• Molecules and Cells
• /
• v.39 no.1
• /
• pp.20-25
• /
• 2016

Photosynthesis is a highly robust process allowing for rapid adjustment to changing environmental conditions. The efficient acclimation depends on balanced redox metabolism and control of reactive oxygen species release which triggers signaling cascades and potentially detrimental oxidation reactions. Thiol peroxidases of the peroxiredoxin and glutathione peroxidase type, and ascorbate peroxidases are the main peroxide detoxifying enzymes of the chloroplast. They use different electron donors and are linked to distinct redox networks. In addition, the peroxiredoxins serve functions in redox regulation and retrograde signaling. The complexity of plastid peroxidases is discussed in context of suborganellar localization, substrate preference, metabolic coupling, protein abundance, activity regulation, interactions, signaling functions, and the conditional requirement for high antioxidant capacity. Thus the review provides an opinion on the advantage of linking detoxification of peroxides to different enzymatic systems and implementing mechanisms for their inactivation to enforce signal propagation within and from the chloroplast.

• Molecules and Cells
• /
• v.20 no.1
• /
• pp.112-118
• /
• 2005

It was previously shown that AtNAP1 is a plastidic SufB protein involved in Fe-S cluster assembly in Arabidopsis. In this study, we investigated the effects of depleting SufB protein from plant cells using virus-induced gene silencing (VIGS). VIGS of NbNAP1 encoding a Nicotiana benthamiana homolog of AtNAP1 resulted in a leaf yellowing phenotype. NbNAP1 was expressed ubiquitously in plant tissues with the highest level in roots. A GFP fusion protein of the N-terminal region (M1-V103) of NbNAP1 was targeted to chloroplasts. Depletion of NbNAP1 resulted in reduced numbers of chloroplasts of reduced size. Mitochondria also seemed to be affected. Despite the reduced number and size of the chloroplasts in the NbNAP1 VIGS lines, the expression of many nuclear genes encoding chloroplast-targeted proteins and chlorophyll biosynthesis genes remained unchanged.

• Horticultural Science & Technology
• /
• v.32 no.5
• /
• pp.607-613
• /
• 2014

Calcium has been associated with improved cold tolerance in many crops. The aim of this study was to investigate the changes in leaf cell $Ca^{2+}$ distribution and cell organelle ultrastructure of loquat (Eriobotrya japonica Lindl.) plants in response to cold stress at $-3^{\circ}C$, using transmission electron microscopy (TEM). Two loquat accessions, Zaozhong 6 (a commercial cultivar) and oakleaf loquat (a wild relative) were used. Cold tolerance, as measured by leaf browning rate, was higher in oakleaf plants, and calcium treatment improved cold tolerance in both species. Cold stress first induced inward transport of $Ca^{2+}$ from the intracellular space. Then, the imported $Ca^{2+}$ was aggregated around the chloroplast membrane, finally entering the chloroplast. This pattern of $Ca^{2+}$ distribution in leaf cells occurred earlier in Zaozhong 6 than in the wild loquat. With increasing time of cold exposure, the chloroplast membranes of Zaozhong 6 leaves were damaged, blurred and even disappeared, while those of wild oakleaf loquat leaves maintained their structure longer. In Zaozhong 6, cold stress induced a clear cavity between poorly structured granal thylakoids and vesicles appearing inside the chloroplast, while in oakleaf leaves cold stress had little effect on the ultrastructure of chloroplasts (although chloroplast membranes looked blurred). Loquat leaves accumulated free calcium ions around chloroplasts in response to cold stress, with earlier calcium accumulation occurring in the cold-sensitive cultivar Zaozhong 6 than in wild oakleaf loquat. These results demonstrate that these two loquat species have differences in both cold tolerance and calcium accumulation dynamics.

• Proceedings of the Plant Resources Society of Korea Conference
• /
• 2010.05a
• /
• pp.14-14
• /
• 2010

Plastid proteomics are essential organelles present in virtually all cells in plants and green algae. Plastids are responsible for the synthesis and storage of key molecules required for the basic architecture and functions of plant cells. The proteome of plastid, and in particular of chloroplast, have received significant amounts of attention in recent years. Various fractionation and mass spectrometry (MS) techniques have been applied to catalogue the chloroplast proteome and its sub-organelles compartments. To better understanding the function of the lumenal sub-organelles within the thylakoid network, we have carried out a systematical analysis and identification of the lumenal proteins in the thylakoid of wheat by using Tricine-SDS-PAGE, and LTQ-ESI-FTICR mass spectrometry followed by SWISS-PROT database searching. We isolation and fractionation these membrane from fully developed wheat leaves using a combination of differential and gradient centrifugation couple to high speed ultra-centrifuge. After collecting all proteins to eliminate possible same proteins, we estimated that there are 407 different proteins including chloroplast, chloroplast stroma, lumenal, and thylakoid membrane proteins excluding 20 proteins, which were identified in nucleus, cytoplasm and mitochondria. A combination of these three programs (PSORT, TargetP, TMHMM, and TOPPRED) was found to provide a useful tool for evaluating chloroplast localization, transit peptide, transmembranes, and also could reveal possible alternative processing sites and dual targeting. Finally, we report also sub-cellular location specific protein interaction network using Cytoscape software, which provides further insight into the biochemical pathways of photosynthesis. The present work helps understanding photosynthesis process in wheat at the molecular level and provides a new overview of the biochemical machinery of the thylakoid in wheat.

