Comparative Physiological and Proteomic Analysis of Two Sugar Beet

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Comparative physiological and proteomic analysis of two sugar beet genotypes with contrasting salt tolerance Yuguang Wang, PIERGIORGIO STEVANATO, Chunhua Lv, Renren Li, and Gui Geng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00244 • Publication Date (Web): 09 May 2019 Downloaded from http://pubs.acs.org on May 10, 2019

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Journal of Agricultural and Food Chemistry

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Comparative physiological and proteomic analysis of two sugar beet genotypes with

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contrasting salt tolerance

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Yuguang Wang1,2, Piergiorgio Stevanato3, Chunhua Lv1,2, Renren Li1,2, Gui Geng1,2*

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1

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of Heilongjiang University, Heilongjiang University, Harbin, 150080 China.

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2

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Heilongjiang University, Harbin, 150080 China.

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3

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Università degli Studi di Padova, Viale dell’Università 16, Legnaro, Padova 35020, Italy.

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Key Laboratory of Sugar Beet Genetic Breeding of Heilongjiang Province, Crop Academy

National Sugar Crop Improvement Centre, Crop Academy of Heilongjiang university,

DAFNAE, Dipartimento di Agronomia, Animali, Alimenti, Risorse Naturali e Ambiente,

*Corresponding author: [email protected].

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ACS Paragon Plus Environment


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Abstract:

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Soil salinity is one of the major constraints affecting agricultural production and crop yield.

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A detailed understanding of the underlying physiological and molecular mechanisms of the

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different genotypic salt tolerance response in crops under salinity is therefore a prerequisite

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for enhancing this tolerance. In this study, we explored the changes in physiological and

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proteome profiles of salt-sensitive (S210) and tolerant (T510) sugar beet cultivars in response

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to salt stress. T510 showed better growth status, higher antioxidant enzymes activities and

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proline level, less Na accumulation and lower P levels after salt-stress treatments. Using

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iTRAQ-based comparative proteomics method, 47 and 56 differentially expressed proteins

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were identified in the roots and leaves of S210, respectively. In T510, 56 and 50 proteins

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changed significantly in the roots and leaves of T510, respectively. These proteins were found

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to be involved in multiple aspects of functions such as photosynthesis, metabolism, stress and

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defense, protein synthesis and signal transduction. Our proteome results indicated that

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sensitive and tolerant sugar beet cultivars respond differently to salt stress. The proteins that

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were mapped to the protein modification, amino acid metabolism, TCA cycle, cell wall

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synthesis and ROS scavenging changed differently between the sensitive and tolerant

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cultivars, suggesting that these pathways may promote salt tolerance in the latter. This work

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leads to a better understanding of the salinity mechanism in sugar beet and provides a list of

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potential markers for the further engineering of salt tolerance in crops.

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Keywords：Sugar beet, Salt stress, iTRAQ, Proteomics, Mass spectrometry

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Introduction

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Salt stress is one of the main constraints affecting agricultural production and crop yield,

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particularly in arid or semi-arid regions 1. More than 20% of cultivated land, which accounts

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for half of the total irrigable land, is currently influenced by salt stress 2. Many crops

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are susceptible to salt stress, with adverse effects on crop production. Improving salt stress

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tolerance in crops has therefore become an urgent priority, and a lot of effort has been made

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to exploit novel salt tolerant genes and explore the molecular mechanism of salt tolerance.

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Salt stress impedes plant growth and development through ion toxicity, oxidative stress,

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osmotic stress and causing nutrient imbalance

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evolved many of different strategies to resist salt stress and developed responses at molecular,

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cellular, tissue and whole plant levels 5. For example, several studies demonstrated that

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selective absorption ions, ion exclusion, increasing osmotic adjustment ability and enhancing

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of antioxidant enzyme activities are implicated in plant salt stress tolerance

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physiological and biochemical processes, numerous genes responsible for ion transport,

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regulating osmotic homeostasis and detoxification have been found to change expression at

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transcriptomic level

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salt-responsive genes have been acquired in some plant species 8-9. These data provide a lot of

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information for understanding salt response pathways and mechanisms of salt-stress tolerance.

