nov.

Effects of ZnO nanoparticles and its bulk form on growth, antioxidant defense system and expression of oxidative stress related genes in Hordeum vulgare L

A comparative analysis of physio-biochemical indices and transcriptional activity of oxidative stress genes in barley (Hordeum vulgare L.) seedlings after 7-days exposure to bulk- and nano-ZnO (300 and 2000 mg/L) was carried out. A dose-dependent reduction in the length and weight of roots and shoots, as well as a significant accumulation of Zn in plant parts, was shown. Alterations in the shape and size of organelles, cytoplasmic vacuolization, and chloroplast and mitochondrial disorganization were also revealed. These processes are particularly pronounced when H. vulgare is exposed to the higher concentrations of nano-ZnO. The study of the antioxidant defense system revealed mainly an increase in the level of reduced glutathione and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione S-transferase (GST). The increases in activity, by 4-fold and 3-fold, was found for glutathione transferase in the roots when exposed to 2000 mg/L bulk- and nano-ZnO, respectively. The study of transcriptional activity demonstrated that in the roots under the influence of bulk- and nano-ZnO, along with Mn-SOD, Fe-SOD is highly expressed, mainly associated with the protection of chloroplasts. Analysis of the Cat 1 and Cat 2 gene expression showed that the main contribution to the increase in catalase activity in treated H. vulgare is made by the CAT-1 isozyme. Generally, in response to the impact of the studied ZnO forms, the antioxidant defense system is activated in H. vulgare, which effectively prevents the progression of oxidative stress in early stages of plant ontogenesis. Nevertheless, with constant exposure to bulk- and nano-ZnO at high concentrations, such activation leads to a depletion of the plant's energy resources, which negatively affects its growth and development. The results obtained could be useful in predicting the risks associated with the further transfer of nano-ZnO to the environment.

Cytological, Histological and Molecular Characterization of Two Isolates of Cucumber Mosaic Virus (CMV) in Egypt

Two isolates of Cucumber mosaic virus (CMV), CMV-wild tobacco (from Alexandria governorate) and CMV-cucumber (from Kafr El-Sheikh governorate) were investigated in this study. Cytological studies on epidermal strips of Nicotiana glutinosa leaves separately infected with each isolate revealed the presence of viral crystalline inclusion bodies within the infected cells. Electron microscopy of ultrathin sections of CMV infected N. glutinosa leaves showed significant alterations in the shape and internal structure of chloroplasts. The cell wall had serrated edges in infected cells but was more severe in cells infected with CMV-wild tobacco isolate compared to those infected with CMV-cucumber isolate. CMV-cucumber isolate was partially purified from systemically infected leaves of N. glutinosa. The ratio A260/ 280 was 1.0 and the concentration of the virus in the preparation was estimated using an extinction coefficient of E260nm0.1%, 1cm = 5. Yield of purified virus was about 2.8 mg/100 g fresh weight of infected N. glutinosa leaves. Electron microscopy of the purified preparation of CMV showed the presence of numerous spherical particles with a mean particle diameter of 28 nm. Amplified real-time reverse transcription-polymerase chain reaction (qRT-PCR) product of coat protein gene of each isolate was purified and sequenced. Sequences of both isolates had been submitted to GenBank Database and ware assigned accession number (LT669766) for CMV-cucumber isolate and (LT706517) for CMV-wild tobacco isolate. The sequences were edited using Chromas Pro. Version 1.34 software and compared with previously subgrouping of 27 isolates of the virus retrieved from the GenBank database. Both CMV-wild tobacco and CMV-cucumber isolates were closely related to the isolate with the accession number AJ585086 with a similarity of 97.07% and 98.54%, respectively, suggesting that the two isolates belong to subgroup II. According to the available literature, this is the first report in Egypt where CMV isolates belonging to subgroup II have been obtained

Ecotoxicological Studies on the Effect of Roundup® (Glyphosate Formulation) on Marine Benthic Microalgae.

