The spectral abundance factor SAF was calculated for each protein. Numerous methods for isolation of intact plastids and sub-fractionation of chloroplast compartments for proteomics studies in Arabidopsis populate our literature today [ 42 , 46 , 84 ].
In addition, robust methods have been developed for isolation of metabolomics- and proteomics-grade chromoplasts of tomato fruit [ 47 , 48 , 49 , 50 , 51 , 58 ] Additional file 1 : Table S1. While protocols for isolating chloroplasts for DNA isolation, enzymatic assays and protein import assays have been described [ 53 , 54 , 55 , 56 , 57 , 85 ], rather surprisingly, few chloroplast large-scale proteomics studies have been reported for tomato leaves [ 52 ] Additional file 1 : Table S1.
Therefore, a high-yield method for intact chloroplast isolation and methods for recovery of the membrane and stromal fractions of chloroplasts for proteomics analyses for tomato was needed. Our protocol for chloroplast isolation builds upon methods developed for Arabidopsis chloroplasts [ 42 ] and incorporates many recommendations from foundational studies in spinach and pea [ 86 , 87 ]. Our methods for proteomics-grade tomato chloroplasts are more similar to those used by van Wijk et al. However, unlike the Arabidopsis protocol, our tissue grinding buffer: 1 did not use the anti-oxidant cysteine; 2 included 1 mM MgCl 2 and 1 mM MnCl 2 similar to several tomato protocols, and 3 included fivefold higher BSA 0.
The general scheme for chloroplast isolation, stroma extraction and proteomics processing and data analysis are provided in Fig. Briefly, the deveined tomato leaf homogenate is centrifuged; the pellet contains both intact and broken chloroplasts. It is clear based on the size of the bands at the two interfaces Fig.
In the process of refining our chloroplast stroma isolation method, we discovered several parameters that markedly increased tomato chloroplast yield. First, only young, undamaged leaves from 4- to 5-week old tomato plants are used. These leaves are tender and many are expanding and therefore provide best yields. Second, several parts of the homogenization protocol are critical for high-yields and intact chloroplasts.
We found that the slurry status of the 1X Grinding Buffer 1 part ice: 1 part liquid enhanced the yield of intact chloroplasts Table 1.
If the Grinding Buffer is too watery, there is excess chloroplast breakage and stroma is lost; if the buffer is too icy, there is insufficient cell breakage and chloroplasts are not released.
Third, the volume of tissue and buffer relative to the blender cup size is critical. If the blender cup is too full, insufficient homogenization occurs; not full enough, foaming protein denaturation and excess plastid breakage occurs. Fourth, as well established in the literature [ 87 ], the duration of the blender pulse is critical and empirical determination of the optimal blender settings are essential.
We found that two 2-sec blender pulses released tomato cell content and retained chloroplast integrity. Another unique feature of our method is that additional leaf tissue was added after the first 2-sec pulse.
Unlike the van Wijk et al. Finally, we also found that by decreasing MgCl 2 in the chloroplast lysis buffer from 5 mM [ 42 ] to 2.
With these modifications, typical chloroplast protein yields from tomato leaves yielded 0. Proteins ranging from over kDa to under 20 kDa were resolved indicating the high quality of proteins recovered at different stages in the tomato leaf chloroplast protocol.
The purified chloroplast extracts were enriched for a subset of the proteins in the total leaf extracts Fig. Furthermore, a majority of the abundant proteins found in intact chloroplasts were also present in the non-soluble, membrane fraction after chloroplast lysis. In contrast, the stromal protein fraction is distinct with a small number of superabundant proteins in the to kDa range. Silver-stained SDS—polyacrylamide gels and immunoblots with protein fractions from the chloroplast stroma isolation protocol.
Masses of molecular weight markers are shown in kDa. The RPS6 antisera cross-reacts with several tomato proteins. The kDa RPS6 protein is solely found the total leaf homogenate; several of its cross-reacting proteins are enriched during the steps used for chloroplast stromal protein isolation.
The mass kDa of each protein is shown. To evaluate the efficacy of our chloroplast and stroma isolation methods, we determined the levels of three proteins that are known to reside in the chloroplast stroma heat shock protein 70; HSP70 , lumen oxygen evolving complex 23; OEC23 , and thylakoid membranes light harvesting complex proteins; LHCP , as well as one cytosolic protein ribosomal protein S6; RPS6.
