Histological, Physiological, and Comparative Proteomic Analyses

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Histological, Physiological, and Comparative Proteomic Analyses Provide Insights into Leaf Rolling in Brassica napus Wenjing Chen, Shubei Wan, Linkui Shen, Ying Zhou, Chengwei Huang, Pu Chu, and Rongzhan Guan J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00744 • Publication Date (Web): 19 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

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Journal of Proteome Research

Histological, Physiological, and Comparative Proteomic Analyses Provide Insights into Leaf Rolling in Brassica napus Wenjing Chen, Shubei Wan, Linkui Shen, Ying Zhou, Chengwei Huang, Pu Chu* and Rongzhan Guan* National Key Laboratory of Crop Genetics and Germplasm Enhancement, Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, No. 1 Weigang, Nanjing, Jiangsu 210095, PR China

ABSTRACT: Moderate leaf rolling is important in ideotype breeding, as it improves photosynthetic efficiency and therefore increases crop yields. To understand the regulatory network of leaf rolling in Brassica napus, a down-curved leaf mutant (Bndcl1) has been investigated. Physiological analyses indicated that the chlorophyll contents and antioxidant enzyme activities were remarkably increased and the photosynthetic performance was significantly improved in Bndcl1. Consistent with these findings, 943 differentially accumulated proteins (DAPs) were identified in the Bndcl1 mutant and its wild-type plants using iTRAQ-based comparative proteomic analyses. Enrichment analysis of proteins with higher abundance in Bndcl1 revealed that the functional category “photosynthesis” was significantly overrepresented. Moreover, proteins associated with oxidative stress response and photosystem II repairing were also up-accumulated in Bndcl1, which might help the mutant to sustain the photosynthetic efficiency under unfavorable conditions. Histological observation showed that the mutant displayed defects in adaxial-abaxial patterning. Important 1

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DAPs associated with leaf polarity establishment were detected in Bndcl1, including ribosomal proteins, proteins involved in post-transcriptional gene silencing and proteins related to brassinosteroid. Together, our findings may help clarify the mechanisms underlying leaf rolling and its physiological effects on plants, and may facilitate ideotype breeding in Brassica napus.

KEYWORDS: Brassica napus, leaf rolling, photosynthesis, adaxial-abaxial polarity, leaf proteome

INTRODUCTION Leaves are the primary organs of photosynthesis in most plants and leaf development affects the formation of crop yield and the establishment of plant architecture.1 Morphogenesis of normal leaves requires the establishment and coordination of proximodistal, mediolateral, and dorsoventral (also known as adaxial-abaxial) polarities.2, 3 The specification of dorsoventral polarity is crucial for flattened lamina formation and alterations to the adaxial-abaxial polarity specification generally result in an upward- or downward-curling leaf phenotype.4 Moderate leaf rolling is considered as a critical element in crop ideotype breeding as it modifies plant architecture, improves photosynthetic efficiency, delays leaf senescence, and alleviates damages from drought, heat and high light stresses.1, 5, 6 Recently, mutants with rolled leaves were isolated and the underlying molecular mechanisms have been intensively studied in Arabidopsis thaliana and Oryza sativa.7 2


