Search

We used serial block-face scanning electron microscopy (SBF-SEM) to study the host-pathogen interface between Arabidopsis cotyledons and the hemibiotrophic fungus Colletotrichum higginsianum . By combining high-pressure freezing and freeze-substitution with SBF-SEM, followed by segmentation and reconstruction of the imaging volume using the freely accessible software IMOD, we created 3D models of the series of cytological events that occur during the Colletotrichum-Arabidopsis susceptible interaction. We found that the host cell membranes underwent massive expansion to accommodate the rapidly growing intracellular hypha. As the fungal infection proceeded from the biotrophic to the necrotrophic stage, the host cell membranes went through increasing levels of disintegration culminating in host cell death. Intriguingly, we documented autophagosomes in proximity to biotrophic hyphae using transmission electron microscopy (TEM) and a concurrent increase in autophagic flux between early to mid/late biotrophic phase of the infection process. Occasionally, we observed osmiophilic bodies in the vicinity of biotrophic hyphae using TEM only and near necrotrophic hyphae under both TEM and SBF-SEM. Overall, we established a method for obtaining serial SBF-SEM images, each with a lateral ( x-y ) pixel resolution of 10 nm and an axial ( z ) resolution of 40 nm, that can be reconstructed into interactive 3D models using the IMOD. Application of this method to the Colletotrichum-Arabidopsis pathosystem allowed us to more fully understand the spatial arrangement and morphological architecture of the fungal hyphae after they penetrate epidermal cells of Arabidopsis cotyledons and the cytological changes the host cell undergoes as the infection progresses toward necrotrophy. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

High growth temperatures negatively affect soybean ( Glycine max (L.) Merr) yields and seed quality. Soybean plants, heat stressed during seed development, produce seed that exhibit wrinkling, discoloration, poor seed germination, and have an increased potential for incidence of pathogen infection and an overall decrease in economic value. Soybean breeders have identified a heat stress tolerant exotic landrace genotype, which has been used in traditional hybridization to generate experimental genotypes, with improved seed yield and heat tolerance. Here, we have investigated the seed protein composition and ultrastructure of cotyledonary parenchyma cells of soybean genotypes that are either susceptible or tolerant to high growth temperatures. Biochemical analyses of seed proteins isolated from heat-tolerant and heat-sensitive genotypes produced under 28/22 °C (control), 36/24 °C (moderate), and 42/26 °C (extreme) day/night temperatures revealed that the accumulation in soybean seeds of lipoxygenase, the β-subunit of β-conglycinin, sucrose binding protein and Bowman-Birk protease inhibitor were negatively impacted by extreme heat stress in both genotypes, but these effects were less pronounced in the heat-tolerant genotype. Western blot analysis showed elevated accumulation of heat shock proteins (HSP70 and HSP17.6) in both lines in response to elevated temperatures during seed fill. Transmission electron microscopy showed that heat stress caused dramatic structural changes in the storage parenchyma cells. Extreme heat stress disrupted the structure and the membrane integrity of protein storage vacuoles, organelles that accumulate seed storage proteins. The detachment of the plasma membrane from the cell wall (plasmolysis) was commonly observed in the cells of the sensitive line. In contrast, these structural changes were less pronounced in the tolerant genotype, even under extreme heat stress, cells, for the most part, retained their structural integrity. The results of our study demonstrate the contrasting effects of heat stress on the seed protein composition and ultrastructural alterations that contribute to the tolerant genotype's ability to tolerate high temperatures during seed development.

Exposure of dark-grown (etiolated) seedlings to light induces the heterotrophic-to-photoautotrophic transition (de-etiolation) processes, including the formation of photosynthetic machinery in the chloroplast and cotyledon expansion. Phytochrome is a red (R)/far-red (FR) light photoreceptor that is involved in the various aspects of de-etiolation. However, how phytochrome regulates metabolic dynamics in response to light stimulus has remained largely unknown. In this study, to elucidate the involvement of phytochrome in the metabolic response during de-etiolation, we performed widely targeted metabolomics in Arabidopsis ( Arabidopsis thaliana ) wild-type and phytochrome A and B double mutant seedlings de-etiolated under R or FR light. The results revealed that phytochrome had strong impacts on the primary and secondary metabolism during the first 24 h of de-etiolation. Among those metabolites, sugar levels decreased during de-etiolation in a phytochrome-dependent manner. At the same time, phytochrome upregulated processes requiring sugars. Triacylglycerols are stored in the oil bodies as a source of sugars in Arabidopsis seedlings. Sugars are provided from triacylglycerols through fatty acid β-oxidation and the glyoxylate cycle in glyoxysomes. We examined if and how phytochrome regulates sugar production from oil bodies. Irradiation of the etiolated seedlings with R and FR light dramatically accelerated oil body mobilization in a phytochrome-dependent manner. Glyoxylate cycle-deficient mutants not only failed to mobilize oil bodies but also failed to develop thylakoid membranes and expand cotyledon cells upon exposure to light. Hence, phytochrome plays a key role in the regulation of metabolism during de-etiolation., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Main Conclusion: While plant irrigation usually increases yield, irrigation also affects seed characteristics with respect to endoreplication level, chemical composition, number of carbonyl bands, and cuticular wax profiles. Seeds of sweet varieties of the narrow-leaved lupin have good nutritional properties; however, these plants are sensitive to water deficit. Irrigation improves lupin yield, but can affect seed characteristics. The purpose of the study was to evaluate irrigation influence on lupin seed features and their chemical composition. Morphological analyses showed worse quality of seeds from the irrigated plants, with regard to their size and weight. This was confirmed by cytophotometric analyses which revealed a lower DNA content in the nuclei of cells from the apical and basal regions of the irrigated seeds. The lower degree of polyploidy of the nuclei entails lower cell sizes and limited space for storage components. Fourier transform infrared spectroscopic analysis demonstrated that protein and cuticular wax profiles of the irrigated seeds were different from the control. The electrophoretic analyses indicated differences in protein profiles including changes in the proportion of lupin storage proteins. Among the various studied elements, only the nitrogen content decreased in the embryo axis of irrigated plants. Although germination dynamics of the irrigated seeds was higher, the seedlings' development rate was slightly lower than in the control. The hydrogen peroxide level in root meristem cells was higher during germination in the control suggesting its regulatory role in seed metabolism/signaling. Our study indicated that irrigation of lupin plant affected seed features and composition.

Seed priming is a pre-sowing method successfully used to improve seed germination. Since water plays a crucial role in germination, the aim of this study was to investigate the relationship between better germination performances of osmoprimed Brassica napus seeds and seed water status during germination. To achieve this goal, a combination of different kinds of approaches was used, including nuclear magnetic resonance (NMR) spectroscopy, TEM, and SEM as well as semi-quantitative PCR (semi-qPCR). The results of this study showed that osmopriming enhanced the kinetics of water uptake and the total amount of absorbed water during both the early imbibition stage and in the later phases of seed germination. The spin⁻spin relaxation time ( T ₂) measurement suggests that osmopriming causes faster water penetration into the seed and more efficient tissue hydration. Moreover, factors potentially affecting water relations in germinating primed seeds were also identified. It was shown that osmopriming (i) changes the microstructural features of the seed coat, e.g., leads to the formation of microcracks, (ii) alters the internal structure of the seed by the induction of additional void spaces in the seed, (iii) increases cotyledons cells vacuolization, and (iv) modifies the expression pattern of aquaporin genes.