Glyphosate is a very effective herbicide and the main active ingredient in Roundup®—the most extensively used herbicide in the world. Since glyphosate is highly water soluble it reaches water bodies easily in surface water runoff. This prompted us to undertake an experiment to evaluate the effects of glyphosate in Roundup® on natural communities of marine microphytobenthos. Microphytobenthos communities were obtained from the environment, and after transporting them to the laboratory and acclimatizing them, they were tested under controlled conditions. Changes in microphytobenthos composition and structure and the deteriorating condition of the cells of community-forming organisms (assessed by analyzing changes in chloroplast shape) were used to assess the impact of Roundup® on endpoints. The tests indicated that microphytobenthic communities were relatively resistant to herbicide. The species richness of the communities probably enabled them to rebuild effectively. Sensitive species were replaced by those more tolerant of glyphosate. Only at the highest glyphosate concentration (8.5 g·dm−3) tested was a strong negative effect noted that limited community abundance and eliminated some of the organisms. The dominant diatoms in the communities were replaced by intensively developing cyanobacteria, which ultimately comprised nearly 60% of all the cells observed in the communities.

Protective roles of salicylic acid in maintaining integrity and functions of photosynthetic photosystems for alfalfa (Medicago sativa L.) tolerance to aluminum toxicity

Aluminum (Al) can be detrimental to plant growth in areas with Al contamination. The objective of this study was to determine whether salicylic acid (SA) can improve plant tolerance to Al stress by mitigating Al toxicity for chloroplasts and photosynthetic systems in alfalfa (Medicago sativa L.). Plants were treated with Al (100 μM) for 3 d in a hydroponic system. The content of Al increased in leaves treated with Al, resulting in damage and deformation of chloroplasts. In Al-damaged leaves, chloroplast envelopes and starch granules disappeared; the lamellae and stroma lamella were loosely arranged and indistinguishable, and the number of grana was reduced; a large number of small plastoglobules appeared. Foliar spraying of 15 μM SA reduced Al content in roots and leaves and alleviated Al damages in chloroplasts. With 15 μM SA treatments, the chloroplast shape returned to a flat ellipsoid, thylakoids were arranged closely and regularly, chloroplasts had intact starch granules, and small plastoglobules disappeared. SA-treated plants had significantly higher aboveground biomass than the untreated control exposed to Al stress. Photosynthetic index and gene expression analyses demonstrated that SA could alleviate adverse effects of Al toxicity by increasing light capture efficiency, promoting electron transport in the photosynthetic electron transport chain and thylakoid lumen deacidification, and promoting synthesis of aenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH). SA played protective roles in maintaining integrity and functions of photosystems in photosynthesis for plant tolerance to Al stress.

Increased ratio of galactolipid MGDG:DGDG induces jasmonic acid overproduction and changes chloroplast shape.

Summary Galactolipids monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) constitute c. 50% and c. 30% of chloroplast membrane lipids, respectively. They are important for photosynthesis and stress tolerance. Mutations in DGD1, the major DGDG‐synthesizing enzyme, severely reduce DGDG content and induce jasmonic acid (JA) overproduction, resulting in stunted growth. However, how DGDG reduction leads to JA overproduction is unknown.We introduced an inducible microRNA (ami‐MGD1) into an Arabidopsis dgd1 mutant to reduce MGDG synthesis, thereby further diminishing galactolipid content, but partially restoring the MGDG : DGDG ratio. Galactolipid and Chl contents, expression of JA‐biosynthesis and JA‐responsive genes, photosystem II (PSII) maximum quantum efficiency, and chloroplast shape were investigated.Expression of JA‐biosynthesis and JA‐responsive genes were reduced in amiR‐MGD1‐transformed dgd1 plants. Stunted growth caused by JA overproduction was also partially rescued, but Chl reduction and PSII impairment remained similar to the original dgd1 mutant. Altered chloroplast shape, which is another defect observed in dgd1 but is not caused by JA overproduction, was also partially rescued.Our results reveal that an increased MGDG : DGDG ratio is the primary cause of JA overproduction. The ratio is also important for determining chloroplast shapes, whereas reduced Chl and photosynthesis are most likely a direct consequence of insufficient DGDG.