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However, transcriptome results do not always correlate with the data of proteomic study.

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Post-transcriptional and post-translational modifications may be the reason for these

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significant differences

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tolerance, it is necessary to investigate temporal protein variations under salinity conditions.

8.

10.

3-4.

Under high salinity conditions, plants have

6-7.

In these

With high-throughput transcriptome sequencing, many of

Therefore, in order to understand the plant responses to salt

3



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Proteomics, the analysis of global protein changes, have been proven as a useful tool for

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identification of key proteins responsible for salt tolerance. For example, proteomic analysis

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of the response of Sesuvium portulacastrum leaves to high salinity was performed

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found that a Na+/H+ antiporter and some ATP synthase subunits showed increasing protein

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abundances under salt stress. These proteins may play a key role in regulating ionic

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homeostasis in true halophytes. Furthermore, Arabidopsis thaliana and Thellungiella

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salsuginea, with different salt stress genotypes, were selected as the experimental materials,

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and the changes of microsomal proteins were analyzed in the leaves of both species by

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two-dimensional fluorescence difference gel electrophoresis. Finally, several salt-responsive

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membrane associated proteins were identified, and this result proved that molecular

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mechanisms primed to deal with salt stress might be the main reason for T. salsuginea

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exhibiting salt stress tolerance 12.

11.

They

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Sugar beet (Beta vulgaris L.) is one of the most important sugar sources and contributes

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about one third of the world’s annual sugar production. It is also used for the production of

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bioethanol as a renewable energy source. Sugar beet is also known as a salt-tolerant plant,

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which can grow in soil containing 140 mM salt 4. Previously, we identified salt stress

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tolerance in differential sugar beet cultivars, and several sugar beet cultivars were classified

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as being either salt-sensitive or salt-tolerant

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salt-tolerant cultivar, while S210 is salt-sensitive. Recently, it was reported that sugar beet

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M14 showed strong salt tolerance, and protein changes in M14 leaves and roots were

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analyzed by proteomic technique under salt stress 14. However, to the best of our knowledge,

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comparing the salt-response proteomes in sugar beet with contrasting genotypes has not been

13.

Our result demonstrated that T510 is a

4


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conducted. In this study, an iTRAQ-based proteomic approach was used to investigate

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proteome changes in sugar beet seedlings with contrasting tolerance during salt stress. Our

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findings offer a comprehensive overview of the functional significance of some differential

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response proteins in the establishment of an adaptive response to salt stress. This study could

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provide insights into the molecular mechanism of salt-stress tolerance in sugar beet.

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Materials and Methods

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Plant Materials and salt treatment

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Seeds of sugar beet T510 (salt-tolerant) and S210 (salt-sensitive) stored in HeilongJiang

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University (Harbin, China) were sterilized by soaking with 0.1% mercuric chloride and 0.2%

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Thiram for 30 min, followed by washing five times with distilled water. The seeds were then

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germinated in sterilized vermiculite at 25 °C. After five days, the uniform sugar beet

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seedlings were transferred to 5 L glass containers containing 1/2 Hoagland nutrient solution

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and grown under the following conditions: 25 °C, relative humidity of 50%-60% and 14 h

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light period/day with photo flux density 450 μmol m−2 s−1. After ten days of growth, one set of

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seedlings from each cultivar was shifted to 1/2 Hoagland nutrient solution containing 280 mM

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NaCl for seven days. The other set grown in solution without NaCl served as controls. The

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solution was replaced every day, to alleviate the effect of evaporation. For proteomic or

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physiological analysis, roots or leaves of four seedlings from each treatment and control were

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pooled as a replicate. Three biological replicates were analyzed for all the experiments.

