International Journal of Plant Biology 2014; volume 5:4887

Proteomic analysis to horizontal controls in cotton and soybean.3,4 Paraheliotropism benefits plants by maintain- Correspondence: Savithiry Natarajan, Soybean of the pulvinus, a heliotropic ing high levels of photosynthetic quantum Genomics and Improvement Laboratory, ARS- tissue, in Glycine max yield under stressed conditions and reducing USDA, 10300 Baltimore Avenue, Beltsville, MD UVB radiation levels.5,6 20705, USA. 1 2 Tel. +1.301.504.5258 - Fax: +1.301.504.5728. Hakme Lee, Wesley M. Garrett, The pulvinus is an enlarged motor organ at E-mail: [email protected] Joseph Sullivan,1 Irwin Forseth,1 the base of leaves found in many leguminous 3 Savithiry S. Natarajan plants. It has been observed to force the move- Key words: soybean, heliotropism, nyctinasty, 1Department of Plant Sciences and ment of leaves in heliotropic, seismonastic, proteomics, LC-MS/MS. Landscape Architecture, University of and nyctinastic patterns.7,8 Unlike pulvini Maryland, College Park, MD; 2USDA-ARS, found in maize and oat that respond to gravity Acknowledgments: we thank Dr. William Kenworthy for providing the seeds for this proj- Animal Bioscience and Biotechnology by permanent growth, the movement of soy ect. This work was carried out with the support of 3 bean pulvini is reversible.8,9 In heliotropism, Laboratory, Beltsville, MD; USDA-ARS, a Cooperative Agreement between the University upon light exposure, an asymmetric turgor Soybean Genomics and Improvement of Maryland, College Park, and USDA-ARS in Laboratory, Beltsville, MD, USA gradient formed between the adaxial and abax- Beltsville, MD, USA. ial motor cells leads to leaf movement. Potassium ion influx coupled with chlorine ion Contributions: the authors contributed equally. is powered by a proton gradient and results in Abstract osmotic influx.7 In addition to heliotropism, Received for publication: 27 May 2013. the pulvinus changes turgor for nyctinastic Revision received: 19 August 2013. Accepted for publication: 23 April 2014. Certain plant species respond to light, dark, leaf folding, and is affected by alterations in and other environmental factors by leaf move- the length of the photoperiod.10 This work is licensed under a Creative Commons ment. Leguminous plants both track and avoid The structure of the pulvinus reveals its spe- Attribution NonCommercial 3.0 License (CC BY- the sun through turgor changes of the pulvi- cialized role in leaf movement. In contrast to NC 3.0). nus tissue at the base of leaves. Mechanisms the stem and petiole, the pulvinus has a rela- only ©Copyright H. Lee et al., 2014 leading to pulvinar turgor flux, particularly tively larger cortex and smaller pith. The motor 11 Licensee PAGEPress srl, Italy knowledge of the proteins involved, are not cells are part of the cortex. Two types of pulv- International Journal of Plant Biology 2014; 5:4887 well-known. In this study we used two-dimen- inar vacuoles found in many species partici- doi:10.4081/pb.2014.4887 sional gel electrophoresis and liquid chro- pate in the volume flux. Tannin-rich vacuolesuse matography-tandom mass spectrometry to sep- have been previously mentioned as a major arate and identify the proteins located in the source of cellular volume change.12 Still other relative size of its pulvinus, and ease in grow- soybean pulvinus. A total of 183 spots were studies have found the primary volume ing samples. After profiling the pulvinus pro- separated and 195 proteins from 165 spots changes to be from the type of vacuole that teome, it was compared to previously identi- were identified and functionally analyzed does not contain tannin.13 fied proteomes of nearby leaf tissue as well as using single enrichment analysis for gene A number of proteins have been linked with guard cells, which are functionally similar to ontology terms. The most significant terms pulvinar heliotropism and nyctinasty. H+- were related to proton transport. Comparison pulvini in that they also change cell shape ATPase activity increases turgor by H+ efflux with guard cell proteomes revealed similar sig- through differences in turgor. and consequent K+ influx, and H+-ATPase nificant processes but a greater number of pul- inhibitors reduce diaheliotropic response in vinus proteins are required for comparable 14 analysis. To our knowledge, this is a novel soybean pulvinus. Blue light is a deactivator report on the analysis of proteins found in soy- of H+-ATPase in Phaseolus vulgaris motor cells Materials and Methods bean pulvinus. These findings provide a better which leads to decreased turgor pressure on understanding of the proteins required for tur- the illuminated region rather than an activator Plant material gor change in the pulvinus. of H+-ATPase on the opposite region.15 Soybean (G. max cv. Clark) seeds were soaked Non-commercialFurthermore, studies on gravitropic grass pul- overnight in tap water before they were planted vini proteins have begun to identify differen- in 6-inch pots (2-3 per pot) with an LC1 soil mix- tially expressed proteins including one demon- ture (Sun Gro Horticulture, Vancouver, BC, Introduction