In immunoblots, all four proteins were readily detected in total leaf extracts Fig. It should be noted that while this antiserum had high specificity for the kDa RPS6 in maize roots [ 66 ], numerous cross-reactive proteins were detected in tomato leaves. However, the kDa RPS6 was the most strongly detected protein and was only identified in leaf homogenates total leaf protein. These immunoblot data indicate that the chloroplasts are largely free of cytosolic protein contamination.
Our proteomics data also supports this result as the cytosolic RPS6 was detected in two of our eight samples with one unique peptide Additional file 3 : Table S3. Small amounts of the lumenal OEC23 were also detected in the non-soluble fraction, consistent with OEC23 being an extrinsic protein that associates with OEC33 and OEC16 at the thylakoid membrane for their role in oxygen evolution [ 90 , 91 ].
Both stromal- and lumen-localized proteins HSP70 and OEC23, respectively were detected in the stromal protein samples via immunoblots [ 90 , 91 , 92 ]. These data suggested that the stromal extract contained soluble lumenal proteins. The chloroplast lumenal proteome is not complex ranging from 80 to proteins [ 93 , 94 , 95 ]; 45 proteins designated as thylakoid peripheral or lumenal proteins in PPDB [ 77 ] were detected in the stromal proteome, representing 3.
In five samples, proteins were acetone precipitated. Manual curation of these proteins was performed using our tomato chloroplast protein Atlas [ 68 ], which predicted tomato protein localization using five published algorithms [ 72 , 73 , 74 , 75 , 76 ].
The proteins identified in the tomato chloroplast stromal extracts are shown based on their designated categories. The chloroplast stromal proteome has chloroplast proteins [ 68 ]. There were co-isolating proteins CIPs that were reproducibly detected. In the eight stromal samples, proteins were detected once with 1 PSM.
Of these proteins, 83 proteins were known to be located within the chloroplast. The remaining proteins had no evidence for chloroplast localization and were classified as low-level contaminants and were removed from further consideration Fig.
Using conservative criteria to identify stromal proteins, we removed proteins identified by one unique peptide Additional file 3 : Table S3; Fig. These proteins had no empirical data to support their localization in the chloroplast based on Arabidopsis homologs PPDB, plprot and SUBA4 evidence or the tomato chloroplast protein Atlas.
In addition, proteins were identified by more than one peptide but were detected sporadically one to three times in our eight samples ; these proteins were designated as low-level contaminants and not considered further Additional file 4 : Table S4; Fig. In some cases, the PSMs for the sporadically identified proteins were high. A summary of the subcellular localization of the proteins identified by one peptide and the sporadically identified proteins is provided in Table 2.
Their distribution in the cytosolic, endomembrane, nuclear, mitochondrial, peroxisomal, and plasma membrane compartments was similar. These CIPs could: 1 be reflective of the inadvertent co-isolation of small quantities of other organelles; 2 report the extensive and dynamic interactions of chloroplasts with other organelles e. We assessed the frequency of detection, abundance, and putative localization of the CIPs Additional file 5 : Table S5.
For example, of the CIPs For perspective, the range of SAFs for the tomato proteins was from 0. CIPs predicted to be localized in the cytosol, peroxisome, nucleus, mitochondrion, and endomembrane system are shown.
Each circle represents a single protein. The predicted subcellular localization of CIPs was imputed based on the tomato chloroplast Atlas, which provided predictions of the locations of the tomato CIPs Additional file 5 : Table S5.
Collectively these data indicated that a majority of the tomato CIP proteins were predicted to reside in the cytosol However, all three of these Arabidopsis proteins were also detected in other subcellular locations and the tomato Atlas did not predict a chloroplast localization, hence the classification as tomato CIPs.
This prediction was not supported by empirical data for the Arabidopsis CIP homologs, as 31 of these Arabidopsis homologs had a non-chloroplast location Additional file 5 : Table S5. Embedded within the cytosol, contamination of chloroplasts with abundant cytosolic proteins is anticipated. In pea, PGKc co-localizes with glyceraldehydeP-dehydrogenase, triose-P-isomerase and aldolase providing an opportunity for direct channeling of substrates between the enzymes [ ].