Impact of salicylic acid on the growth and physiological activities of parsley plants under lead toxicity.

Impact of spraying 50µM salicylic acid (SA), lead nitrate soil treatments [1 and 2mM Pb (NO3)2] and their combinations on parsley leaves (Petroselinum crispum L.) for 3 weeks was studied to evaluate leaf symptoms, photosynthetic pigments, anthocyanin, ultrastructure, malondialdehyde (MDA), soluble proteins, phenolic compounds, and guaiacol peroxidase activity (GPOX). Under Pb effect, parsley leaves showed chlorosis and decline in the content of photosynthetic pigments chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoid (Car) with increasing Pb treatments compared to the control. SA spraying helped to reduce chlorosis and increase photosynthetic pigments of Pb-treated plants compared to that of Pb treatment alone. Leaf anthocyanin content of SA-sprayed plants significantly increased compared to the control. On contrast, the anthocyanin content of Pb-treated plants with or without SA treatment decreased compared to the control. Parsley leaf chloroplasts were characterized by many and large starch grains. Deformations of chloroplast shape, increasing formation of plastoglobules and degeneration of chloroplast grana thylakoids were observed in Pb-treated plants. MDA and total phenolic compound contents increased in Pb-treated plants compared to the control. In contrast, soluble protein content decreased in Pb-treated plants. The decrease in leaf photosynthetic pigments and increase MDA contents was Pb-concentration dependent. The results as indicated by increasing lipid peroxidation confirmed Pb treatments generated reactive oxygen species (ROS) which caused oxidative stress. In contrast, SA application declined the extent of detrimental and harmful influence of Pb toxicity as indicated by the decrease MDA content, and increase in photosynthetic pigments, anthocyanin and phenolic compound contents of parsley leaves.

Selenium modulates cadmium-induced ultrastructural and metabolic changes in cucumber seedlings.

Intensive insight into the potential mechanisms of Se-induced Cd tolerance in cucumber seedlings is essential for further improvement of vegetable crop cultivation and breeding to obtain high yields and quality in Cd-contaminated soil. To reveal the ultrastructural and metabolic differences in Se-induced Cd tolerance, we examined the ultrastructures of chloroplasts and root cells and characterised 155 differentially expressed metabolites under Cd and/or Se stress using gas chromatography-mass spectrometry (GC-MS)-based metabolomics. Exogenous Se greatly relieved Cd-caused injuries to the ultrastructures of cucumber leaves and roots; for example, the shapes of chloroplasts treated with Cd + Se improved or even began to return to normal, the nuclei of root cells began to regenerate better and the chromatin was well-distributed compared with plants treated with Cd alone. Metabolite profiling revealed several intermediates of glycolysis and the tricarboxylic acid (TCA) cycle; also, some amino acids were up-accumulated in Cd + Se-treated cucumber seedlings and down-accumulated in Cd-treated cucumber seedlings, such as pyruvic acid, galactose, lactose, glutaric acid and alanine in leaves, glucose-6-phosphate and serine in roots, and lactic acid and glycine in both leaves and roots. These metabolites may play dominant roles in developing Se-mediated Cd tolerance. Moreover, a high level of sugars and polyols, amino acids and organic acids were up-accumulated in Cd-treated plants. Meanwhile, our data suggest that high accumulation of fructose, α-ketoglutaric acid, shikimic acid, fumaric acid and succinic acid in roots is a Cd-specific response, indicating that these metabolites are vital for cucumbers to develop Cd resistance. This study extends the current understanding of the mechanisms of Se in abating Cd contamination in cucumber and demonstrates that metabolomics profiling provides a more comprehensive view of the response of plants to heavy metals.

Effects of zinc oxide nanoparticles on enzymatic and nonenzymatic antioxidant content, germination, and biochemical and ultrastructural cell characteristics of Portulaca oleracea L.