strating MAPK-like activity.16 Canada). The plants were grown in a growth While studies have identified a number of chamber at the University of Maryland, College Plants respond to light, gravity, touch, and genes involved in a variety of tropic responses, Park, set to a 16:8 photoperiod, temperatures of other environmental signals by both temporary there remains a great dearth of knowledge on 25:20 C day:night, with a PAR of 500 mol m–2 s–1 and permanent differential growth.1,2 The the gene products and their expression pat- and 60% relative humidity. The plants were directional growth of plants as a response to an terns. The purpose of this study was to map the watered to avoid water stress and received 100 external stimulus is called tropism. The move- proteome of the soybean (Glycine max) pulvi- ppm 20:20:20 fertilizer once a week. The plants ment of leaves by sunlight, or heliotropism, nus using trichloroacetic acid/acetone extrac- were harvested after the appearance of six or can angle leaf lamina both toward (diaheliotro- tion, 2-dimensional gel electrophoresis (2-DE) seven trifoliolate leaves (between six and eight pism) and away from (paraheliotropism) the and identification by liquid chromatography- weeks). The terminal and lateral pulvini from light depending on the intensity of the irradi- tandom mass spectrometry (LC-MS/MS). This the second through sixth trifoliolate leaves were ance, circadian rhythms (nyctinasty), and map would highlight the molecular and func- separately excised with a razor one to two hours environmental stresses. Diaheliotropic move- tional characterization of the pulvinus at the into the light period and frozen in liquid nitro- ment has been shown to increase water use protein level. The soybean has been selected gen. The pulvini were stored in a −80°C freezer efficiency and carbon assimilation compared because of its importance as a food crop, the until further use.

[page 8] [International Journal of Plant Biology 2014; 5:4887] Article

Protein extraction excised and rinsed twice with 50% methanol, tein clusters at 100%, 90%, and 50% homology Trichloroacetic acid (TCA)/acetone precipi- ten minutes each. The gel pieces were recon- as curated by UniRef. Proteins without a name tation, described previously by Natarajan et al. stituted and subsequently dehydrated in solu- at the 50% homology level remained uncharac- was used to extract pulvinar protein.17 For tions of 25 mM ammonium bicarbonate and terized. each of three biological replicates approxi- acetonitrile respectively, by placement on a Biological process gene ontology (GO) mately 2.0 g of pulvinus were ground into a shaker for ten minutes per solution. The prior terms as listed in the Gene Ontology powder using a mortar and pestle with liquid step was repeated for a second time. The spots Annotation (GOA) Database, a collaboration nitrogen, then extracted with a 10% TCA were further dried in a speed vac concentrator with the UniProt Knowledgebase, were for about 15 minutes. Each gel piece was then (w/v)/0.07% β-mercaptoethanol (v/v) in ace- retrieved for the proteins and input into the tone mixture. Following a minimum of one reswollen with a 20 μL aliquot of 10 ng/μL agriGO version 1.2 GO analysis program hour incubation at −20°C and centrifugation porcine trypsin (sequencing grade, Promega, (China Agricultural University, Beijing, China) at 14,000 g in 4°C for 20 minutes, the super- Madison, WI, USA) in 25 mM ammonium for single enrichment analysis. For this analy- natant was discarded. The pellet was rinsed bicarbonate and refrigerated for one hour in sis, the abundance of the GO terms found in 4°C before overnight incubation at 37°C. with 0.07% β-mercaptoethanol in acetone identified pulvinar proteins were compared to The excess trypsin solution surrounding the solution followed by centrifugation at 14,000 g a background list of soybean genes. Single gel spot was transferred to new tubes and the (4°C) for 20 minutes; the rinsing and centrifu- enrichment analysis indicates the dominant remaining peptides were extracted from the gation steps were repeated until the super- biological processes in the examined tissue gel pieces by 50% acetonitrile/5% trifluo- natant was clear. After vacuum drying, the pel- against the entire plant species. It is one of roacetic acid (TFA)(v/v). 50 L of the extrac- let was resolubilized in a 7 M urea, 2 M μ several available methods to compare the sig- tion mixture was added to each gel piece and thiourea, 4% CHAPS, 1% DTT solution and nificance of each GO term. In addition, the placed in a shaker for an hour. The super- sonicated on ice for 45 minutes. The super- analysis used as a default, a hypergeometric natants were added to the original trypsin natant was collected after centrifugation at statistical method with the Benjamini- digests and the extraction was repeated once 14,000 g (4°C) for 20 minutes and the protein Yekutieli correction method and a minimum more with another 50 L aliquot. The super- concentration was quantified using the μ five terms for significance.22 The reference GO natants were then dried for up