• Korean Journal of Plant Tissue Culture
• /
• v.27 no.5
• /
• pp.407-409
• /
• 2000

Outer envelope membrane proteins of chloroplasts encoded by the nuclear genome are transported without the N-terminal transit peptide. Here, we investigated the targeting mechanism of AtOEP7, an Arabidopsis homolog of small outer envelope membrane proteins in vivo. AtOEP7 was expressed transiently in protoplasts or stably in transgenic plants as fusion proteins with GFP. In both cases AtOEP7:GFP was targeted to the outer envelope membrane when assayed under a fluorescent microscope or by Western blot analysis. Except the transmembrane domain, deletions of the N- or C-terminal regions of AtOEP7 did not affect targeting although a region closed to the C-terminal side of the transmembrane domain affected the targeting efficiency. Targeting experiments with various hybrid transmembrane mutants revealed that the amino acid sequence of the transmembrane domain determines the targeting specificity The targeting mechanism was further studied using a fusion protein, AtOEP7：NLS：GFP, that had a nuclear localization signal. AtOEP7：NLS：GFP was efficiently targeted to the chloroplast envelope despite the presence of the nuclear localization signal. Taken together, these results suggest that the transmembrane domain of AtOEP7 functions as the sole determinant of targeting specificity and that AtOEP7 may be associated with a cytosolic component during translocation to the chloroplast envelope membrane.

• ALGAE
• /
• v.18 no.2
• /
• pp.121-127
• /
• 2003

The pyrenoid ultrastructure and distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase in the chloroplasts of Chlamydomonas reinhardtii was studied using the immunogold localization technology with electron microscopy. There were several tubular thylakoids invading in the pyrenoid matrix to form several spokewise channels. The connections between pyrenoid matrix and stroma of chloroplast were the partial of channels. The starch sheath surrounding the pyrenoid was separated into several parts by the connections in transection. Some thylakoids were packed together near the connections in one side of the pyrenoid. Those special structures might be used to transport substance between pyrenoid and stroma of chloroplasts. With the antibody raised against the large subunits of Rubsico from C. protothecoides, the result of the gold immunolocalization of Rubisco in Chlamydomonas reinhardtii showed most of the gold particles heavily labeled the pyrenoid matrix, as well as the starch sheath matrix, and very few in the stroma of chloroplasts. The gold particle density was 880.00 $\pm$ 164.32, 190.00 $\pm$ 152.39 and 9.60 $\pm$ 5.37 ${\mu}m^{-2}$ in pyrenoid matrix, starch sheath and stroma region of chloroplast respectively (background: 5.67 $\pm$ 1.53 ${\mu}m^{-2}$). 99.59% of the total Rubiscos was calculated to be concentrated in the pyrenoid matrix and starch sheath by spatial densities. The gold immunolocalization of Rubisco activase also showed that Rubisco activase was mainly concentrated in the periphery of the pyrenoid and the starch sheath (the density was as high as 229.69 $\pm$ 96.96 ${\mu}m^{-2}$). There were very few gold particles located in the stroma of chloroplasts. These results indicated that pyrenoid surface and starch sheath was the site for Rubisco activation and $CO_2$ fixation, which supported the suggestion that pyrenoids perform photosynthesis function.

• Journal of Plant Biotechnology
• /
• v.48 no.3
• /
• pp.165-172
• /
• 2021

The solubilization of isolated proteins into the adequate buffer containing of surfactants is primary step for proteomic analysis. Particularly, sodium dodecyl sulfate (SDS) is the most widely used surfactant, however, it is not compatible with mass spectrometry (MS). Therefore, it must be removed prior to MS analysis through rigorous washing, which eventually results in inevitable protein loss. Recently, photocleavable surfactant, 4-hexylphenylazosulfonate (Azo), was reported which can be easily degraded by UV irradiation and is compatible with MS during proteomic approach using animal tissues. In this study, we employed comparative label-free proteomic analysis for evaluating the solubilization efficacies of the Azo and SDS surfactants using rice leave proteins. This approach led to identification of 3,365 proteins of which 682 proteins were determined as significantly modulated. Further, according to the subcellular localization prediction in SDS and Azo, proteins localized in the chloroplast were the major organelle accounting for 64% of the total organelle in the SDS sample, while only 37.5% of organelle proteins solubilized in the Azo were predicted to be localized in chloroplast. Taken together, this study validates the efficient solubilization of total protein isolated from plant material for bottom-up proteomics. Azo surfactant is suitable as substitute of SDS and promising for bottom-up proteomics as it facilitates robust protein extraction, rapid washing step during enzymatic digestion, and MS analysis.