This report focuses on the application of zinc oxide nanoparticles (ZnO NPs) carrying phycomolecule ligands as a novel plant growth promoter aimed at increasing the crop productivity of purslane (<em>Portulaca oleracea</em> L.). Experiments were performed under controlled greenhouse conditions using a completely randomized design with nine replications. Purslane seeds were treated with four concentrations of ZnO NPs (0, 10, 100, and 500 mg L<sup>−1</sup>) and four concentrations of bulk ZnO (0, 10, 100, and 500 mg L<sup>−1</sup>). The ultrastructural characteristics of the leaves of the plants treated with of 500 mg L<sup>−1</sup> ZnO NPs were determined using transmission electron microscopy (TEM). The results indicated that the treatment with ZnO NPs increased the content of chlorophyll <em>a</em> and chlorophyll <em>b</em>, carotenoids, and total phenolic and flavonoid compounds significantly more than the treatment with bulk ZnO. Our findings also showed that the application of high concentrations of ZnO NPs is the most effective strategy to considerably induce the antioxidant capacity and enzymes of purslane plants. Furthermore, the seed germination percentage and sprout growth rates were significantly higher in the plants treated with 500 mg L<sup>−1</sup> of ZnO NPs (100% ±0.00), compared to the control plants (93.33% ±1.66). The TEM images revealed the concentration of ZnO NPs and cell membrane rupture, as well as a deformation in the shape of chloroplasts and a decrease in their number in the plants treated with 500 mg L<sup>−1</sup> ZnO NPs, compared to the control plants. Owing to their toxicity, high concentrations of ZnO NPs lead to oxidative stress in plants. Thus, our findings provide a new alternative strategy for increasing crop productivity, i.e., the application of ZnO NPs as a novel plant growth booster, in comparison with the bulk ZnO treatment.

Three-dimensional ultrastructural change of chloroplasts in rice mesophyll cells responding to salt stress.

Excess salinity inhibits the metabolism of various systems and induces structural changes, especially in chloroplasts. Although the chloroplast body seems to swell under salinity stress as observed by conventional transmission electron microscopy, previous studies are limited to 2-D data and lack quantitative comparisons because specimens need to be sliced into ultrathin sections. This study shows three-dimensionally the structural changes in a whole mesophyll cell responding to salinity stress by serial sectioning with a focused ion beam scanning electron microscope (FIB-SEM) and compares the differences in chloroplast structures based on reconstructed models possessing accurate numerical voxel values. Leaf blades of rice plants treated with 100 mm NaCl or without (control) for 4 d were fixed chemically and embedded in resin. The specimen blocks were sectioned and observed using the FIB-SEM, and then the sliced image stacks were reconstructed into 3-D models by image processing software. On the transverse sections of rice mesophyll cells, the chloroplasts in the control leaves appeared to be elongated meniscus lens shaped, while those in the salt-treated leaves appear to be expanded oval shaped. The 3-D models based on serial sectioning images showed that the chloroplasts in the control cells spread like sheets fitted to the shape of the cell wall and in close contact with the adjacent chloroplasts. In contrast, those in the salt-stressed cells curled up into a ball and fitted to cell protuberances without being in close contact with adjacent chloroplasts. Although the shapes of chloroplasts were clearly different between the two treatments, their volumes did not differ. The 3-D reconstructed models of whole rice mesophyll cells indicated that chloroplasts under salt stress conditions were not swollen but became spherical without increasing their volume. This is in contrast to findings of previous studies based on 2-D images.

Cell and chloroplast anatomical features are poorly estimated from 2D cross-sections.

Leaf function is intimately related to the size, shape, abundance and position of cells and chloroplasts. Anatomy has long been assessed and quantified in two dimensions with 3D structure inferred from 2D micrographs. Serial block face scanning electron microscopy (SBF-SEM) was used to reconstruct 95 cells and 1173 chloroplasts from three wheat and nine chickpea leaves (three samples each from three chickpea genotypes). Wheat chloroplast volume was underestimated by 61% in mesophyll cells and 45% in bundle sheath cells from 2D micrographs, whereas chickpea mesophyll chloroplast volume was underestimated by 60% using simple geometrical models. Models of chickpea spongy and palisade cells both under- and overestimated surface area and volume by varying degrees. These models did not adequately capture irregular shapes such as flattening of chloroplasts or lobed spongy mesophyll cells. It is concluded that simple geometrical models to estimate chloroplast and cell 3D volume and surface area from 2D micrographs are inadequate, and that SBF-SEM has strong potential to contribute to improved understanding of leaf form and function.