to two hours on 18 term list was the soybean locus genome provid- Bradford method. a speed vac concentrator. The peptides were only ed by Phytozome (US Department of Energy, solubilized in a 20 L solution containing 5% μ Joint Genome Institute, Walnut Creek, CA, 2D gel electrophoresis acetonitrile /0.1% formic acid. For first dimension electrophoresis, 500 µg USA). GO terms significantly present in high- of protein in a solution of 7 M urea, 2 M use er numbers in the pulvinus tissue were dis- Mass spectrometry cussed. thiourea, 4% CHAPS, 50 mM DTT, 1% IPG The peptides were run through an LTQ buffer (pH 4-7), and 0.002% bromophenol blue Orbitrap XL hybrid linear ion trap Orbitrap were loaded onto 13 cm IPG strips, pH 4-7, and mass spectrometer (ThermoFisher Scientific, run on a flatbed Ettan IPGphor II (GE San Jose, CA, USA) with reverse-phase chro- Results and Discussion Healthcare, Piscataway, NJ, USA) under condi- matography on a 100×0.18 mm BioBasic-18 tions described earlier by Natarajan et al.: 30 V column and 3 μL/min flow rate. The 30-minute for 13 hours, 500 V for one hour, 1000 V for one linear gradient was 5-40% acetonitrile in a Protein extraction, identification, hour, 8000 V gradually for 1:30 hours, 8000 V 0.1% formic acid solution. The resolution sur- and functional analysis for 24000 Vhr, and 5000 V for ten hours.17 The vey scan was over the range 400-1600 m/z To investigate pulvinus protein functional final step was truncated if the protein (4=30000 at m/z 400) and for each polypeptide analysis, 2-DE was used to separate the appeared to be sufficiently separated. Prior to gel spot, the MS/MS spectra of the five most extracted proteins. A total of 183 spots were second dimension SDS-PAGE the IPG strips abundant ions were recorded. The electrospray then excised for LC-MS/MS analysis (Figure were equilibrated twice to reduce the disulfide voltage was 3.5 kV with normalized collision 1). MS/MS data run through Scaffold resulted bridges, first in DTT and then in iodoac- energy set to 30% and a minimum ion count of in the identification of 195 proteins; 18 spots etamide (IAA), 15 minutes each in equilibra- 5000. Mascot Distiller version 2.3.00 was of the 183 total either did not contain any pep- tion buffer (50 mM Tris-HCl pH 8.8, 6 M urea, employed for producing searchable peak lists. tides or had a protein identification probabili- 30% glycerol, 2% SDS, 0.002% Non-commercial bromophenol ty <95%. Fifty-five proteins were named direct- blue, 1% DTT). The strips were loaded onto Data analysis ly as a result of the Scaffold analysis workflow. 12.5% polyacrylamide gels using a Hoefer SE MS/MS data were analyzed by the Scaffold An additional 116 proteins were named 600 Ruby electrophoresis unit and run for 15 toolkit version 3 (Proteome Software, Portland, through the UniRef sequence clusters at the mA per gel for 30 minutes and 25 mA per gel OR). Scaffold searches MS/MS data against 100%, 90%, or 50% homology level. The for up to five hours. The gels were stained for several database search engines and computes remaining 24 proteins were not named even at two days using Coomassie Blue G-250. Gels a peptide probability incorporating similar the 50% homology level. Of the 195 proteins, were scanned on an ImageScanner III (GE results among the search engines. Based on 129 had GO terms for biological processes. Healthcare), and analyzed using Progenesis the peptide distribution, a protein probability Protein or cluster names, UniProtKB accession SameSpots software (Nonlinear Dynamics, is computed and the peptides are identified as numbers, UniRef accession numbers, molecu- Durham, NC, USA). Gels were stored in a parts of the computed protein.21 The MS/MS lar weight, corresponding gel spots, and genet- 17.5% aqueous ammonium sulfate solution data was searched against the UniProt ic information are listed in Supplementary until further use. Knowledgebase. The results were limited to G. Table 1. max, with minimum values of two significant AgriGO single enrichment analysis of 129 In-gel digestion peptide matches, 80% peptide identification proteins compared to a background soybean Trypsin digestion of selected spots was probability, and 95% protein identification genome locus identified 74 significant GO based on methods by Shevchenko et al. (1996) probability. Uncharacterized proteins were terms (data not shown). Most of the 74 terms and Gharahdaghi et al. (1999).19,20 Spots were identified by examining the homologous pro- fell under three broad parent terms: nucle-

[International Journal of Plant Biology 2014; 5:4887] [page 9] Article obase, nucleoside, and nucleotide metabolic processes (23 child GO terms), nitrogen com- pound metabolic processes (13 child GO terms), and transport (11 child GO terms). All terminal significant child GO terms are listed in Supplementary Table 2. The top two signifi- cant biological process GO terms were related to proton transport. The third significant term was a negative regulator of proton transport (oxidative phosphorylation, GO:0006119). Photosynthesis and respiration were also enriched compared to the reference back- ground.