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Measurement of Physiological index

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Leaf area was measured by LI-3000C area meter (LI-COR Biosciences). Leaf relative

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water content (LRWC) was measured using the following formula: LRWC (%) = [(fresh 5



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weight−dry weight)/(turgid weight−dry weight)]×100. Chlorophyll (a+b) content was

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detected using the method reported by Kaur et al 15, and net photosynthetic rate of leaves were

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measured

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Determination of free amino acids and proline in root were conducted with the method of

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ninhydrin colorimetric and spectrometric, respectively 4.

by

an

LC4

photosynthesis

tester

(ADC Bio

Scientific).

116

The detailed processes for determination of ascorbate peroxidase (APX) and superoxide

117

dismutase (SOD) activity were published earlier by the laboratory 4. The malondialdehyde

118

(MDA) level and relative electric conductivity in leaves were assessed as previously reported

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by Chołuja et al

120

Shanghai Precision & Scientific Instrument) 4. N, P and Cl- content were conducted using

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Kjeldahl method, ammonium molybdate and ultraviolet spectrophotometer method,

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respectively, as reported by Wang et al 4.

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Protein extraction

16.

Na+ and K+ levels were measured by flame spectrophotometer (FP640,

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Samples were ground to a fine powder in liquid nitrogen and transferred to a 2 mL tube

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with 1.5 mL precooled 90% acetone containing 10% TCA and 0.07% DTT. The extract was

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vortexed and incubated 2 h at -20 °C. After centrifugation at 10,000 g for 30 min at 4 °C, the

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precipitate was collected and washed with precooled acetone containing 0.07% DTT for 1.5 h

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at 4 °C. The mixture was then centrifuged at 10,000 g for 30 min at 4 °C, and the pellet was

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saved. The washing step was repeated three times. After centrifugation at 10,000 g for 30 min

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at 4 °C, the pellet was solubilized in lysis buffer (8 M urea, 2 M thiourea, 4% Chaps, 50 mM

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DTT) and vortexed for 5 min. The solution was centrifuged as described above and the

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supernatants were saved for further analysis. Protein concentrations were measured using a 6


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Bradford Kit.

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Trypsin digestion, iTRAQ labeling and high pH RPLC Separation

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Each sample, containing 100 μg of protein were reduced using 10 mM DTT for 30 min at

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56 °C, and alkylated with 50 mM iodoacetamide at 25 °C in the dark for 50 min. The proteins

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were then precipitated with acetone, and dissolved in 100 mM triethylammonium bicarbonate

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buffer containing 1 M urea. Protein samples were digested with trypsin at 20:1 (protein:

139

trypsin on a weight basis) at 37 °C for 15 h. Subsequently, the peptide mixture was labeled

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using the iTRAQ Reagent 8-plex Kit (AB Sciex Inc., USA) according to the manufacturer’s

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instructions. The replicate samples of T510 roots under 0 mM NaCl condition were labeled

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with 115, 116 and 117; and reagent 118, 119 and 121 for T510 roots at 280 mM NaCl. In

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addition, the replicate samples of T510 leaves under 0 mM NaCl condition were labeled with

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117, 118 and 119; and reagent 113, 114 and 121 for T510 leaves at 280 mM NaCl. The

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replicates of S210 roots under 0 mM NaCl condition were labeled with 113, 119 and 121, and

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the salt-treated samples labeled with tags 114, 115 and 116, respectively. The untreated S210

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leaves replicates were labeled with 113, 114 and 115, and the salt treated samples labeled

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with tags 116, 117 and 118. Samples were fractionated with the high pH reverse phase

149

separation method as previously reported 17. Finally, ten fractions per sample were acquired

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for subsequent experiments.