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A complex gene regulatory network is implicated in the determination of adaxial-abaxial polarity,8 which plays an essential role in the molecular mechanism controlling leaf rolling. Components specifying the adaxial cell identity include the Myb domain gene ASYMMETRIC LEAVES1 (AS1),9 the LATERAL ORGAN BOUNDARIES domain gene AS2,10, 11 the trans-acting short interfering RNAs (ta-siRNAs),12 the HOMEODOMAIN-LEUCINE ZIPPER Class III (HD-ZIP III) genes13,14 and a HD-ZIP IV gene Roc5 in rice.15 Abaxial determinants include the KANADI (KAN) family genes, KAN1-3 in Arabidopsis16 and SHALLOT-LIKE1(SLL1) in rice,6 the AUXIN RESPONSE FACTOR (ARF) genes,17 the YABBY (YAB) family genes,18 and two microRNAs (miR165 and miR166).19 The mutual antagonistic interactions between these determinants, which contribute to the acquisition and maintenance of leaf polarity,20 are critical for asymmetric leaf development.21 Moreover, the production and regulation of miR165/miR166 and tasiRNAs requires the participation of ARGONAUTE1 (AGO1), RNA-DEPENDENT POLYMERASE6 (RDR6), SUPPRESSOR OF GENE SILENCING3 (SGS3), AGO7 and DICER-LIKE 4 (DCL4), 22-24 which explains their importance in the determination of leaf polarity. In addition to the transcription factor families and small RNAs, accumulating evidence has illustrated that hormones, especially brassinosteroid (BR), are related to the leaf morphology regulation. Mutants impaired in BRs biosynthesis or response usually exhibit dwarf phenotypes with rolled or erect leaves. BRASSINOSTEROID INSENSITIVE 1 (BRI1) is essential in BR signal transduction, and bri1 mutant in Arabidopsis displayed a severely dwarfed phenotype with shorter petioles and curled 3



rosette leaves.25 Similar to the Arabidopsis mutant bri1, the tomato BR-deficient mutant dumpy (dpy) and BR-insensitive mutant curl-3 (cu-3) showed extreme dwarfism and downward curling leaves.26 ROLLED and ERECT LEAF 1 (REL1) affects leaf morphology through its participation in BR signaling transduction and rel1 dominant mutant displayed pleiotropic phenotypes including dwarfism and adaxially rolled leaves.27 OsDWARF4 encodes a cytochrome P450 (CYP90B1) which catalyzes the rate-determining step of BR biosynthesis, and BR deficiency led to the erect leaf phenotype of osdwarf4, which improved its biomass production and grain yield.28 Rapeseed (Brassica napus) is one of the world’s most important oilseed crops. Few mutants with curly leaves have been identified in rapeseed, and the mechanisms underlying leaf rolling in B. napus remain to be explored. In our previous studies, a down-curved leaf B. napus mutant (Bndcl1) with reduced plant height and shorter petioles was isolated by ethyl methanesulfonate (EMS) treatment, and the dominant mutant gene has been mapped to chromosome C05 of B. napus, within a 175 kb region.29 Here, the protein profiles of Bndcl1 and its corresponding wild-type (WT) plants were compared using the isobaric tags for relative and absolute quantitation (iTRAQ) technology and important differentially accumulated proteins (DAPs) responding to Bndcl1 mutation were screened. The biological processes potentially related to the altered phenotypes of Bndcl1 were analyzed with the aim to reveal the effects and the mechanisms of leaf rolling in B. napus.

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EXPERIMENTAL SECTION Plant Materials The Brassica napus down-curved leaf mutant Bndcl1 was isolated from EMS-treated Brassica napus cv. ‘NJ7982’. Seeds were planted in pots and grown as previously described.30 Seven weeks after sowing, the phenotypic differences between the Bndcl1 mutant and the corresponding WT plants became evident, and samples were taken at this time point. The fifth leaves from Bndcl1 and the WT plants were collected, frozen in liquid nitrogen immediately and kept at -80°C until use. The plants used in each experiment were grown in parallel under identical conditions, and four biological replicates were prepared for the subsequent analyses. Histology and Microscopy Histology analyses were performed as described before 31, 32 with minor changes. Samples from mature leaf blades were fixed in FAA (10% formaldehyde, 5% acetic acid, 50% ethanol) at 4 °C for 24 h, dehydrated through a graded ethanol series, cleared in dimethylbenzene and embedded in paraffin. Transverse-sections were cut using a microtome (Leica RM2016), and stained in toluidine blue. Bright-field photographs were taken using the Olympus BX53 microscope. Pigment Determination, Chlorophyll Fluorescence Measurement and Photosynthetic Characteristics The fully expanded leaves of Bndcl1 and the WT plants were used for pigment determination, in situ Chlorophyll (Chl) fluorescence and gas-exchange measurements. Chl were extracted and their contents were determined as described 5