Proton transport The two most significant (lowest e-values) GO terms were related to the transport of pro- tons through hydrolysis and synthesis of ATP (Supplementary Table 2). This would indicate Figure 1. Representative SDS-PAGE gel of soybean lateral pulvinus, pH 4-7. The lateral a higher demand for proton transport in the and terminal pulvinus did not significantly differ from one another (data not shown). pulvinus relative to the overall soybean plant The numbers correspond to the proteins identified through LC-MS/MS and listed in and confirms our understanding of turgor Supplementary Table 1. Eighteen spots did not produce significant hits. change in pulvinar motor cells. The changes in the different turgor pressures between the adaxial and abaxial motor cells that result in leaf movement are triggered by K+ and Cl- flux nus the dissociated anion of malic acid, tein only type identified by significant GO terms, across the plasma membranes. Proton concen- malate, is believed to serve as an additional studies have found actin (spots 53, 55, 57) to trations dictate the level of activity in ion chan- source of charge balance in the cell, as the co- be more integral to pulvinar bending. The GO nels and consequent osmotic movement.7 transport of Cl- does not account for all the pos- terms for actin proteins in the soybean pulvi- Vacuolar H+-ATPases are believed to also itive charge associated with K+ movement.usenus, however, have yet to be annotated and assist in Ca2+ accumulation through the gen- Malic acid appears in higher concentrations were therefore unable to be included in the eration of the electrochemical gradient across during the day in the whole pulvinus of P. coc- analysis. Kanzawa et al. and Yao et al. detected the fragmentation of actin filaments in the tonoplast.23 In their examination of cineus motor cells than at night, and increased + response to cold and electrical stimuli, respec- apoplast pH levels in the nyctinastic S. saman concentrations correspond to regions of K tively in Mimosa pudica pulvini; Kanzawa et al. pulvini, Lee and Satter confirmed that swelling accumulation. The majority of malic acid also detected microtubule fragmentation but of motor cells corresponded to proton extru- occurs through de novo synthesis in situ microtubule modulators did not appear to sion, and the same light treatment resulted in (~80%), not by transport between the abaxial affect pulvinar bending while actin modulators opposing proton fluxes between the adaxial and adaxial regions.25 Malate is also known to did.1,27 Yao et al. noted that both actin and abaxial motor cells.24 Okazaki found simi- be an important counter-ion in guard cells.26 rearrangement and the presence of intracellu- lar pH changes in P. vulgaris pulvini and noted Four of the five proteins with the malate lar Ca2+ were required for bending.27 Several that an increase of extracellular pH were metabolism annotation were various isoforms 2+ accompanied by inhibition of plasma mem- of malate dehydrogenase (spots 83-85, 87). other proteins with Ca -dependent activity were detected in the pulvinus, including inosi- brane H+-ATPase activity which would lead to The fifth, malic enzyme (spot 13), is involved tol 3-phosphate synthase (spot 31), apyrase turgor decrease.15 Unlike Lee and Satter, in the synthesis of malic acid. A precursor to (spots 33 and 91), and annexin (spot 110). Okazaki found that blue light increased extra- malate is sucrose, believed to be present in These three proteins could function as early cellular pH in both adaxial and abaxial proto- motor cells by import from nearby tissues and Non-commercial participants in signal-transduction pathways plasts and speculated that the relative differ- also by the degradation of starch. One protein that lead to ion movement across the plasma ences in turgor between the two regions identified as the starch degrading enzyme membrane. resulted from the differences in the amount of beta-amylase was detected in various amounts blue light received. It is important to remem- across eight spots in the pulvinus gel (spots 15-17, 28-30, 51 and 62). Ion transporters and aquaporins ber that the responses measured by the two Along with proton ATPases was the expecta- groups of researchers were not the same; Lee Cytoskeletal processes tion of finding aquaporins and K+ transport and Satter were looking at the leaf movements Two significant GO terms, protein polymer- proteins in the pulvinus. Previous studies have of a nyctinastic plant while Okazaki was exam- ization and microtubule-based movement, suggested