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LC-MS/MS analysis and proteomics data analysis

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Each fraction was lyophilized and re-suspended with 30 μL solvent A (5% acetonitrile, 0.1%

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formic acid), separated by Thermo Easy nLC-1000 system and analyzed by a

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quadrupole-Orbitrap-linear ion trap mass spectrometer (Orbitrap Fusion Lumos Tribrid) 7



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(Thermo Fisher Scientific). A 10 μL peptide sample was separated on the analytical column

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(Homemade C18 150 mm x 75 μm, 3 μm) with a linear gradient, from 2% buffer B (95%

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acetonitrile, 0.1% formic acid) to 30% for 72 min. The detailed parameter for setting LC

158

system and fusion mass spectrometer was taken from the literature 18. The MS/MS data were

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searched

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(uniprot-taxonomy_3555_20160412) by proteome discoverer program version 2.1 (Thermo

161

Fisher Scientific). The search parameters were listed as follows: precursor mass tolerance was

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set at 10 ppm, and fragment mass tolerance was 50 mmu. Trypsin was chosen as the enzyme

163

with an allowance for two missed cleavages. Carbamidomethylation at cysteine was set as the

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fixed modification, and modifications of oxidation at Met were set as variable modifications

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19.

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lower than 1%. The cutoff ion score for peptide identification was set as 17. At least two

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unique peptides were used for protein quantification, and the method of normalization on

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protein median was applied to correct experimental bias. The differentially expressed proteins

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were identified based on t-test with p-value less than 0.05 and fold change should be greater

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than 1.3 or less than 0.7. The differentially expressed proteins were categorized with Gene

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Ontology (GO) database, UniProt database and Clusters of Orthologous Groups of Proteins

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System (COG) software.

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Quantitative real-time PCR analysis

in

the

Beta

vulgaris

UniProtKB/Swiss-Prot

database

The target-decoy approach was used to make peptide level false discovery rates (FDR)

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Ten proteins were selected from root and leaves to verify the differential expression at

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RNA level. Total RNA from two sugar beet cultivars was extracted by TRIZOL reagent (life

176

technology). For synthesis of the first strand of cDNA, a reverse transcription kit from 8


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Toyobo company was used. Quantitative real-time PCR was performed by Bio-Rad

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Quantitative PCR system, and 18s rRNA gene was selected as an internal gene. The PCR

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reaction system, the PCR reaction procedure and dosage of SYBR GREEN were consistent

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with our previous report 10. All primer sequences were referred to Table S1, and each PCR

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reaction was conducted twice with three replicates.

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Results

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Morphological and physiological changes in two genotypes of sugar beet under salt

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stress

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Two sugar beet genotypes, T510 and S210, which were identified from 60 sugar beet

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cultivars, differed greatly in salt-stress tolerance. The exposure of sugar beet seedlings to 280

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mM NaCl led to many morphological and physiological changes. Under salt treatment, both

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genotypes exhibited growth retardation as compared with their controls. However, the effect

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of salt stress was more significant in S210 than T510 (Figure 1a). The dry weight

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measurements showed that under salt stress T510 reduced by 31.03% and S210 by 46.87% as

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compared to control conditions (Figure 1b). Furthermore, sugar beet salt tolerant genotype

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T510 exhibited larger total leaf area and root length than salt sensitive genotype S210,

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respectively (Figure 1c, d). Consistently with the morphological response, RWC also

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exhibited a decreasing trend upon exposure to NaCl, and the effect was more significant in

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S210 (Figure 1e). In addition, chlorophyll content is a key indicator of plant photosynthesis.

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As shown in Figure 2a and b, after salt treatment, T510 also showed higher chlorophyll

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content and net photosynthetic rate, suggesting that salt-tolerant T510 acquired superior

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photosynthesis ability in response to salt stress. Proline is a kind of osmotic adjustment 9



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substance, which can accumulate in plants under various stress conditions. Moreover, proline

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level is closely related to plant stress tolerance. Similarly, our study found that proline content

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in T510 root was significantly higher than that in S210 under salinity. However, there was no

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significant difference in the content of free amino acids between the roots of two sugar beet

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cultivars under control or salt-stress conditions.