Background: Soybean is a globally important oil seed crop. Both the high protein and oil content of soybean seeds make this crop a lucrative commodity. As in higher eukaryotic species with available genomes, the functional annotation of most of soybean's genes still remains to be investigated. A major hurdle in the functional genomics of soybean is a rapid method to test gene constructs before embarking on stable transformation experiments., Results: In this paper we describe the morphology and composition of the persistent single-cell aleurone layer that derives from the endosperm of developing soybean seeds. Its composition compared to cotyledonary tissue indicates the aleurone layer plays a role in both abiotic and biotic stress. The potential utility as the aleurone layer as a transient expression system in soybean was shown. As a near transparent single-cell layer it can be used as a transient expression system to study transgene expression and inter- and intra-cellular targeting as it is amenable to microscopic techniques., Conclusion: The transparent single cell aleurone layer was shown to be compositionally comparable to cotyledonary tissue in soybean with an enrichment in oxidative response proteins and shown to be a potential transient expression platform.

Premise of the Study: Tetraena simplex is an independently evolved C 4 species in the Zygophylloideae (Zygophyllaceae) and a characteristic forb of saline flats in hot and sandy desert habitats. During early ontogeny, the species had a morphological shift from planar cotyledons (dorsiventral symmetry) to terete, succulent leaves (radial symmetry). We tested whether this shift had a corresponding change in internal Kranz anatomy and tissue patterning., Methods: For a comprehensive characterization of C 4 photosynthesis across early ontogeny in T. simplex, structural and ultrastructural anatomical properties and localization patterns, activities, and immunoblotting of key C 4 photosynthetic enzymes were compared in mesophyll and bundle sheath tissues in cotyledons and leaves., Key Results: Cotyledons and leaves possessed different types of Kranz anatomy (atriplicoid type and a "Tetraena" variant of the kochioid type, respectively), reflecting the change in leaf morphology. In bundle sheath cells, key differences in ultrastructural features included increased organelle numbers and chloroplast thylakoid stacking. C 4 enzymes had strict tissue-specific localization patterns within bundle sheath and mesophyll cells in both cotyledons and leaves. The decarboxylase NAD-ME maintained the highest activity, increasing from cotyledons to leaves. This classified T. simplex as fully C 4 across ontogeny and a strictly NAD-ME biochemical subtype., Conclusions: Tetraena simplex cotyledons and leaves showed differences in Kranz type, with associated progression in ultrastructural features, and differing activities/expression levels of C 4 enzymes. Furthermore, leaves characterized a new "Tetraena" variation of the kochioid Kranz anatomy., (© 2018 Botanical Society of America.)

Isolated bean cells were used to understand the contribution of cell wall and cytoplasmic matrix on starch digestibility. Cotyledon cells were treated enzymatically and mechanically to reduce the level of cell intactness. SEM and chemical characterization revealed that enzymatic treatment modified cell wall thickness and porosity without altering the cytoplasmic matrix, whereas mechanical treatment completely disrupted cell structure. Decreasing cell intactness increased the rate but not the extent of starch digestion in-vitro. It was concluded that cell wall serves as a permeable barrier limiting the access of digestive enzymes. Cytoplasmic matrix, on the other hand, reduced further the accessibility of amylase to starch affecting its hydrolysis rate. In addition, it was proven that cell structural changes, if any, occurring during digestion had no effect on starch hydrolysis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vacuoles have been shown to undergo deep modifications in relation to plant developmental stages and in the maintaining the cellular homeostasis. In this context, we studied the variations of the vacuolar membrane size and α-TIP aquaporin distribution at early and advanced seed stages of maturation, germination and embryo growth in Vicia faba cotyledon storage cells., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Brassicaceae is at the forefront of evolution because of its frequent hybridization. Hybridization is responsible for the induction of widespread genetic and phenotype changes, making it important in agricultural production. In this study, we obtained resynthesized allotetraploid Brassica napus by performing interspecific crossing of B. rapa × B. oleracea combined with embryo rescue. We applied light microscopy and electronic microscopy to analyze the microstructure and ultrastructure of seeds of diploid parents and their allotetraploid progeny. Results showed that pigments in the seed coat were mainly distributed in the palisade layer. B. rapa presented the highest amount of pigment followed by B. napus and B. oleracea. B. napus had the thickest palisade layer followed by B. rapa and B. oleracea. The seed coat microsculpturing in B. rapa and B. napus was characterized as reticulate or reticulate-foveate, whereas that in B. oleracea was observed to be rugose and sulcate. The area index of the protein body was higher in central meristematic cells than in parenchyma cells. By contrast, the area index of the oil body was the lowest in central meristematic cells. Protein bodies were found to be heterogeneous with crystal globoids in two diploid parents and resynthesized allotetraploid progenies. Oil bodies consisted of large and small oil bodies, the sizes of which differed between two parents and allotetraploid progenies. Small oil bodies were spheroid, whereas large oil bodies were ovoid in shape. The quantity of oil bodies indicated that oil bodies were spheroid in two parents, ranging in size from 0.12 to 1.18 μm. In comparison, the size of large oil bodies in allotetraploid progenies exceeds 2.0 μm. These findings suggest that the anatomy of resynthesized allotetraploid seeds remarkably differs from that of two diploid parents, and these differences definitely affect the nutritional components of rapeseeds.

Plants in a temperate climate are often subject to different environmental factors, chilling stress among them, which influence the growth especially during early stages of plant development. Chloroplasts are one of the first organelles affected by the chilling stress. Therefore the proper biogenesis of chloroplasts in early stages of plant growth is crucial for undertaking the photosynthetic activity. In this paper, the analysis of the cotyledon chloroplast biogenesis at different levels of plastid organization was performed in cucumber, one of the most popular chilling sensitive crops. Influence of low temperature on the ultrastructure was manifested by partial recrystallization of the prolamellar body, the formation of elongated grana thylakoids and a change of the prolamellar body structure from the compacted "closed" type to a more loose "open" type. Structural changes are strongly correlated with galactolipid and carotenoid content. Substantial changes in the galactolipid and the carotenoid composition in dark-chilled plants, especially a decrease of the monogalactosyldiacylglycerol to digalactosyldiacylglycerol ratio (MGDG/DGDG) and an increased level of lutein, responsible for a decrease in membrane fluidity, were registered together with a slower adaptation to higher light intensity and an increased level of non-photochemical reactions. Changes in the grana thylakoid fluidity, of their structure and photosynthetic efficiency in developing chloroplasts of dark-chilled plants, without significant changes in the PSI/PSII ratio, could distort the balance of photosystem rearrangements and be one of the reasons of cucumber sensitivity to chilling., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Dicot leaves are composed of a heterogeneous mosaic of jigsaw puzzle piece-shaped pavement cells that vary greatly in size and the complexity of their shape. Given the importance of the epidermis and this particular cell type for leaf expansion, there is a strong need to understand how pavement cells morph from a simple polyhedral shape into highly lobed and interdigitated cells. At present, it is still unclear how and when the patterns of lobing are initiated in pavement cells, and one major technological bottleneck to addressing the problem is the lack of a robust and objective methodology to identify and track lobing events during the transition from simple cell geometry to lobed cells. We developed a convex hull-based algorithm termed LobeFinder to identify lobes, quantify geometric properties, and create a useful graphical output of cell coordinates for further analysis. The algorithm was validated against manually curated images of pavement cells of widely varying sizes and shapes. The ability to objectively count and detect new lobe initiation events provides an improved quantitative framework to analyze mutant phenotypes, detect symmetry-breaking events in time-lapse image data, and quantify the time-dependent correlation between cell shape change and intracellular factors that may play a role in the morphogenesis process., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