However, these additional enzymes were not identified as stromal CIPs suggesting that this complex does not exist in tomato or the protein associations are labile. Of the remaining cytosolic CIPs, proteins associated with numerous functions were identified. The Arabidopsis AKR2A homolog At2g works with a cytosolic HSP17 to target membrane proteins to the plastid outer membrane [ , , ]; however, the tomato cytosolic HSP17 homolog was not detected in any of our stromal samples. The most highly represented cytosolic CIPs were those associated with translation, with four elongation factors, two initiation factors, two ribosomal protein subunits, and five tRNA synthetases Additional file 5 : Table S5.
When the lists of sporadically identified proteins and proteins identified with one unique peptide were examined, an additional 38 ribosome subunits, five initiation factors and three elongation factors were also identified Additional file 3 : Table S3, Additional file 4 : Table S4. Chloroplasts, peroxisomes, and mitochondria participate in the photorespiratory pathway that catabolizes the products produced by the oxygenation reaction of ribulose-1,5-bisphosphate carboxylase [ ].
Electron microscopy and in situ laser analyses have shown that in the light, peroxisomes and mitochondria have intimate and dynamic interactions with chloroplasts and with each other [ , ]. Chloroplasts may also interact with peroxisomes via dynamic peroxisome membrane extensions called peroxules [ ].
Therefore, it is not surprising that 19 peroxisomal proteins and 52 mitochondrial proteins were CIPs Additional file 5 : Table S5, Fig. Hydroxypyruvate reductase Solyc01g , Glutamate:glyoxylate aminotransferase Solyc05g , and Serine:glyoxylate aminotransferase Solyc12g were identified in all eight samples and were abundant proteins with NSAF scores of 0.
A byproduct of photorespiration is hydrogen peroxide, which is dissipated by a robust peroxisomal ROS-scavenging system [ ]. Reumann et al. Although we are extrapolating between two species, the substantial differences in NSAF values for peroxisomes determined by Reumann et al. In tomato, ICL is detected in fruits and leaves [ ] and has been correlated with the peroxisome to glyoxysome transition during leaf senescence [ ].
It is noteworthy that other enzymes of the glycolytic cycle e. NSAFs ranged from 1. Proteins associated with the TCA cycle 14 proteins and amino acid biosynthesis or catabolism 14 proteins were enriched in the CIPs. There is substantial evidence that nuclei and plastids interact [ ].
Chloroplasts can be found directly appressed to nuclear envelopes and connected to nuclei via stromules. These direct and yet dynamic communication channels may allow for the exchange of metabolites, H 2 O 2 , and, perhaps, proteins.
Twelve chromatin-associated proteins i. Finally, there is a well-established biochemical continuity between the endoplasmic reticulum and the chloroplast [ 31 ]. Therefore, it is not surprising that there were 45 endomembrane system proteins that were identified as CIPs Table 2 , Additional file 5 : Table S5, Fig.
Tomato is the most cultivated horticultural crop worldwide, with over 4. In addition, tomato is a model system for the study of the induction of plant defenses associated with wounding, herbivory and pathogen attack [ ]. As chloroplasts are key regulators of stress perception and signal transduction [ 5 , 33 ] and the site of production of secondary metabolites and plant hormones involved in defense, an understanding of the dynamics of the chloroplast leaf proteome is needed.
The protocol provided here provides a detailed method to assure high quality and high yields of intact chloroplasts from tomato leaves suitable for proteomics analysis. As a number of yield-limiting steps were identified in this protocol, the methods can be adapted to virtually any plant species. In conjunction with the tomato nuclear and plastid genome sequences [ 56 , 71 ], evaluation of changes to the tomato chloroplast proteome, and its sub-organellar fractions, in response to cues during development, as well as abiotic and biotic stress are now possible.
Future confirmation of CIP localization using fluorescent reporter fusion proteins will determine if these proteins are imported and localized in more than one organelle or if their co-isolation with chloroplasts solely reflects the known tight apposition of ER, peroxisomes, mitochondria, and nuclei with chloroplasts [ , , , ].