Chemically synthesized silver nanoparticles induced physio-chemical and chloroplast ultrastructural changes in broad bean seedlings

This study was conducted to explore the effects of priming of seven-year-old aged seeds with different concentrations of silver nanoparticles (AgNPs) on growth of broad bean (Vicia faba L.). Seeds were primed with different concentrations of AgNPs for 6 h before growing in the plastic trays. Different growth parameters like growth attributes, photosynthetic pigments, carbohydrates, antioxidant enzymes and chloroplast ultrastructure were estimated after 14 days of germination. Priming with AgNPs affected the root and shoot growth attributes as compared with control depending upon concentrations of AgNPs. In all treatments, photosynthetic pigments increased significantly above control levels, but total soluble sugars decreased in 10 and 50 ppm AgNPs and slightly increased in 100 ppm AgNPs as compared with control. Starch accumulation was apparent in all treated seedlings above that of control levels. Mesophyll cells of all treated seedlings were altered with electron dense particles than control. Priming with AgNPs affected the chloroplast structure which appeared in the form of less stacking of Greene, formation of protrusions and extensions, irregular shape of chloroplasts as compared with spindle shaped regular chloroplasts of control. In all treatments, total phenols were slightly affected as compared with control. The antioxidant enzyme activities in seedlings varied with the dose and type of antioxidants. Overall, AgNPs adversely affected the chloroplast ultrastructure, but increased growth of seedlings and starch accumulation. Further studies are required to explore the effects of AgNPs on the long-term on crop productivity of aged seeds.

Participation of actin filaments, myosin and phosphatidylinositol 3-kinase in the formation and polarisation of tetraspore germ tube of Gelidium floridanum (Rhodophyta, Florideophyceae).

This study aimed to examine the evidence of direct interaction among actin, myosin and phosphatidylinositol 3-kinase (PI3K) in the polarisation and formation of the tetraspore germ tube of Gelidium floridanum. After release, tetraspores were exposed to cytochalasin B, latrunculin B, LY294002 and BDM for a period of 6h. In control samples, formation of the germ tube occurred after the experimental period, with cellulose formation and elongated chloroplasts moving through the tube region in the presence of F-actin. In the presence of cytochalasin B, an inhibitor of F-actin, latrunculin B, an inhibitor of G-actin, and BDM, a myosin inhibitor, tetraspores showed no formation of the germ tube or cellulose. Spherical-shaped chloroplasts were observed in the central region with a few F-actin filaments in the periphery of the cytoplasm. Tetraspores treated with LY294002, a PI3K inhibitor, showed no formation of the tube at the highest concentrations. Polarisation of cytoplasmic contents did not occur, only cellulose formation. It was concluded that F-actin directs the cell wall components and contributes to the maintenance of chloroplast shape and elongation during germ tube formation. PI3K plays a fundamental role in signalling for the asymmetric polarisation of F-actin. Thus, F-actin regulates the polarisation and germination processes of tetraspores of G.floridanum.

Simultaneous chloroplast, mitochondria isolation and mitochondrial protein preparation for two-dimensional electrophoresis analysis of Ice plant leaves under well watered and water-deficit stressed treatments