The transition from etiolated to green seedlings involves the conversion of etioplasts into mature chloroplasts via a multifaceted, light-driven process comprising multiple, tightly coordinated signaling networks. Here, we demonstrate that light-induced greening and chloroplast differentiation in tomato (Solanum lycopersicum) seedlings are mediated by an intricate cross talk among phytochromes, nitric oxide (NO), ethylene, and auxins. Genetic and pharmacological evidence indicated that either endogenously produced or exogenously applied NO promotes seedling greening by repressing ethylene biosynthesis and inducing auxin accumulation in tomato cotyledons. Analysis performed in hormonal tomato mutants also demonstrated that NO production itself is negatively and positively regulated by ethylene and auxins, respectively. Representing a major biosynthetic source of NO in tomato cotyledons, nitrate reductase was shown to be under strict control of both phytochrome and hormonal signals. A close NO-phytochrome interaction was revealed by the almost complete recovery of the etiolated phenotype of red light-grown seedlings of the tomato phytochrome-deficient aurea mutant upon NO fumigation. In this mutant, NO supplementation induced cotyledon greening, chloroplast differentiation, and hormonal and gene expression alterations similar to those detected in light-exposed wild-type seedlings. NO negatively impacted the transcript accumulation of genes encoding phytochromes, photomorphogenesis-repressor factors, and plastid division proteins, revealing that this free radical can mimic transcriptional changes typically triggered by phytochrome-dependent light perception. Therefore, our data indicate that negative and positive regulatory feedback loops orchestrate ethylene-NO and auxin-NO interactions, respectively, during the conversion of colorless etiolated seedlings into green, photosynthetically competent young plants., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

The arils of Bixa orellana L. seeds contain carotenoid storage cells (CSCs). The main compounds in these cells include bixin and norbixin, which are important pigments in the food and pharmaceutical industries. Although many studies have been conducted on these chemical constituents, the cellular events that occur during the development of the carotenoid-accumulating cells in the arils and their relationship with the final carotenoid accumulation in the vacuoles remain unknown. In this study, the development of the CSCs in B. orellana arils was analyzed by light and transmission electron microscopy. Carotenoids formed in specialized cells, whose number and size increased during aril development. At various stages of development, the cytoplasm of the CSCs contained chromoplasts that held an extensive network of tubules and plastoglobules. Next to the chromoplasts, lipid droplets may fuse one another to form osmiophilic bodies. In addition, vesicles were observed next to the tonoplast. At the final stages of development, both the osmiophilic bodies and vesicles, which became quadrangular or rectangular, were stored in the vacuoles of the CSCs. This study reported for the first time the occurrence of different storage unit types within the vacuole of carotenoid storage cells.

Because of the weak penetrating power of electrons, the signal-to-noise ratio of a transmission electron micrograph (TEM) worsens as section thickness increases. This problem is alleviated by the use of the scanning transmission electron microscopy (STEM). Tomography analyses using STEM of thick sections from yeast and mammalian cells are of higher quality than are bright-field (BF) images. In this study, we compared regular BF tomograms and STEM tomograms from 500-nm thick sections from hypertrophied Golgi stacks of alfalfa root cap cells. Due to their thickness and intense heavy metal staining, BF tomograms of the thick sections suffer from poor contrast and high noise levels. We were able to mitigate these drawbacks by using STEM tomography. When we performed STEM tomography of densely stained chloroplasts of Arabidopsis cotyledon, we observed similar improvements relative to BF tomograms. A longer time is required to collect a STEM tilt series than similar BF TEM images, and dynamic autofocusing required for STEM imaging often fails at high tilt angles. Despite these limitations, STEM tomography is a powerful method for analyzing structures of large or dense organelles of plant cells.

Background: Non-photosynthetic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins are distributed in Brassicaceae plants. Brassica oleracea WSCP (BoWSCP) and Lepidium virginicum WSCP (LvWSCP) are highly expressed in leaves and stems, while Arabidopsis thaliana WSCP (AtWSCP) and Raphanus sativus WSCP (RshWSCP) are highly transcribed in floral organs. BoWSCP and LvWSCP exist in the endoplasmic reticulum (ER) body. However, the subcellular localization of AtWSCP and RshWSCP is still unclear. To determine the subcellular localization of these WSCPs, we constructed transgenic plants expressing Venus-fused AtWSCP or RshWSCP., Results: Open reading frames corresponding to full-length AtWSCP and RshWSCP were cloned and ligated between the cauliflower mosaic virus 35S promoter and Venus, a gene encoding a yellow fluorescent protein. We introduced the constructs into A. thaliana by the floral dip method. We succeeded in constructing a number of transformants expressing Venus-fused chimeric AtWSCP (AtWSCP::Venus) or RshWSCP (RshWSCP::Venus). We detected fluorescence derived from the chimeric proteins using a fluorescence microscope system. In cotyledons, fluorescence derived from AtWSCP::Venus and RshWSCP::Venus was detected in spindle structures. The spindle structures altered their shape to a globular form under blue light excitation. In true leaves, the number of spindle structures was drastically reduced. These observations indicate that the spindle structure was the ER body., Conclusions: AtWSCP and RshWSCP have the potential for ER body targeting like BoWSCP and LvWSCP.

(Phaseolus vulgaris). The hardening of Phaseolus vulgaris beans stored at high temperature and high relative humidity is one of the main constraints for consumption. The objective of this research was to evaluate by scanning electron microscopy, structural changes in cotyledons and testa of the hardened beans. The freshly harvested grains were stored for twelve months under two conditions: 5 ° C-34% RH and 37 ° C-75% RH, in order to promote hardening. The stored raw and cooked grains were lyophilized and fractured. The sections of testa and cotyledons were observed in an electron microscope JSM-6390. After twelve months, grains stored at 37 ° C-75% RH increased their hardness by 503%, whereas there were no significant changes in grains stored at 5 ° C-34% RH. At the microstructural level, the cotyledons of the raw grains show clear differences in appearance of the cell wall, into the intercellular space size and texture matrix protein. There were also differences in compaction of palisade and sub-epidermal layer in the testa of raw grains. After cooking, cotyledon cells of the soft grains were well separated while these ofhard grains were seldom separated. In conclusion, the found differences in hard and soft grains showed a significant participation of both structures, cotyledons and testa, in the grains hardening.

Recent advances in the acquisition of large-scale datasets of transmission electron microscope images have allowed researchers to determine the number and the distribution of subcellular ultrastructures at both the cellular level and the tissue level. For this purpose, it would be very useful to have a computer-assisted system to detect the structures of interest, such as organelles. Using our original image recognition framework CARTA (Clustering-Aided Rapid Training Agent), combined with procedures to highlight and enlarge regions of interest on the image, we have developed a successful method for the semi-automatic detection of plant organelles including mitochondria, amyloplasts, chloroplasts, etioplasts, and Golgi stacks in transmission electron microscope images. Our proposed semi-automatic detection system will be helpful for labelling organelles in the interpretation and/or quantitative analysis of large-scale electron microscope imaging data.