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Springer: Heidelberg; Purification and proteomic analysis of chloroplasts and their sub-organellar compartments. Preparation of stroma, thylakoid membrane, and lumen fractions from Arabidopsis thaliana chloroplasts for proteomic analysis. In: Jarvis RP, editor. In the upper part of Figure 4 a , there is another chloroplast with several grana inside. Similarly, in the corresponding region of Figure 4 b , the fluorescence lifetime becomes smaller due to the higher concentration of PSII.
In the corresponding region of Figure 4 c , SHG spots with very short lifetime are observed, showing the existence of grana. The intensity profile and lifetime profile corresponding to the dashed line in the highlighted area are shown in the right-hand side. The color bar shows the corresponding lifetime. We have demonstrated a noninvasive method to identify the distribution of grana and starch inside a live mesophyll cell without any special specimen labeling or handling. There are two types of SHG structures inside a chloroplast of a plant cell.
One is collocated with strong 2PF, and the other is complementary to 2PF. The former correspond to grana while the latter correspond to starch.
The structure identification is further proved by fluorescence lifetime measurements. The nonlinear nature of the multiphoton process provides useful intrinsic optical sectioning capability and is less likely to cause damage in live sample, enabling observation of organelle dynamics during plant growth.
Our technique will be useful to study granal structural variation among different plant specie [ 33 ], and can be used in the field of botanical evolutionism. The leaf we used here was detached from a fresh ferns, Macrothelypteris torresiana Gaud. Ching , which belongs to shaded plants with large grana [ 33 — 37 ]. The leaf was mounted in water between a coverslip and a glass slide.
The edges of the coverslip were sealed by nail varnish. The glass slide was placed on the microscope stage for observation. The experimental setup is shown in Figure 5 , which is similar to our previous reports [ 23 , 38 ]. This setup allows the simultaneous measurement of SHG and 2PF in the forward and backward directions. The laser source is a mode-locked Yb:fiber laser, whose central wavelength is nm. The pulse width, repetition rate, and maximal average power are fs, 48 MHz, and 5 W, respectively.
The excitation light was directed into an Olympus FV system with a pair of X-Y galvanometric mirrors to achieve raster scanning. The average laser power at sample position is about 60 — 70 mW. The 2PF signals were epi-collected by the same objective while the SHG signals were collected by a condenser in the forward direction.
Schematic diagram of the experiment set-up. For intensity measurement, both backward and forward channels are used to detect 2PF and SHG signals, respectively. M1, M2: mirrors. A high-speed photodetector synchronize the laser repetition rate to the photon counting system. During lifetime measurement, corresponding filters are placed in front of the photon counting PMT to allow either 2PF or SHG detection without cross talk.
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Aust J Plant Physiol. Photosynth Res. Year B Carnegie Inst Wash. Download references. You can also search for this author in PubMed Google Scholar. Correspondence to Shi-Wei Chu. All authors read and approved the final manuscript. Additional file 1: Macrothelypteris torresiana Gaud. Ching leaf. AVI 2 MB. Reprints and Permissions. Chemiosmosis: An energy coupling mechanism that uses energy stored in the form of a hydrogen ion gradient across a membrane to drive cellular work, such as the synthesis of ATP.
Light reactions: The steps in photosynthesis that occur on the thylakoid membranes of the chloroplast and that convert solar energy to the chemical energy of ATP and NADPH, creating oxygen in the process. Photoautotroph: An organism that harnesses light energy to drive the synthesis of organic compounds from carbon dioxide. Photosynthesis: The conversion of light energy to chemical energy that is stored in glucose or other organic compounds; occurs in plants, algae and certain prokaryotes such as cyanobacteria.
Photosystem I: One of two light-harvesting units of a chloroplast's thylakoid membrane; it uses the P reaction-center chlorophyll. Photosystem II: One of two light-harvesting units of a chlorplast's thylakoid membrane: it uses the P reaction center chlorophyll.
Adhering junction: A type of junction between cells forming tissues that are subjected to stretching and pulling, such as the skin. This type of junction provides very tight contact between adjacent cells and allows the cells to function as a unit.
Collagen: A glycoprotein in the extracellular matrix that forms strong fibers found extensively in connective tissue and bone.
Cytoplasmic streaming: A circular flow of cytoplasm, involving myosin and actin filaments, that speed the distribution of materials within cells. Extracellular Matrix: The material outside cells which provides support and structure to tissue.
It consists of ground substance and fibers.
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