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Furthermore, Na+ and Cl- levels increased significantly in both genotypes as compared to

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control conditions. Importantly, S210 showed greatly increased Na+ and Cl contents under

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salt stress, which were almost 1.10 and 1.18 fold more than that measured in T510 (Figure 3a,

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b), respectively. The K+ concentrations in leaves were significantly decreased for both

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genotypes under salinity conditions, and T510 had higher leaf K+ levels than S210 under

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stress or control conditions (Figure 3c). The Na+/ K+ ratio dramatically increased under salt

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stress, but T510 could limit it from reaching as high a level as S210 (Figure 3d). The

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concentrations of other macroelements (N and P) in leaves were determined in both genotypes.

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N levels were found higher in T510 under control or salt-stress conditions (Figure 4a).

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However, P levels were found to be the same in both T510 and S210 under control conditions

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but increased significantly in S210 after stress (Figure 4b). Obviously, these data

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demonstrated that T510 could withstand saline conditions.

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MDA content is usually one of the important indicators of stress-caused plasma membrane

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injury. In our study, MDA levels in leaves were measured. As shown in Figure 5a, the

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contents for both genotypes increased under salt stress, which suggested that damage to the

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plasma membrane was enhanced by the exposure of seedlings to 280 mM NaCl. However,

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MDA levels were found to be increased significantly in S210 after salt stress. In addition, 10


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relative electric conductivity was found to be the same in both T510 and S210 under

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non-stress conditions but increased significantly in S210 under salt stress (Figure 5b).

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Furthermore, the activities of antioxidant enzymes implicated in detoxification of reactive

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oxygen species (ROS) are accepted as indicators for plant tolerance to salt stress. In order to

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confirm the role of antioxidant system in salt stress, APX and SOD activity in leaves was

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detected under normal or salt-stress conditions. In this study, the activity of SOD and APX

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was found to be higher in T510 than S210 under 280 mM NaCl, although activity of these

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enzymes did not exhibit a significant difference between the two genotypes under control

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conditions (Figure 5c, d). These results indicated ROS scavenging capacities in T510 were

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higher in comparison to S210 under salinity conditions, which may contribute to salt-stress

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tolerance of T510.

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The influence of salt stress on leaf and root proteomes of the two sugar beet genotypes

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To investigate the molecular mechanisms governing salinity tolerance in sugar beet

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seedlings, a comprehensive proteomics analysis of salt-sensitive S210 and salt-tolerant T510

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genotypes was developed under control (0 mM NaCl) and salt stress (280 mM NaCl) using

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the iTRAQ LC-MS/MS based approach. A total of 4,872 proteins were identified in sugar

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beet S210, and differential expression proteins were selected based on fold change was

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greater than 1.3 or less than 0.7 and a p-value less than 0.05. In sugar beet S210, 56 proteins

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(Table 1) in leaves as well as 47 proteins (Table 2) in roots were differentially accumulated

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under salt stress. Among the 4,182 proteins in sugar beet T510 leaves and roots, 50 (Table 1)

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and 56 (Table 2) proteins belonging to leaves and roots samples, respectively, were found as

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exhibiting significantly changed in abundance following salt stress. 11



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Functional classification of differentially expressed proteins

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To explore which proteins participate in the process of plant salt responses, all identified

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differentially expressed proteins in leaves and roots were divided into 10 functional categories:

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metabolism, stress and defense, protein synthesis, photosynthesis, transcription related protein,

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protein folding and degradation, signaling transduction, transport related protein, others and

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unknown (Figure 6). In both S210 and T510, the most abundant group belonged to the

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metabolism category. This accounted for 34% of identified proteins in S210 and T510 roots,

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and 21% and 22% in S210 and T510 leaves, respectively. The second highly enriched

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functional category was protein synthesis (18%) and photosynthesis (14%) in S210 and T510

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leaves, respectively. However, the next category was stress and defense in the roots of the two

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sugar beet genotypes (15% in S210 and 13% in T510). In addition, a larger proportion of

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proteins related to protein synthesis were found in leaves of the sensitive genotype compared

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to that of the tolerant one (18% vs 4%, respectively), whereas, signaling transduction proteins

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were found in a smaller proportion in leaves and roots of S210 compared to that of T510 (5%

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vs 8%, 9% vs 13%, respectively). Moreover, the proportion of differentially expressed

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proteins related to metabolism and photosynthesis were obviously different between the

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leaves and roots in the two genotypes.