Purine nucleotides can be fully catabolized by plants to recycle nutrients. We have isolated a urate oxidase (uox) mutant of Arabidopsis thaliana that accumulates uric acid in all tissues, especially in the developing embryo. The mutant displays a reduced germination rate and is unable to establish autotrophic growth due to severe inhibition of cotyledon development and nutrient mobilization from the lipid reserves in the cotyledons. The uox mutant phenotype is suppressed in a xanthine dehydrogenase (xdh) uox double mutant, demonstrating that the underlying cause is not the defective purine base catabolism, or the lack of UOX per se, but the elevated uric acid concentration in the embryo. Remarkably, xanthine accumulates to similar levels in the xdh mutant without toxicity. This is paralleled in humans, where hyperuricemia is associated with many diseases whereas xanthinuria is asymptomatic. Searching for the molecular cause of uric acid toxicity, we discovered a local defect of peroxisomes (glyoxysomes) mostly confined to the cotyledons of the mature embryos, which resulted in the accumulation of free fatty acids in dry seeds. The peroxisomal defect explains the developmental phenotypes of the uox mutant, drawing a novel link between uric acid and peroxisome function, which may be relevant beyond plants., (© 2014 American Society of Plant Biologists. All rights reserved.)

Phytoextraction is an established method of removal of heavy metals from contaminated soils worldwide. Phytoextraction is most efficient if local plants are used in the contaminated site. We propose that Prosopis pubescens (Screw bean mesquite) would be a successful phytoextractor of copper in our local soils. In order to determine the feasibility of using Screw bean mesquite, we utilized inductively-coupled plasma-optical emission spectroscopy (ICP-OES) and elemental analysis to observe the uptake of copper and the effects on macro and micro nutrients within laboratory-grown seedlings. We have previously shown that P. pubescens is a hyperaccumulator of copper in soil-grown seedlings. Light and transmission electron microscopy demonstrated death of root cells and ultrastructural changes due to the presence of copper from 50 mg/L - 600 mg/L. Ultrastructural changes included plasmolysis, starch accumulation, increased vacuolation and swollen chloroplasts with disarranged thylakoid membranes in cotyledons. Inductively coupled plasma-optical emission spectroscopy analyses of macro- and micro-nutrients revealed that the presence of copper sulfate in the growth medium of Petri-dish grown Prosopis pubescens seedlings resulted in dramatic decreases of magnesium, potassium and phosphorus. At 500-600 mg/L of copper sulfate, a substantial increase of sulfur was present in roots.

The Orange (Or) gene is a gene mutation that can increase carotenoid content in plant tissues normally devoid of pigments. It affects plastid division and is involved in the differentiation of proplastids or non-colored plastids into chromoplasts. In this study, the de-etiolation process of the wild type (WT) cauliflower (Brassica oleracea L. var. botrytis) and Or mutant seedlings was investigated. We analyzed pigment content, plastid development, transcript abundance and protein levels of genes involved in the de-etiolation process. The results showed that Or can increase the carotenoid content in green tissues, although not as effectively as in non-green tissues, and this effect might be caused by the changes in biosynthetic pathway genes at both transcriptional and post-transcriptional levels. There was no significant difference in the plastid development process between the two lines. However, the increased content of antheraxanthin and anthocyanin, and higher expression levels of violaxanthin de-epoxidase gene (VDE) suggested a stress situation leading to photoinhibition and enhanced photoprotection in the Or mutant. The up-regulated expression levels of the reactive oxygen species (ROS)-induced genes, ZAT10 for salt tolerance zinc finger protein and ASCORBATE PEROXIDASE2 (APX2), suggested the existence of photo-oxidative stress in the Or mutant. In summary, abovementioned findings provide additional insight into the functions of the Or gene in different tissues and at different developmental stages.

In willow seeds, photooxidative damage is mainly restricted to the outer cotyledonary tissues, significantly reducing normal germination. Here we analyzed the damage generated in cotyledonary tissues and investigated whether the increase in reactive oxygen species (ROS) generation in seedlings from photooxidized seeds can affect the morphogenetic capacity of the shoot apical meristem. Seeds were photooxidized under different light intensities and the evolution of the damage during seedling growth was studied by light and transmission electron microscopies. The level of lipid peroxidation and changes in antioxidant capacity were measured following the time course of superoxide dismutase, catalase, ascorbate peroxidase and guaiacol peroxidase enzyme activities, and the effect of photooxidative stress on the genesis of new leaf primordia and lateral roots was examined. Early and active endocytosis and autophagy, changes in chloroplast morphology, as well as the accumulation and diffusion of ROS all play important roles in the early cell death observed in cotyledonary tissues. Following germination, seedlings from photooxidized seeds anticipated the emergence of first leaves, which complemented the altered functionality of the damaged cotyledons., (© 2013 Scandinavian Plant Physiology Society.)

Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Inga vera, native to South America, is an important leguminous species used for ecological restoration of riparian forests and its seeds are among the most recalcitrant ones described up to date. In this work, we analysed the metabolic profile, cell ultrastructure as well as cell wall polysaccharides of I. vera seeds in order to better understand its maturation, which allows embryo germination without a quiescent phase. Increased amounts of citric, glutamic, pyroglutamic, and aspartic acids from stages I to II (120 and 129 days after flowering (DAF)) corroborate the hypothesis of high metabolism, shifting from fermentative to aerobic respiration at seed maturity. This phase was characterized by an extensive vacuolization of embryonic cells, which also indicate high metabolic activity. The proportion of arabinose in the cell walls of embryonic axis (approx. 20%) was lower than those found in some orthodox seeds (nearly 40%), suggesting that arabinose-containing polysaccharides, which are thought to provide more flexibility to the cell wall during natural drying, are less abundant in I. vera seeds. Taken together, our results provide evidence that the major changes occurred during early stages of seed maturation of I. vera, indicating that the rapid temporary metabolic shift observed between stages I and II may be related to the lack of desiccation phase, moving directly to germination., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The plastid has its own translation system, and its ribosomes are assembled through a complex process in which rRNA precursors are processed and ribosomal proteins are inserted into the rRNA backbone. DEAD-box proteins have been shown to play roles in multiple steps in ribosome biogenesis. To investigate the cellular and physiological roles of an Arabidopsis DEAD-box protein, RH3, we examined its expression and localization and the phenotypes of rh3-4, a T-DNA insertion mutant allele of RH3. The promoter activity of RH3 is strongest in the greening tissues of 3-day and 1-week-old seedlings but reduced afterwards. Cotyledons were pale and seedling growth was retarded in the mutant. The most obvious abnormality in the mutant chloroplasts was their lack of normal ribosomes. Electron tomography analysis indicated that ribosome density in the 3-day-old mutant chloroplasts is only 20% that of wild-type chloroplasts, and the ribosomes in the mutant are smaller. These chloroplast defects in rh3-4 were alleviated in 2-week-old cotyledons and true leaves. Interestingly, rh3-4 seedlings have lower amounts of abscisic acid prior to recovery of their chloroplasts, and were more sensitive to abiotic stresses. Transcriptomic analysis indicated that nuclear genes for chloroplast proteins are down-regulated, and proteins mediating chloroplast-localized steps of abscisic acid biosynthesis are expressed to a lower extent in 1-week-old rh3-4 seedlings. Taken together, these results suggest that conversion of eoplasts into chloroplasts in young seedlings is critical for the seedlings to start carbon fixation as well as for maintenance of abscisic acid levels for responding to environmental challenges., (© 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.)

Circadian clocks are biochemical timers regulating many physiological and molecular processes according to the day/night cycle. The small GTPase LIGHT INSENSITIVE PERIOD1 (LIP1) is a circadian clock-associated protein that regulates light input to the clock. In the absence of LIP1, the effect of light on free-running period length is much reduced. Here, we show that in addition to suppressing red and blue light-mediated photomorphogenesis, LIP1 is also required for light-controlled inhibition of endoreplication and tolerance to salt stress in Arabidopsis (Arabidopsis thaliana). We demonstrate that in the processes of endoreplication and photomorphogenesis, LIP1 acts downstream of the red and blue light photoreceptors phytochrome B and cryptochromes. Manipulation of the subcellular distribution of LIP1 revealed that the circadian function of LIP1 requires nuclear localization of the protein. Our data collectively suggest that LIP1 influences several signaling cascades and that its role in the entrainment of the circadian clock is independent from the other pleiotropic effects. Since these functions of LIP1 are important for the early stages of development or under conditions normally experienced by germinating seedlings, we suggest that LIP1 is a regulator of seedling establishment.