previously.33 The leaves of each plant were measured five times and all data were collected between 09:00 am and 11:00 am. The maximal and effective quantum yield of photosystem II (PSII), photochemical quenching (qP) and non-photochemical quenching parameters (NPQ) were determined using a Mini-PAM photosynthesis yield analyzer (Walz, Effeltrich, Germany) following the manufacturer’s instruction and previous reports.34, 35 Plants were kept in darkness for at least 20 min before the maximal quantum yields were measured. The photosynthetic parameters were determined with a Li-Cor 6400 portable photosynthesis system (Li-Cor Inc. Lincoln, NE, USA) at 23°C as described before 30. Each experiment has four biological replicates. Assay of Antioxidant Enzymes The leaf samples (0.5 g) were grounded with 5mL of 0.05 M potassium phosphate buffer (pH 7.8) and centrifuged at 12,000 g for 20 min at 4 °C. The supernatants were collected for further analyses. The enzyme activity of peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) were measured spectrophotometrically as previously described.36, 37. Each experiment has four biological replicates. Determination of Superoxide Anion Radical (O2• −), Hydrogen peroxide (H2O2) and Thiobarbituric Acid Reactive Substances (TBARS) The concentration of the O2• −was determined spectrophotometrically as previously described.38 H2O2 contents were measured by using commercial reagent kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The TBARS assay was performed for the evaluation of lipid peroxidation as previously described.39, 40 Each 6


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experiment has four biological replicates. Protein Extraction, Digestion and iTRAQ analysis Protein extraction, digestion and iTRAQ analysis of leaf tissue from WT and Bndcl1 plants were performed as previously described with some changes.33 Prepared samples were finely grounded and dissolved with 200µl triethylammonium bicarbonate (TEAB) dissolution buffer (8 M urea, 100 mM TEAB, pH 8.0). The mixture was centrifuged for 20 min at 12000 r/min, and the supernatant collected was precipitated by pre-cooled acetone containing 10mM dithiothreitol (DTT) at −20◦C for 2 hours. Samples were then reduced with 10mM DTT at 56◦C for 1h. The precipitate was collected after centrifugation for 20 min at 12000r/min, and then dissolved with 100 µl TEAB buffer. The protein content was quantified using the Bradford assay. 41 After reduction and alkylation, trypsin (Promega) was added in protein samples at a ratio of 1:50 (w: w) and incubated overnight at 37 °C for protein digestion. Samples were then labeled with iTRAQ 8-plex kits (AB Sciex UK Limited, Warrington, Cheshire, UK). Four biological replicates were used for each sample. Peptides were labeled with iTRAQ tags 113, 114, 115 and 116 for the WT samples, while 117, 118, 119 and 121 for the mutant samples. The labeled samples were mixed in equivalent amount and lyophilized. The peptide mixture was dried, dissolved in buffer A (20 mM ammonium formate, pH 10.0) and fractionated using high-performance liquid chromatography (HPLC) system (Thermo Fisher, DINOEX Ultimate 3000 BioRS) with a Durashell C18 column (Welch Materials, Inc., 5 um, 100 Å, 4.6×250 mm). Peptides were eluted with a gradient of 3%-8% buffer B (20 7



mM ammonium formate, 80% acetonitrile, pH 10.0) for 5 min, 8%-18% buffer B for 12 min, 18%-32% buffer B for 11 min, 32%-45% buffer B for 5 min, 45%-80% buffer B for 5 min, and 80% buffer B for 5 min, at a flow rate of 1 ml/min. The collected fractions were vacuum dried and finally combined into 12 pools, then desalted and subjected to liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI MS/MS) analysis. LC-ESI MS/MS Analysis An ekspertTM nanoLC 400 HPLC system coupled to the Triple TOF 5600 plus (AB SCIEX, USA) was used for LC-ESI-MS/MS analysis as previously described 42. Data was acquired using following settings: ion spray voltage 2.5 kV, curtain gas 30 PSI, nebulizer gas 5 PSI, and interface heater temperature 150 °C. MS1 spectra were collected in the range 350-1500 m/z for 250 ms. For precursor ion selection, the mass tolerance was set to 50 mDa. The 40 most intense precursors which exceeded 150 counts per second with a 2+ to 5+ charge state were selected for fragmentation. MS2 spectra were collected in the range 50-2000 m/z for 100 ms. Dynamic exclusion was set for 12 s. Protein Identification and Quantification MS/MS data were searched using ProteinPilot Software v4.5 (AB SCIEX, USA) against the ‘Brassica napus’ subset of the NCBI non-redundant sequence (NR) databases (176703 entries, update in October 2015) for protein identification, as previously described 33. The search parameters used were as follows: iTRAQ 8-plex peptide label, trypsin enzyme, cysteine modified with iodoacetamide, thorough search 8