• Journal of Plant Biotechnology
• /
• v.36 no.1
• /
• pp.68-74
• /
• 2009

We have previously demonstrated that overexpression and characterization of Brassica rapa type-l metallothionein gene (BrMT1) in Arabidopsis which showed enhanced resistance to cadmium and ROS. Here, we present the consistent study of our previous report about BrMTs. BrMT3 expressing DTY167 cells showed resistance to Zn and Pb as well as Cd. Thus, we have developed the BrMT3 overexpression Arabidopsis to enhance capacity for metal stresses. Successful expression and localization were achieved using the rubisco transit peptides of RbcS-BrMT3-GFP protein, which was confirmed by western blot analysis with the GFP antibody and green fluorescence signal from the chloroplast. BrMT3 overexpression Arabidopsis plants exhibited a higher resistance to cadmium compared to control plants. This result indicates that BrMT3 would be applicable to the development of plants with enhanced resistance against heavy metal stresses.

• Journal of Environmental Science International
• /
• v.5 no.6
• /
• pp.813-826
• /
• 1996

The toxic effects of aluminium (Al) on growth, chlorophyll content, $\delta-aminolevulinic$ acid dehydratase (ALAD) activity and anatomy of root and shoot were investigated in 7-day-old azuki bean (Vigna angularis) seedlings. Significant depressions in root elongation was observed in the low concentrations of Al (50, 100 $\muM)$ and increasing Al concentrations caused a sharp decline of root and shoot growth. The degree of inhibition was dependent upon Al supply. Exposure to 50 $\muM$ Al or more inhibited root elongation within 1 day. In the 50 $\muM$ Al treatments, a recovery of root growth was seen after 7 days exposure. In contrast, lateral root initials was little affected by Al exposure. Al toxicity symptoms and growth responses were more well developed in the roots than in the shoots. Analysis of Al localization in root cells by hematoxylin stAlning showed that Al entered root apices and accumulated in the epidermal and cortical cells immeadiately below the epidermis. There was a good positive correlation between the level of chlorophyll and ALAD activity. Increasing Al concentrations caused a decrease in total chlorophyll contents, accompanied by proportional changes in ALAD activity, suggesting a cootr-dinated reduction of a photosynthetic machinery. Al exerted specific influence on the morphology of root ann shoot. At higher concentrations of Al the roots induced drastic anatomical changes. The epidermal cells were disorganized or destructed while the cortical cells exhibited distortion of cell shape and/or disintegration. The diameter of root and transectional area of cortical cells decreased considerably with Al treatment. In the shoot Al also enhanced reduction of diameter of shoot and cell size. Gross anatomy of leaves treated with Al did not differ significantly from the controls, except for fewer and smaller chloroplast. Our results indicate that toxic effect of Al appear to be manifested primarily in roots and secondarily on shoots, and changes in root morphology are related to changes in the root growth patterns. Results are further discussed in re181ion to the findings in other plant species, and it is concluded that Al causes morphological, structural and, presumably, functional damage to the roots of the species investigated.

• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.67 no.4
• /
• pp.211-221
• /
• 2022

Recently, the demand for the cultivation of upland soil has been increasing, and the rate of conversion of paddy soil into upland soil is also increasing. Theincrease in uneven precipitation due to climate change has resulted in dramatic effects of waterlogging stress on upland crops. Therefore, the present study was conducted to investigate the changes in growth characteristics and the expression patterns of proteins at the two-leaf stage of adzuki beans. The domestic cultivar, Arari (Miryang No. 8), was used to test waterlogging stress. At the two-leaf stage of adzuki beans, plant height slightly decreased androot fresh weight showed significant changes after 3 days of waterlogging treatment. Chlorophyll content was also significantly different after 3 days of waterlogging treatment compared to its content in control plants. Using two-dimensional gel electrophoresis, more than 400 protein spots were identified. Twenty-one differentially expressed proteins from the two-leaf stage were analyzed using linear trap quadrupole-Fourier transform-ion cyclotron resonance mass spectrometry. Of these 21 proteins, 9 were up-regulated and 12 were down-regulated under waterlogging treatment. Protein information resource (https://pir.georgetown.edu/) categories were assigned to all 49 proteins according to their molecular function, cellular component localization, and biological processes. Most of the proteins were found to be involved in the biological process, carbohydrate metabolism and were localized in chloroplasts.