In plants, the trans-Golgi network and early endosomes (TGN/EE) function as the central junction for major endomembrane trafficking events, including endocytosis and secretion. Here, we demonstrate that the KEEP ON GOING (KEG) protein of Arabidopsis thaliana localizes to the TGN/EE and plays an essential role in multiple intracellular trafficking processes. Loss-of-function keg mutants exhibited severe defects in cell expansion, which correlated with defects in vacuole morphology. Confocal microscopy revealed that KEG is required for targeting of plasma membrane proteins to the vacuole. This targeting process appeared to be blocked at the step of multivesicular body (MVB) fusion with the vacuolar membrane as the MVB-associated small GTPase ARA6 was also blocked in vacuolar delivery. In addition, loss of KEG function blocked secretion of apoplastic defense proteins, indicating that KEG plays a role in plant immunity. Significantly, KEG was degraded specifically in cells infected by the fungus Golovinomyces cichoracearum, suggesting that this pathogen may target KEG to manipulate the host secretory system as a virulence strategy. Taking these results together, we conclude that KEG is a key component of TGN/EE that regulates multiple post-Golgi trafficking events in plants, including vacuole biogenesis, targeting of membrane-associated proteins to the vacuole, and secretion of apoplastic proteins.

Using immunocytochemical methods, at both the light and electron microscopic level, we have investigated the spatial and temporal distribution of lipid transfer protein 1 (LTP1) epitopes during the induction of somatic embryogenesis in explants of Arabidopsis thaliana. Immunofluorescence labelling demonstrated the presence of high levels of LTP1 epitopes within the proximal regions of the cotyledons (embryogenic regions) associated with particular morphogenetic events, including intense cell division activity, cotyledon swelling, cell loosening and callus formation. Precise analysis of the signal localization in protodermal and subprotodermal cells indicated that cells exhibiting features typical of embryogenic cells were strongly labelled, both in walls and the cytoplasm, while in the majority of meristematic-like cells no signal was observed. Staining with lipophilic dyes revealed a correlation between the distribution of LTP1 epitopes and lipid substances within the cell wall. Differences in label abundance and distribution between embryogenic and non-embryogenic regions of explants were studied in detail with the use of immunogold electron microscopy. The labelling was strongest in both the outer periclinal and anticlinal walls of the adaxial, protodermal cells of the proximal region of the cotyledon. The putative role(s) of lipid transfer proteins in the formation of lipid lamellae and in cell differentiation are discussed. Key message Occurrence of lipid transfer protein 1 epitopes in Arabidopsis explant cells accompanies changes in cell fate and may be correlated with the deposition of lipid substances in the cell walls.

Although seeds are a sink organ, chlorophyll synthesis and degradation occurs during embryogenesis and in a manner similar to that observed in photosynthetic leaves. Some mutants retain chlorophyll after seed maturation, and they are disturbed in seed storability. To elucidate the effects of chlorophyll retention on the seed storability of Arabidopsis (Arabidopsis thaliana), we examined the non-yellow coloring1 (nyc1)/nyc1-like (nol) mutants that do not degrade chlorophyll properly. Approximately 10 times more chlorophyll was retained in the dry seeds of the nyc1/nol mutant than in the wild-type seeds. The germination rates rapidly decreased during storage, with most of the mutant seeds failing to germinate after storage for 23 months, whereas 75% of the wild-type seeds germinated after 42 months. These results indicate that chlorophyll retention in the seeds affects seed longevity. Electron microscopic studies indicated that many small oil bodies appeared in the embryonic cotyledons of the nyc1/nol mutant; this finding indicates that the retention of chlorophyll affects the development of organelles in embryonic cells. A sequence analysis of the NYC1 promoter identified a potential abscisic acid (ABA)-responsive element. An electrophoretic mobility shift assay confirmed the binding of an ABA-responsive transcriptional factor to the NYC1 promoter DNA fragment, thus suggesting that NYC1 expression is regulated by ABA. Furthermore, NYC1 expression was repressed in the ABA-insensitive mutants during embryogenesis. These data indicate that chlorophyll degradation is induced by ABA during seed maturation to produce storable seeds.

• The cell and developmental biology of zygotic embryogenesis in the model legume Medicago truncatula has received little attention. We studied M. truncatula embryogenesis from embryo sac until cotyledon maturation, including oil and protein body biogenesis. • We characterized embryo development using light and electron microscopy, measurement of protein and lipid fatty acid accumulation and by profiling the expression of key seed storage genes. • Embryo sac development in M. truncatula is of the Polygonum type. A distinctive multicellular hypophysis and suspensor develops before the globular stage and by the early cotyledon stage, the procambium connects the developing apical meristems. In the storage parenchyma of cotyledons, ovoid oil bodies surround protein bodies and the plasma membrane. Four major lipid fatty acids accumulate as cotyledons develop, paralleling the expression of OLEOSIN and the storage protein genes, VICILIN and LEGUMIN. • Zygotic embryogenesis in M. truncatula features the development of a distinctive multicellular hypophysis and an endopolyploid suspensor with basal transfer cell. A clear procambial connection between the apical meristems is evident and there is a characteristic arrangement of oil bodies in the cotyledons and radicle. Our data help link embryogenesis to the genetic regulation of oil and protein body biogenesis in legume seed., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

Here, a large fraction of plant mitochondrial actin was found to be resistant to protease and high-salt treatments, suggesting it was protected by mitochondrial membranes. A portion of this actin became sensitive to protease or high-salt treatment after removal of the mitochondrial outer membrane, indicating that some actin is located inside the mitochondrial outer membrane. The import of an actin-green fluorescent protein (GFP) fusion protein into the mitochondria in a transgenic plant, actin:GFP, was visualized in living cells and demonstrated by flow cytometry and immunoblot analyses. Polymerized actin was found in mitochondria of actin:GFP plants and in mung bean (Vigna radiata). Notably, actin associated with mitochondria purified from early-developing cotyledons during seed germination was sensitive to high-salt and protease treatments. With cotyledon ageing, mitochondrial actin became more resistant to both treatments. The progressive import of actin into cotyledon mitochondria appeared to occur in concert with the conversion of quiescent mitochondria into active forms during seed germination. The binding of actin to mitochondrial DNA (mtDNA) was demonstrated by liquid chromatography-tandem mass spectrometry analysis. Porin and ADP/ATP carrier proteins were also found in mtDNA-protein complexes. Treatment with an actin depolymerization reagent reduced the mitochondrial membrane potential and triggered the release of cytochrome C. The potential function of mitochondrial actin and a possible actin import pathway are discussed.