the presence of aquaporins, and at ining the heliotropic leaf movement of P. vul- were both annotated to eight tubulin proteins least two K+ transporters for inward and out- garis. The apparent differences in pH response (spots 18-21, 24, 25, 27, 38, 39 and 43). The ward ion movement.28,29 The channels, con- to blue light may be a result of a photoreceptive multiple tubulin proteins are an example of the ventionally labeled as K and K for hyperpolar- signaling pathway unique to heliotropic plants. H D tetraploid nature of soybean; despite having ized and depolarized K+ channels, respectively, Malate metabolism over 86% sequence homology five tubulin beta- correspond to inward and outward ion move- Malate metabolism was the fifth most-com- 2 chain proteins are encoded by genes located ment. However, Yu et al. found evidence for a on five different chromosomes (data not mon significant GO term as calculated by voltage-independent KH channel not affected agriGO (Supplementary Table 2). In the pulvi- shown). While tubulin is the cytoskeletal pro- by extracellular pH levels that appears to serve

[page 10] [International Journal of Plant Biology 2014; 5:4887] Article not a minor role in K+ influx.30 Fluerat-Lessard Pulvinus proteome compared to guard cells to the pulvinus conceivably skews the GO et al. detected high levels of a 23 kDa aquapor- Besides the pulvinus motor cell, another cell analysis. Additional comparisons of the two in in addition to V-H+ATPases in the aqueous demonstrating rapid turgor changes is the proteomes would require a higher amount of vacuole of mature M. pudica pulvini compared guard cell. Like the pulvinus, guard cells protein extraction from soybean pulvinus, and to juvenile, non-functioning pulvini.31 At least require H+-ATPase activity triggered by light care must be taken to compare the soybean two aquaporin-like gene products were suc- for its ion transport and subsequent water pulvinus with the guard cell profile of another cessfully cloned from S. saman pulvini; one of movement. In guard cells H+-ATPase activity in tetraploid organism. the two, SsAQP2, also demonstrated diurnal the plasma membrane increases when and circadian patterns and was not found to an exposed to blue light, with a corresponding appreciable degree in nearby leaf tissue.28 decrease of extracellular pH and turgor Unfortunately we did not detect any other increase.26 However the opposite response Conclusions transporters (other than ferritin, spot 147) or occurred in Okazaki’s examination of P. vul- any proteins localized to the plasma membrane garis motor cells, where blue light illumination To our knowledge, this is a novel report on in the soybean pulvinus. There could be sever- led to the inhibition of H+-ATPase.15 This sug- the analysis of protein in soybean pulvinus. al explanations for the lack of these proteins. gests that, despite functional similarities, These findings provide a better understanding of the molecular basis of pulvinar protein func- For one, aquaporins have an isoelectric point guard cells and heliotropic pulvinus motor cells tion. In summary, 195 proteins were extracted (~8) higher than the range that was used in have distinct mechanisms for their turgor and positively identified from 165 spots from this study. Furthermore, plasma membrane responses. It could be that guard cells, more the pulvinus gel. Gene ontology analysis of sig- proteins typically are not recovered from tradi- often located on the abaxial leaf surface, have nificant terms found the top dominant GO bio- tional protein extraction methods, requiring a similar photoreceptive mechanism to abaxial logical processes were related to proton trans- an additional partitioning step and special motor cells of nyctinastic plants such as S. 32 + port, malate metabolism, and cytoskeletal extraction buffers. V-type H -ATPases, such saman. Therefore comparing the proteomes of movement, which are in good agreement with as the three detected in the soybean pulvinus, the two tissues could assist in determining the previous studies on pulvinar response. are not only localized in vacuoles but may also degree of similarity of guard cells to pulvini of Regrettably many highly basic proteins were be located in the plasma membrane. The GO either heliotropic or nyctinastic plants. notonly detected, including proteins at isoelectric annotation for cellular component of the three Zhao et al. examined the diploid Arabidopsis points above pH 7 and plasma membrane pro- V-H+ATPases did not specify plasma mem- 35 guard cell proteome. Their top GO