This paper presents a simultaneous isolation of pure, intact chloroplasts and mitochondria from mature leaves of Ice plant (Mesembryanthemum crystallinum) and mitochondrial protein preparation for two-dimensional electrophoresis (2DE) analysis under well watered and water -deficit stressed treatments. The washed chloroplasts and mitochondria were purified with Percoll gradients prepared using a Master flex R pump. The chloroplast and mitochondrial proteins were extracted in lysis buffer containing a protease inhibitor mix supplemented with 1 μM Leupeptin and 1 μM E64, followed by precipitation with ice-cold acetone. The protein contents were determined by an EZQ protein quantitation kit. The results show that chloroplast and mitochondria isolated from Ice plant leaves via this protocol have pure and intact. The shape of chloroplast and mitochondria observed by microscopy were clear and sharp. This procedure was employed for assessing the significant differences in mitochondrial protein expression patterns from the well watered and water-deficit stressed treatment leaves collected at dawn (6 a.m.) and dusk (6 p.m.). The results showed 71 and 20 differentially abundant spots between control and CAM for 6 a.m. and 6 p.m., respectively. In addition, 32 protein spots were differentially abundant for 6 a.m. control compared with 6 p.m. control, and 45 protein spots were differentially abundant for 6 a.m. CAM compared with 6 p.m. CAM. Spots that displayed differential abundance for control compared with CAM likely included proteins involved in mitochondrial processes necessary for CAM function. Through further analysis, these proteins will be identified and characterized in the near future using mass-spectrometry-based techniques.

Alterations in photosynthetic capacity and morpho-histological features of leaf in alfalfa plants subjected to water deficit-stress in different soil types

Water deficit can greatly influence plant growth and development. A pot trial was conducted to study the impact of various levels of water deficit on alfalfa (Medicago sativa L.) plants grown in different soil types. The results elucidated that increasing drought level and/or sand proportion in the cultivation soil reduced leaf growth vigor as indicated by declined number of leaves per plant as well as less cumulative leaf biomass, area, specific area, relative water content and degree of succulence with corresponding increase in the cumulative degree of leaf sclerophylly. Leaf anatomical analysis revealed that drought increased leaf thickness and ground tissue thickness but reduced phloem, xylem and whole vascular bundle areas, as well as the number of xylem vessels and rays. In addition, transmission electron microscopic examination of leaves cleared that the shape of chloroplasts was irregular when droughted. Also, stress reduced the number of both chloroplasts per cell and starch grains per chloroplast as well as the area of individual chloroplasts and starch grains. Moreover, severe water deficit resulted in the appearance of plastoglobules within chloroplasts. Concerning pigment fractions, chlorophyll a, b and a + b as well as total pigments and photosystem II activity were all declined in water-unsatisfied plants. Meanwhile, stress enhanced the production of carotenoids and also increased the calculated chlorophyll a/b ratio. The results also showed that water deficit increased glucose, fructose, trehalose, sucrose and total soluble sugars but reduced the amount of polysaccharides and total carbohydrates in alfalfa plants.

Cytogenetic Analyses of PSL1 Mutant, a Novel Low‐Temperature‐Sensitive Purple‐Striped Leaf Color Mutant in Wheat

Low‐temperature‐sensitive leaf color mutants are valuable materials for investigating the mechanisms related to chloroplast development and chlorophyll biosynthesis under low‐temperature conditions. PSL1 is a spontaneous low‐temperature‐sensitive purple‐striped leaf mutant of wheat (Triticum aestivum L.). The progeny of PSL1 exhibited three leaf color phenotypes under low‐temperature conditions (i.e., purple and albino leaf [pur], purple‐striped and albino‐striped leaf [pst], and normal green leaf [gre]). The leaf color of pur and pst displayed normal green under high temperature conditions. The leaf color of gre remained normal green under cold or high temperatures. The critical low temperature for leaf color variations in pur and pst was ∼5°C for >35 d. The shapes of chloroplasts in the purple and albino leaf sectors were irregular in pur and pst, where the thylakoid lamellar was undeveloped and no grana formed. The chlorophyll and carotenoid contents of pur and gre were remarkably reduced in the purple and albino periods, but the anthocyanin contents accumulated greatly in the same period. The photosynthetic capacities of the flag leaves in pur, pst, and gre were the same as that in the Xinong1125 during the heading stage. Genetic analysis showed that the leaf color mutations in PSL1 in response to temperature were controlled by cytoplasmic inheritance. Quantitative real‐time polymerase chain reaction showed that the psbA and psbC genes were downregulated most in the newly developed leaf tissues in pur and pst under low temperatures. This indicated that the psbA and psbC genes may be involved in chloroplast development under low‐temperature conditions in wheat.