Mobilization of seed storage reserves is essential for seed germination and seedling establishment. Here, we report that AtDSEL, an Arabidopsis thalianaDAD1-like Seedling Establishment-related Lipase, is involved in the mobilization of storage oils for early seedling establishment. AtDSEL is a cytosolic member of the DAD1-like acylhydrolase family encoded by At4g18550. Bacterially expressed AtDSEL preferentially hydrolyzed 1,3-diacylglycerol and 1-monoacylglycerol, suggesting that AtDSEL is an sn-1-specific lipase. AtDSEL-overexpressing transgenic Arabidopsis plants (35S:AtDSEL) were defective in post-germinative seedling growth in medium without an exogenous carbon source. This phenotype was rescued by the addition of sucrose to the growth medium. In contrast, loss-of-function mutant plants (atdsel-1 and atdsel-2) had a mildly fast-growing phenotype regardless of the presence of an exogenous carbon source. Electron microscopy revealed that 5-day-old 35S:AtDSEL cotyledons retained numerous peroxisomes and oil bodies, which were exhausted in wild-type and mutant cotyledons. The impaired seedling establishment of 35S:AtDSEL was not rescued by the addition of an exogenous fatty acid source, and 35S:AtDSEL seedling growth was insensitive to 2,4-dichlorophenoxybutyric acid, indicating that β-oxidation was blocked in AtDSEL-overexpressers. These results suggest that AtDSEL is involved in the negative regulation of seedling establishment by inhibiting the breakdown of storage oils., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Electron microscopic images of mitochondrial nucleoids isolated from mung bean seedlings revealed a relatively homogeneous population of particles, each consisting of a chromatin-like structure associated with a membrane component. Association of F-actin with mitochondrial nucleoids was also observed. The mitochondrial nucleoid structure identified in situ showed heterogeneous genomic organization. After pulsed-field gel electrophoresis (PFGE), a large proportion of the mitochondrial nucleoid DNA remained in the well, whereas the rest migrated as a 50-200 kb smear zone. This PFGE migration pattern was not affected by high salt, topoisomerase I or latrunculin B treatments; however, the mobility of a fraction of the fast-moving DNA decreased conspicuously following an in-gel ethidium-enhanced UV-irradiation treatment, suggesting that molecules with intricately compact structures were present in the 50-200 kb region. Approximately 70% of the mitochondrial nucleoid DNA molecules examined via electron microscopy were open circles, supercoils, complex forms, and linear molecules with interspersed sigma-shaped structures and/or loops. Increased sensitivity of mtDNA to DNase I was found after mitochondrial nucleoids were pretreated with high salt. This result indicates that some loosely bound or peripheral DNA binding proteins protected the mtDNA from DNase I degradation.

The respiration rates R (oxygen uptake per min) and body mass M (mg per individual) of sunflower (Helianthus annuus L.) seedlings were measured for populations raised in the dark (scotomorphogenesis) and for plants subsequently grown in white light (photomorphogenesis) to determine the allometric (scaling) relationship for R vs. M. Based on ordinary least squares and reduced major axis regression protocols, cellular respiration rates were found to increase non-linearly as a 'broken-stick' curve of increasing M. During germination, the scaling was ca. 7.5-fold higher than after the emergence of the cotyledons from the seed coat, which can be attributed to the hypoxic conditions of the enclosed embryo. During seedling development, R was found to scale roughly as the 3/7 power of body mass (i.e., R ~ M(-3/7)), regardless of whether plants were grown in the dark or subsequently in white light. The numerical value of 3/7 statistically significantly differs from that reported across field- or laboratory-grown plants (i.e., R ~ M(-1.0)). It also differs from the expectations of recent allometric theory (i.e., R ~ M(-0.75) to M(-1.0)). This difference is interpreted to be the result of species-specific tissue-compositions that affect the volume fractions of metabolically active and less active cells. These findings, which are supported by cytological and ultrastructural observations (i.e., scanning- and transmission electron micrographs), draw attention to the need to measure R of developing plants in a tissue- or organ-specific context.

Background and Aims: Sclerotinia sclerotiorum can attack >400 plant species worldwide. Very few studies have investigated host-pathogen interactions at the plant surface and cellular level in resistant genotypes of oilseed rape/canola (Brassica napus)., Methods: Infection processes of S. sclerotiorum were examined on two B. napus genotypes, one resistant cultivar 'Charlton' and one susceptible 'RQ001-02M2' by light and scanning electron microscopy from 2 h to 8 d post-inoculation (dpi)., Key Results: The resistant 'Charlton' impeded fungal growth at 1, 2 and 3 dpi, suppressed formation of appresoria and infection cushions, caused extrusion of protoplast from hyphal cells and produced a hypersensitive reaction. At 8 dpi, whilst in 'Charlton' pathogen invasion was mainly confined to the upper epidermis, in the susceptible 'RQ001-02M2', colonization up to the spongy mesophyll cells was evident. Calcium oxalate crystals were found in the upper epidermis and in palisade cells in susceptible 'RQ001-02M2' at 6 dpi, and throughout leaf tissues at 8 dpi. In resistant 'Charlton', crystals were not observed at 6 dpi, whereas at 8 dpi they were mainly confined to the upper epidermis. Starch deposits were also more prevalent in 'RQ001-02M2'., Conclusions: This study demonstrates for the first time at the cellular level that resistance to S. sclerotiorum in B. napus is a result of retardation of pathogen development, both on the plant surface and within host tissues. The resistance mechanisms identified in this study will be useful for engineering disease-resistant genotypes and for developing markers for screening for resistance against this pathogen.

Portulacaceae is one of 19 families of terrestrial plants in which species having C(4) photosynthesis have been found. Representative species from major clades of the genus Portulaca were studied to characterize the forms of photosynthesis structurally and biochemically. The species P. amilis, P. grandiflora, P. molokiniensis, P. oleracea, P. pilosa, and P. umbraticola belong to the subgenus Portulaca and are C(4) plants based on leaf carbon isotope values, Kranz anatomy, and expression of key C(4) enzymes. Portulaca umbraticola, clade Umbraticola, is NADP-malic enzyme (NADP-ME)-type C(4) species, while P. oleracea and P. molokiniensis in clade Oleracea are NAD-ME-type C(4) species, all having different forms of Atriplicoid-type leaf anatomy. In clade Pilosa, P. amilis, P. grandiflora, and P. pilosa are NADP-ME-type C(4) species. They have Pilosoid-type anatomy in which Kranz tissues enclose peripheral vascular bundles with water storage in the centre of the leaf. Portulaca cf. bicolor, which belongs to subgenus Portulacella, is an NADP-ME C(4) species with Portulacelloid-type anatomy; it has well-developed Kranz chlorenchyma surrounding lateral veins distributed in one plane under the adaxial epidermis with water storage cells underneath. Portulaca cryptopetala (clade Oleracea), an endemic species from central South America, was identified as a C(3)-C(4) based on its intermediate CO(2) compensation point and selective localization of glycine decarboxylase of the photorespiratory pathway in mitochondria of bundle sheath cells. The C(4) Portulaca species which were examined also have cotyledons with Kranz-type anatomy, while the stems of all species have C(3)-type photosynthetic cells. The results indicate that multiple structural and biochemical forms of C(4) photosynthesis evolved in genus Portulaca.