term was teins. In order to recover the latter, methods brane or the tonoplast; given the dearth of related to stress response (response to cold), other plasma membrane proteins it seems such as two-phase partitioning and the use of with energy categories making up threeuse of the special buffers would be necessary to recover more likely that the H+ATPases detected were top eight. In the soybean pulvinus GO terms for the integral membrane proteins. Finally, the from endomembranes. photosynthesis and respiration were less sig- number of proteins recovered from the pulvi- nificant than in the Arabidopsis guard cell. The nus will have to increase for future compara- Pulvinus proteome compared to most abundant protein found in the tive studies with proteins of the guard cell. the root, leaf, and guard cell Arabidopsis guard cell was the stress response The pulvinus proteome profiled here was protein TGG1, which functions as a defense compared with the leaf proteome identified by against pathogen attack.35 In the soybean pul- Xu et al. to verify differences between the two vinus protein abundance was not measured References tissues.33 One advantage of comparing the two and so no similar observations could be made. tissues is that the protein extraction process The different rankings of significant GO terms 1. Kanzawa N, Hoshino Y, Chiba M, et al. as well as the soybean cultivar examined were may reflect different mechanisms between the Change in the actin cytoskeleton during the same in both studies. The distribution of two. Zhu et al. compared mesophyll and guard seismonastic movement of Mimosa pudi- pulvinar proteins in the various functional cat- cells from the tetraploid crop species Brassica ca. Plant Cell Physiol 2006;47:531-9. egories were expected to differ from the leaf napus using a combination of quantitative GO 2. Herrera R, Krier C, Lalanne C, et al. because of the dominant biological functions analysis and a qualitative functional approach Keeping the stem straight: a proteomic distinct for type of tissue. Xu et al. identified a following the functional categories listed by analysis of maritime pine seedlings under- large number of soybean leaf proteinsNon-commercial involved Bevan et al. for the Arabidopsis genome. 34,36 going phototropism and gravitropism. in energy production.33 Several of those pro- Zhu et al. found the most highly enriched BMC Plant Biol 2010;10:217. teins were also identified in the pulvinus, guard cell proteins relative to the mesophyll 3. Zhang YL, Zhang HZ, Feng GY, et al. Leaf including rubisco large subunit, rubisco acti- cell fell under the categories of energy, photo- diaheliotropic movement can improve car- vase, and oxygen-evolving enhancer proteins 1 synthesis, membrane and transport, metabo- bon gain and water use efficiency and not and 2, among others. The pulvinus proteome lism, and stress response. Categorization of intensify photoinhibition in upland cotton also contained ATP synthases and two other the soybean pulvinus proteins using the Bevan (Gossypium hirsutum L.). Photosynthetica proteins involved in nucleoside phosphate et al. method found similar results for the 2009;47:609-15. biosynthesis which did not appear in the leaf major groups (data not shown). In contrast to 4. Rakocevic M, Neumaier N, Oliveira GM, et proteome. While the separation methods for the guard cell proteome, cytoskeletal and other al. Heliotropic responses of soybean culti- the two tissues were largely the same, Xu et al. structural proteins appeared to factor in more vars at three phenological stages and used the method of Bevan et al. for classifying strongly in the pulvinus. Both Zhao et al. and under two water regimes. Pesqui Agropecu the proteins without reporting significant GO Zhu et al. recovered around 10-fold more pro- Bras 2010;45:661-70. terms.34 As a result, direct side by side compar- teins than recovered in soybean pulvinus pro- 5. Kao WY, Forseth IN. Diurnal leaf move- ison of significant molecular processes cannot teins, including many of the plasma membrane ment, chlorophyll fluorescence and carbon be undertaken. In general, the pulvinus pro- proteins that were not extracted using the assimilation in soybean grown under dif- teome contained many more stress-response methodology of this study. The magnitude of ferent nitrogen and water availabilities. proteins than the leaf. detected proteins in the guard cells compared Plant Cell Environ 1992;15:703-10.

[International Journal of Plant Biology 2014; 5:4887] [page 11] Article

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