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Transcriptional analysis of genes for some differentially expressed proteins

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In order to validate the change levels of differentially expressed proteins, qRT-PCR

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analyses were applied to examine 10 genes (5 genes for leaves and 5 for roots) with different

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salt stress expression profiles in the iTRAQ data (Figure 7). Previously, these proteins were

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reported to be involved in plant response to salt or abiotic stresses in other plant species. The 12


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expression patterns of most genes investigated in our study are consistent with the abundance

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changes in protein levels. These results demonstrated that the expression of these proteins was

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regulated at transcription level. However, the only exception to the same patterns between

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transcripts and proteins is jasmonate-induced protein homolog (JIPH). JIPH showed

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significant decrease in transcript level in the salt-sensitive genotype S210 under salt stress,

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but no changes in its protein abundance (Figure 7c, d).

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Discussion

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Salinity has wide-ranging impacts on plants, including ion imbalance and osmotic

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homeostasis. To resist salt stress, plants had complex regulatory mechanisms, including salt

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responsive signaling transduction and metabolism changes

275

and physiological changes were observed in both sugar beet cultivars, which demonstrated

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that the salt-tolerant cultivar T510 exhibited stronger tolerance than the sensitive cultivar

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S210 under salt stress. Plant growth as biomass or leaf areas production is dependent on net

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photosynthesis and, thus, one of adverse effects that salt stress causes, plant growth inhibition,

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is attributed to a significant decrease in photosynthesis. This study showed that chlorophyll

280

content in the two cultivars was significantly decreased under salt stress, as previously

281

reported 4. Moreover, other reports also showed that chlorophyll level not only reflects the

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capacity of plant photosynthesis, but also is widely used as an indicator for assessing plant

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salt tolerance 21. Thus, the higher chlorophyll content and net photosynthesis rate in T510 may

284

partially contribute to salt-stress tolerance.

20.

In this study, morphological

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Cell membrane stability is generally accepted as indicator to evaluate the extent of cellular

286

damages induced by various abiotic stresses. An obvious increase in relative electric 13



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conductivity was observed for two cultivars under salt stress, indicating an enhancing of

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membrane permeability. Yet, the relative electric conductivity in salt-tolerant T510 was lower

289

than in salt-sensitive S210 subjected to salinity, indicating that T510 membrane permeability

290

was less affected by salt stress. Our results are in agreement with another report

291

found that salt-tolerant wheat genotype had relatively higher membrane stability than

292

salt-sensitive genotype under a salinity treatment.

293

widely thought as an index for lipid peroxidation caused by salt stress. In our study, a more

294

significant increase in MDA level in salt-sensitive S210 than in salt-tolerant T510 subjected

295

to salt stress suggested a higher level of lipid peroxidation for S210 induced by salt stress, and

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salt-tolerant T510 may have better protection against oxidative damage as evidenced by

297

higher antioxidant enzyme activities, such as SOD and APX. The present results are the same

298

as Hu et al 23, who showed that salt-tolerant Lolium perenne had a more efficient antioxidative

299

system.

22.