Synthesis of 5-aminolevulinic acid (ALA) represents a rate limiting step in the tetrapyrrole biosynthetic pathway, and is regulated by metabolic feedback control of glutamyl-tRNA reductase (GluTR) activity. The FLU protein has been attributed to this regulation. Later in the biosynthetic pathway, reduction of protochlorophyllide (Pchlide), catalyzed by protochlorophyllide oxidoreductase (POR), ensures another important regulatory step in the chlorophyll biosynthesis. In the present work, we investigated the expression and cellular abundance of nuclear-encoded and plastid-encoded proteins involved in ALA synthesis and Pchlide reduction in Norway spruce (Picea abies L. Karst.) as a representative of plant species with high ability to synthesize chlorophyll in the dark. Using dark-grown, light/dark-grown and gabaculine-treated seedlings, we demonstrated that gabaculine-impaired etiochloroplast and chloroplast development has no negative effect on GluTR accumulation in the cotyledons. However, in contrast to control plants, the relative amount of GluTR was similar both in the dark-grown and light/dark-grown gabaculine-treated seedlings. We identified a partial sequence of the FLU-like gene in Norway spruce, and using antibodies against the FLU-like protein (FLP), we showed that FLP accumulated mostly in the dark-grown control seedlings and gabaculine-treated seedlings. In contrast to nuclear-encoded GluTR and FLP, accumulation of plastid-encoded light-independent POR (DPOR) was sensitive to gabaculine treatment. The levels of DPOR subunits were substantially lower in the light/dark-grown control seedlings and gabaculine-treated seedlings, although the corresponding genes chlL, chlN and chlB were expressed. Since we analyzed the samples with different plastid types, plastid ultrastructure and physiological parameters like Pchlide and chlorophyll contents, in vivo chlorophyll fluorescence and photosynthetic efficiency of the seedlings were characterized. Apart from etiochloroplast-specific accumulation of the DPOR subunits, we described, in some detail, additional specific features of chlorophyll biosynthesis in the spruce seedlings that differ from those known in angiosperms., (Copyright 2010 Elsevier GmbH. All rights reserved.)

The mobilization of storage reserves, with particular emphasis on storage proteins of Mucuna pruriens (L.) DC., cotyledons, and embryo was investigated from the ultrastructural and biochemical points of view. Proteins and starch were the two main storage substances in cotyledons, and proteins and lipids were the main ones in the embryo. Embryo protein bodies were smaller and fewer in number than those of cotyledons. Structural and ultrastructural data determined between 24 and 48 h after imbibition and between 48 and 72 h after imbibition, the end of significant embryo and cotyledon protein mobilization, respectively, indicating more precocious storage protein mobilization in the axis than cotyledons. Moreover, storage protein mobilization in embryo and cotyledons occurred before the end of germination. Water soluble proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, producing 29 bands with molecular weights from 14 to 90 KDa. Embryo extract contained more proteins than cotyledon extract, contained seven characteristic bands, and showed a higher variability of the optical density trend than cotyledon.

Transfer cells (TCs) trans-differentiate from differentiated cells by developing extensive wall ingrowths that enhance plasma membrane transport of nutrients. Here, we investigated transcriptional changes accompanying induction of TC development in adaxial epidermal cells of cultured Vicia faba cotyledons. Global changes in gene expression revealed by cDNA-AFLP were compared between adaxial epidermal cells during induction (3 h) and subsequent building (24 h) of wall ingrowths, and in cells of adjoining storage parenchyma tissue, which do not form wall ingrowths. A total of 5795 transcript-derived fragments (TDFs) were detected; of these, 264 TDFs showed epidermal-specific changes in gene expression and a further 207 TDFs were differentially expressed in both epidermal and storage parenchyma cells. Genes involved in signalling (auxin/ethylene), metabolism (mitochondrial; storage product hydrolysis), cell division, vesicle trafficking and cell wall biosynthesis were specifically induced in epidermal TCs. Blockers of auxin action and vesicle trafficking inhibited ingrowth formation and marked increases in cell division accompanied TC development. Auxin and possibly ethylene signalling cascades induce epidermal cells of V. faba cotyledons to trans-differentiate into TCs. Trans-differentiation is initiated by rapid de-differentiation to a mitotic state accompanied by mitochondrial biogenesis driving storage product hydrolysis to fuel wall ingrowth formation orchestrated by a modified vesicle trafficking mechanism.

Sucrose binding proteins (SBPs) were predicted to be membrane-associated, but have been shown to localize in the lumen of protein storage vacuoles of various seeds. In this study, a new 64 kDa SBP has been identified from developing mung bean (Vigna radiata) seeds (here termed VrSBP1) via MS/MS analysis and N-terminal amino acid sequencing analysis and specific antibodies were generated using purified VrSBP1 proteins. Western blot analysis with the new VrSBP1 antibodies showed that, similar to most seed storage proteins, VrSBP1 proteins accumulated during seed development and were subsequently mobilized once the mung bean seeds germinated. Immunogold electron microscope (EM) studies on ultra-thin sections of high-pressure freezing/frozen substituted developing mung bean cotyledons demonstrated that VrSBP1 was localized specifically to the tonoplast of the protein storage vacuole and to the limiting membrane of a novel putative prevacuolar compartment. Biochemical and subcellular fractionation studies further demonstrated that VrSBP1 proteins were membrane-associated in developing mung beans, consistent with their tonoplast localization. This study thus shows convincing evidence of tonoplast-localization of a plant SBP for its future functional characterization and provides a model of studying non-integral membrane proteins associated with the tonoplasts in plant cells.

Using RNAi, the seed oil body protein 24-kDa oleosin has been suppressed in transgenic soybeans. The endoplasmic reticulum (ER) forms micro-oil bodies about 50 nm in diameter that coalesce with adjacent oil bodies forming a hierarchy of oil body sizes. The oil bodies in the oleosin knockdown form large oil body-ER complexes with the interior dominated by micro-oil bodies and intermediate-sized oil bodies, while the peripheral areas of the complex are dominated by large oil bodies. The complex merges to form giant oil bodies with onset of seed dormancy that disrupts cell structure. The transcriptome of the oleosin knockdown shows few changes compared to wild-type. Proteomic analysis of the isolated oil bodies of the 24-kDa oleosin knockdown shows the absence of the 24-kDa oleosin and the presence of abundant caleosin and lipoxygenase. The formation of the micro-oil bodies in the oleosin knockdown is interpreted to indicate a function of the oleosin as a surfactant.

The plastid-localized methylerythritol phosphate (MEP) pathway synthesizes the isoprenoid precursors for the production of essential photosynthesis-related compounds and hormones. We have identified an Arabidopsis thaliana mutant, rif1, in which posttranscriptional upregulation of MEP pathway enzyme levels is caused by the loss of function of At3g47450, a gene originally reported to encode a mitochondrial protein related to nitric oxide synthesis. However, we show that nitric oxide is not involved in the regulation of the MEP pathway and that the encoded protein is a plastid-targeted homolog of the Bacillus subtilis YqeH protein, a GTPase required for proper ribosome assembly. Consistently, in rif1 seedlings, decreased levels of plastome-encoded proteins were observed, with the exception of ClpP1, a catalytic subunit of the plastidial Clp protease complex. The unexpected accumulation of ClpP1 in plastids with reduced protein synthesis suggested a compensatory mechanism in response to decreased Clp activity levels. In agreement, a negative correlation was found between Clp protease activity and MEP pathway enzyme levels in different experiments, suggesting that Clp-mediated degradation of MEP pathway enzymes might be a mechanism used by individual plastids to finely adjust plastidial isoprenoid biosynthesis to their functional and physiological states.

Under phosphate deprivation, higher plants change their lipid composition and recycle phosphate from phospholipids. A phospholipase D, PLDzeta2, is involved in this recycling and in other cellular functions related to plant development. We investigated the localization of Arabidopsis PLDzeta2 by cell fractionation and in vivo GFP confocal imaging. AtPLDzeta2 localizes to the tonoplast and the Nter regulatory domain is sufficient for its sorting. Under phosphate deprivation, AtPLDzeta2 remains located in the tonoplast but its distribution is uneven. We observed PLDzeta2-enriched tonoplast domains preferentially positioned close to mitochondria and beside chloroplasts. In absence of PLDzeta2, membrane developments were visualized inside vacuoles.