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mode, and biological modifications. The False Discovery Rate (FDR) was estimated using the Proteomics System Performance Evaluation Pipeline (PSPEP) algorithm integrated into the ProteinPilot and only data with a FDR < 5% were used for further analysis. Proteins with at least one unique peptide identified and the unused value > 1.3 were considered for subsequent analysis.42 For protein quantification, a protein ratio was automatically calculated by ProteinPilot software using weighted average of log-transformed peptide ratios. Shared peptides and missing values were discarded and were not taken into account for protein quantification. The normalization method was set as median and the data were normalized by bias correction and background correction. A 1.5-fold cutoff was used for fold-change analyses, with a p-value (provided by student's t-test, corrected by the Benjamini-Hochberg approach) < 0.05 present in at least three replicates 43. Bioinformatics and Enrichment Analyses All sequences of proteins identified were used for searching against the NCBI NR database, with the e-value < 1.0E-5. Functional annotation of the proteins identified was then conducted by searching against the Gene Ontology (GO) database, 44 the Clusters of Orthologous Groups of proteins (COG) database 45 and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. 46, 47 For GO and KEGG pathway enrichment analysis, an FDR-adjusted p value less than 0.05 was used as the threshold.48 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

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Plant samples for proteomics experiments were used for qRT-PCR analyses. Total RNA extraction and qRT-PCR were conducted according to the method described before.39 Gene-specific primers were designed according to the corresponding sequences of the selected genes in Brassica napus genome (http://www.genoscope.cns.fr/brassicanapus/cgi-bin/gbrowse/colza/) and the primer sequences used are listed in Table S1. The Actin2-like (BnaC06g08720D) was used as an internal control for the normalization, as the iTRAQ data indicated that its level is not affected by Bndcl1 mutation. Three biological replicates were used.

RESULTS Histological Analyses of Bndcl1 leaves Compared with the WT plants, the Bndcl1 mutant exhibited compact phenotype at seedling stage, with down-curved leaves and shorter petioles (Figure 1A and B). To uncover the cellular basis of this mutant phenotype, histological analyses were conducted. Reduced asymmetry was noticed in Bndcl1 leaves. The number of chloroplasts was significantly greater in Bndcl1, especially in the spongy mesophyll cells (Figure S2), and intercellular spaces between spongy mesophyll cells were smaller in the mutant than those in the WT leaves (Figure 1C and D). Moreover, cells were fewer in the abaxial phloem tissue and the abaxial epidermal cells below the leaf vein were significantly enlarged (Figure 1E and F) in Bndcl1 mutant, accompanied by a considerably low extent of incurvature of this section. Improved Photosynthetic Performance in Bndcl1 Mutant 10


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To assess the effects of leaf rolling on photosynthetic performance in Bndcl1 mutant, the Chl contents, Chl fluorescence and gas exchange parameters were determined. Compared to the WT plants, the contents of Chl a, Chl b, total Chl and the ratio of Chl a/b in Bndcl1 were significantly increased (Table 1). The maximal quantum yield (dark-adapted Fv/Fm) of PSII, photochemical quenching (qP), and non-photochemical quenching (NPQ) remained unaffected in Bndcl1 (Table 2). However, the effective quantum yield of PSII (ΦPSII) was significantly increased in the mutant, due to the 8.6% increase of the maximal fluorescence in light-adapted leaves (Fm’). Table 1. The chlorophyll contents of WT and Bndcl1 mutant. Data are presented as means ± standard deviation (SD), n=4. * P < 0.05. Genotype Chl a (mg·g-1FW) Chl b (mg·g-1FW) Total Chl (mg·g-1FW) Chl a/Chl b