They

In addition, the MDA level has been

300

Salt stress causes high accumulation of Na+ and Cl- in different sugar beet tissues, which

301

affect normal metabolic activities and leads to a decline in yield. In the present study,

302

salt-tolerant T510 has lower leaf Na+ and Cl- concentrations than salt-sensitive S210,

303

speculating that salt-tolerant T510 may alleviate the effects on plant growth and development

304

induced by salt stress through reducing leaf Na+ and Cl- levels. In addition, it has been

305

reported that salt tolerance is related with K concentrations, and regulation of K uptake and

306

maintaining desirable Na+/ K+ ratio are important strategies to withstand salt stress

307

results also demonstrated that salt-tolerant T510 had the capacity to maintain a lower Na+/ K+

308

ratio, so Na+/ K+ might be used as an indicator to assess plant salt-stress tolerance. Moreover, 14


24.

Our

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we explored the effects of salinity stress on several nutrients. It has been reported that high

310

accumulation of P in sugar beet leaves may be a main cause of plant growth inhibition under

311

salt stress 4. Therefore, low concentrations of P in salt-tolerant T510 leaves may be one of the

312

main reasons for alleviating effects on salinity inhibiting plant growth.

313

Changes of gene expression and protein abundance play a vital role in plant responses to

314

salt stress. Previously, sugar beet M14 containing wild beet one chromosome showed strong

315

salt stress tolerance, and soluble or membrane protein changes in M14 leaves and roots were

316

analyzed by proteomic technique at 200 mM and 400 mM NaCl 14, 25. Unlike previous studies,

317

in order to further explore salt tolerance mechanisms in sugar beet, we investigated proteomic

318

changes of both sugar beet cultivars contrasting with genotypes at 280 mM NaCl, some new

319

proteins related to salt tolerance in sugar beet were identified in this study. Our research

320

revealed that the difference in salt tolerance and sensitivity of sugar beet might be attributed

321

to the differential accumulation of some salt-stress responsive proteins. Most of the

322

differentially expressed proteins were taking part in metabolism, stress and defense, protein

323

folding and degradation, protein synthesis, photosynthesis, transcription related protein,

324

signaling transduction and transport related protein. The insights into the regulatory

325

mechanisms involved in salt-stress tolerance of sugar beet are discussed below.

326

Metabolism related proteins

327

Metabolism plays an essential role in the plant stress response process that maintains living

328

cells. In our study several metabolic proteins involved in energy and carbohydrate metabolism,

329

amino acid metabolism and others were differentially accumulated under salt stress in the two

330

sugar

beet

genotypes.

Malate

dehydrogenase 15


(MDH)

reversibly


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331

catalyzes the oxidation of malate to oxaloacetate in the tricarboxylic acid (TCA) cycle and is

332

also a key enzyme controlling the malate valve

333

NADP-malate dehydrogenase in Arabidopsis thaliana can confer tolerance to salt stress,

334

which is mainly due to better maintaining the cellular redox environment

335

dehydrogenase was obviously down-regulated under salt stress in the roots and leaves of S210,

336

but not in the tissue of T510. Overall, the results indicated T510 had a more stable TCA cycle

337

and cellular redox environment under salt stress than S210. Moreover, sucrose synthase (Sus)

338

recognized as a key enzyme in sucrose metabolism in plants was only down regulated after

339

salt treatment in S210 roots. It is reported that abiotic stress can conspicuously induce HbSus5

340

expression in Hevea brasiliensis

341

osmotic balance under salt stress, but also provides energy required for maintaining cellular

342

processes. Therefore, compared with T510, S210 decreased the protein levels of sucrose

343

synthase in root under salinity stress, which may lead to a lower accumulation of compatible

344

solutes and less energy supply to cope with the stress in the salt-sensitive cultivar.

345

28.

26.

In addition, overexpression of

27.

Malate

Sucrose acts not only as osmoprotectant for regulating

On exposure to salt stress, the synthesis of many amino acids such as glutamine,

346

methionine and cysteine in plants may be affected

347

(SAMS) takes part in synthesizing S-adenosyl methionine (SAM) from methionine and ATP,

348

and SAM is used as a precursor in the biosynthesis of polyamines (PAs) and involved in

349

plant growth regulation, as well as stress response. A previous report indicated that

350

overexpression of a sugar beet SAMS in Arabidopsis lead to increase salt and H2O2 tolerance

351

through regulating polyamine metabolism 30. In this study, proteomic analysis also showed

352

that SAMS were only up-regulated in the roots or leaves of the salt-tolerant genotype T510

29.