Cucumber seedlings were grown under three doses of supplemental ultraviolet-B (UV-B) irradiation to examine the effects on the surface structure of the cotyledons. Medium and high doses of irradiation induced glazing (formation of translucent, glossy layers) on the adaxial surfaces of cotyledons, especially those exposed to a high dose of UV-B. Observation with a scanning electron microscope revealed that the adaxial surfaces of cotyledons exposed to a medium dose of UV-B and controls became rough in appearance, but unevenness of the surface was not apparent in cotyledons irradiated with a high dose of UV-B. UV-B irradiation affected the types and amounts of alkanes and primary alcohols, the main components of cucumber cuticular wax. Based on cotyledon area, the amounts of these components were significantly higher in cotyledons irradiated with a medium dose of UV-B than in controls. This effect could be a consequence of small cotyledon area and constant wax production in the cotyledons irradiated with a medium dose of UV-B. The distribution patterns of homologs within the alkane and primary alcohol fractions shifted during growth to longer alkyl chain length in the control cotyledons. UV-B irradiation repressed these changes, suggesting that UV-B acts on cuticular wax biosynthetic pathways.

Following on from previous work, the temporal and spatial accumulation of the aspartic proteinases (EC 3.4.23) cardosin A and cardosin B during postembryonic seed development of cardoon (Cynara cardunculus) was studied, mRNA and protein analyses of both cardosins suggested that the proteins accumulate during seed maturation, and that cardosin A is later synthesised de novo at the time of radicle emergence. Immunocytochemistry revealed that the precursor form of cardosin A accumulates in protein bodies and cell walls. This localisation in seeds is different from that previously described for cardoon flowers, suggesting a tissue-dependent targeting of the protein. It is known that procardosins are active and may have a role in proteolysis and processing of storage proteins. However, the presence of procardosin A in seeds could be related to the proposed role of the plant-specific insert in membrane lipid conversion during water uptake and solute leakage in actively growing tissues. This is in accordance with the recently proposed bifunctional role of aspartic proteinase precursor molecules that possess a membrane-destabilising domain in addition to a protease domain. Mature cardosin B, but not its mRNA, was detected in the first hours after seed imbibition and disappeared at the time of radicle emergence. This extracellular aspartic protease has already been implicated in cell wall loosening and remodelling, and its role in seed germination could be related to loosening tissue constraints for radicle protusion. The described pattern of cardosin A and B expression suggests a finely tuned developmental regulation and prompts an analysis of their possible roles in the physiology of postembryonic development.

The ability to combine nucleic acid hybridisation or immunospecific reactions with structural and ultrastructural analysis of virus-infected tissues has provided the opportunity to resolve the spatial details of infection with respect to the production of virus-specific products and the nature of the host response. These technologies may seem lengthy and complex but offer high rewards in terms of revealing the details of host-virus interactions not otherwise accessible.

Phototropin 1 (phot1) is a blue-light Ser/Thr receptor kinase that contains two LOV domains. It is a plasma membrane-associated protein that mediates phototropism, blue-light induced chloroplast movement, and stomatal opening. The aim of the present work was to analyze the intracellular localization of phot1 protein in Ipomoea nil seedlings. In cotyledon and hypocotyl cells of etiolated seedlings, phot1 was specifically localized in the plasma membrane regions, whereas in light-treated seedlings, it was homogeneously distributed throughout the whole cytoplasm, excluding cell nuclei and vacuoles. Phot1 was also localized in cotyledon epidermal and guard cells. Such a localization pattern suggests a light-dependent intracellular distribution of phot1 in Ipomoea nil. On the basis of the spatial distribution, the possible role of phot1 is also discussed.

Background and Aims: Cotyledon number has long been a primary morphological feature distinguishing monocots from other angiosperms. Recent placement of Hydatellaceae near the early-divergent angiosperm order Nymphaeales, rather than in the monocot order Poales, has prompted reassessment of seedling morphology in this poorly known family., Methods: Seedlings of six species representing all eco-geographical groups of Hydatellaceae are described using light and scanning electron microscopy., Key Results: Two seedling types were discovered. Material examined of Trithuria submersa, T. bibracteata, T. austinensis and T. filamentosa possess a transparent bilobed sheathing structure that surrounds the main axis below the first foliage leaf. The seed coat is attached to the sheathing structure. Seedlings of Trithuria lanterna and T. konkanensis lack a sheathing structure, and the seed coat is attached to a short, narrow lateral outgrowth on the main axis of the seedling., Conclusions: The sheathing structure that is present in seedlings of some Hydatellaceae could be homologized with the two united cotyledons of water lilies. It also resembles the single cotyledon of some monocots, and hence demonstrates a possible pathway of the origin of a monocot-like embryo, though no homology is implied. The sheathing structure is reduced in Trithuria lanterna and T. konkanensis, and the short, narrow outgrowth of its seedling could represent a single cotyledon. This synapomorphy suggests that the only Indian species of Hydatellaceae, T. konkanensis, is closer to the northern Australian T. lanterna than to the south-western Australian T. bibracteata.

Background and Aims: Seedlings of monocots are much more diverse than those of other angiosperms, often with very derived character states. This makes morphological interpretation difficult. The morphology of seedlings of most of the 16 families of the Poales alliance are only incompletely known. The present study aims first to develop an unambiguous terminology for the description of monocotyledonous seedlings. This makes possible clear morphological comparisons and the use of homologous terms for organs. Finally, plotting of well defined characters onto a molecular tree allows the polarization of character states., Methods: Seedlings were grown in Petri dishes on moist filter paper under permanent light conditions and analysed using light and scanning electron microscopy. Only seeds collected at natural habitats or from plants with a well documented source were used. Seedling vouchers are deposited in the alcohol collection of Monocot seedlings in the Botanische Staatssammlung München (M)., Key Results: Based on an unambiguous terminology, seedlings of a great number of genera are described and presented as figures, representing all families of Poales except Ecdeiocoleaceae. Seedlings of Rapateaceae, Joinvilleaceae and Mayacaceae are described for the first time. Morphological comparisons reveal a plausible interpretation of even very modified organ structures, including those of the grass seedling., Conclusions: This study demonstrates that detailed studies of seedling morphology can provide interesting morphological insights and also new facts for phylogenetic analyses. However, the morphological diversity of seedlings in the monocots is as yet incompletely known, and in some, e.g. Alismatales or Zingiberales, the seedling structure is particularly poorly understood in terms of comparative morphology.

Chloroplast development in cotyledons differs in a number of ways from that in true leaves, but the cotyledon-specific program of chloroplast biogenesis has not been clarified. The cyo1 mutant in Arabidopsis thaliana has albino cotyledons but normal green true leaves. Chloroplasts develop abnormally in cyo1 mutant plants grown in the light, but etioplasts are normal in mutants grown in the dark. We isolated CYO1 by T-DNA tagging and verified that the mutant allele was responsible for the albino cotyledon phenotype by complementation. CYO1 has a C(4)-type zinc finger domain similar to that of Escherichia coli DnaJ. CYO1 is expressed mainly in young plants under light conditions, and the CYO1 protein localizes to the thylakoid membrane in chloroplasts. Transcription of nuclear photosynthetic genes is generally unaffected by the cyo1 mutation, but the level of photosynthetic proteins is decreased in cyo1 mutants. Recombinant CYO1 accelerates disulfide bond reduction in the model substrate insulin and renatures RNase A, indicating that CYO1 has protein disulfide isomerase activity. These results suggest that CYO1 has a chaperone-like activity required for thylakoid biogenesis in cotyledons.