WT 3.448±0.588 1.995±0.079 5.443±0.668 1.723±0.226

Bndcl1 5.686±0.550 * 2.278±0.103 * 7.965±0.651 * 2.492±0.130 *

Table 2. Determination of chlorophyll fluorescence parameters in WT and Bndcl1 mutant. Data are presented as means ± SD, n=4. * P < 0.05. Genotype WT Dark-adapted F0 213.00±13.23 Fm 1251.30±98.01 Fv/Fm 0.829±0.007 Light-adapted leaves F 138.50±6.84 Fm’ 595.45±21.22 ΦPSII 0.766±0.007 qP 1.185±0.033 qN 0.582±0.028 NPQ 0.920±0.094

Bndcl1 218.85±7.39 1289.95±26.77 0.830±0.006 140.40±6.14 646.55±25.92 * 0.783±0.006 * 1.286±0.081 0.627±0.043 1.039±0.132

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Gas exchange measurements (Table 3) showed that compared to the WT plants, net photosynthesis and transpiration rate were increased by 36% and 62% in the mutant, respectively, while no significant difference was observed for the stomatal conductance and intercellular CO2 concentration between Bndcl1 and its corresponding WT plants. These results taken together demonstrated that photosynthetic performance was improved in the Bndcl1 mutant. Table 3. Leaf gas exchange measurements of WT and Bndcl1 mutant. Data are presented as means ± SD, n=4. * P < 0.05, ** P < 0.01. Genotype

Net photosynthetic rate (µmol CO2 m-2 s-1)

Stomatal conductance (mol H2O m-2 s-1)

Intercellular CO2 concentration (µmol CO2 mol-1)

Transpiration rate (mmol H2O m-2 s-1)

WT Bndcl1

9.04±0.21 12.30±1.06**

0.12±0.03 0.16±0.03

265.25±25.20 256.50±17.94

2.32±0.44 3.78±0.53**

Increased Antioxidant Enzyme Activities and Reduced Oxidative Damages in Bndcl1 Mutant The generation of reactive oxygen species (ROS) is inevitable during the light reaction of photosynthesis and the PSII is vulnerable to light-induced damage (photoinhibition). O2•− is involved in the photoinhibitory process and free H2O2 is generated by dismutation of O2•−.49 Interestingly, the O2•− level and the H2O2 content were reduced by 39% and 41% in Bndcl1, respectively (Figure 2A and B), suggesting that the ROS levels were significantly decreased in Bndcl1. Moreover, Bndcl1 showed approximately 20% lower TBARS concentration than the WT plants (Figure 2C), implying reduced level of membrane lipid peroxidation in the mutant. In consistent with these observations, statistically significant increase of antioxidant enzyme 12


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activity was noticed in Bndcl1. The activities of the POD, SOD and CAT were increased by 34%, 30% and 43% in Bndcl1 leaves, respectively, as compared with the corresponding WT plants (Figure 2D-F). Characterization of DAPs in Bndcl1 Mutant To explore the leaf proteome profile and to identify DAPs in response to Bndcl1 mutation, iTRAQ-based quantitative proteomic analysis was performed. The repeatability of the replicates were estimated by coefficient of variation (CV) analysis. The cumulative fraction of CV values for the WT and Bndcl1 samples were 76% and 80%, respectively (Figure S1A), with the CV threshold set at 20%. Meanwhile, the median CVs of the WT and Bndcl1 replicate samples were 14% and 13%, respectively (Figure S1B), suggesting a good biological reproducibility of this iTRAQ analysis. Overall, 367,283 spectra were generated, of which 124661 spectra were identified (≥95% confidence) and 26,986 spectra matched known peptides (Table S2). In total, 5019 proteins were identified with a FDR

Histological, Physiological, and Comparative Proteomic Analyses

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