S-adenosylhomocysteine synthase

16


Page 17 of 55


353

under salt stress, but not in the salt-sensitive S210. These results indicated that salt-tolerant

354

T510 could enhance the synthesis of PA synthesis regulated by SAMS to increase salinity

355

tolerance. Furthermore, another protein, cysteine proteinase inhibitor, also has specially

356

increasing abundance in the leaves of T510. The main function of cysteine proteinase

357

inhibitor is to regulate endogenous proteolytic activities in the process of plant growth and

358

development. Previously, we demonstrated that a cysteine proteinase inhibitor in sugar beet

359

was also involved in plant response to various abiotic stresses 31. Over-expressing cysteine

360

proteinase inhibitor in Arabidopsis could increase plant salt stress tolerance. Overall,

361

differentially accumulated proteins participating in metabolic processes may make a

362

concerted effort for plant survival in high salinity conditions.

363

Stress and defense-related proteins

364

As plant response to salt stress is often accompanied by the induction of many stress

365

responsive proteins, it was expected that some of these particularly change abundance in the

366

tolerant sugar beet. We found a significant up-regulation of several antioxidant enzymes,

367

L-ascorbate peroxidase 6 and peroxidase, under salt stress in the tolerant genotype T510 root,

368

whereas these protein levels were unchanged in the sensitive cultivar. Moreover, L-ascorbate

369

oxidase homolog showed specific down-regulation in the sensitive genotype S210 root. Salt

370

stress usually elevates the producing of ROS, which lead to oxidative injury to plant cells by

371

membrane damage and attack macromolecules. It is reported that salt-tolerance in plants is

372

related to the increasing of antioxidant enzymes and antioxidant activity 32-33. The high level

373

of antioxidant enzymes under salt stress is involved in preventing oxidative damage.

374

Late embryogenesis abundant (LEA) proteins are implicated in protecting plant 17



Page 18 of 55

375

membrane system and macromolecules from damage. LEA proteins can be dramatically

376

induced by ABA and various abiotic stresses including salinity 34. Overexpression of a LEA

377

protein gene AtLEA14 can confer salt-stress tolerance in Arabidopsis

378

overexpressing of SiLEA14 from Setaria italic in Arabidopsis enhanced transgenic seedlings

379

tolerance to salt and osmotic stress

380

abundant proteins, LEA47 and LEA14, were dramatically increasing their abundance in

381

tolerant genotype T510, but this phenomenon did not appear in the sensitive genotype S210.

382

It is reported that lignin contents in the IbLEA14-overexpressing calli were increased

383

compared with control calli 37. We thus speculated that higher abundance of LEA proteins

384

may participate in regulating lignin production for increasing salt tolerance.

36.

35.

Moreover,

Interestingly, in leaves, two late embryogenesis

385

Furthermore, plants have evolved cross-tolerance mechanisms to be able to cope with

386

different stresses. Some proteins related to pathogenesis such as dirigent proteins 1 and 23

387

were found to be down-regulated in the roots of salt-sensitive cultivar S210 under salt stress

388

but not in salt-tolerant T510. Dirigent-like family proteins were implicated in lignifcation

389

and play a crucial role in response to pathogens in plants. Consistent with our results, several

390

dirigent protein genes in sugar cane and pepper (Capsicum annuum L.) are reported to be

391

involved in the response to abiotic stresses of drought, salinity and oxidatives

392

previous study demonstrated that dirigent proteins may direct stereo-selective phenolic

393

coupling reactions in the lignin formation process 40. Therefore, the down-regulation of these

394

dirigent proteins in S210 may partially affect lignin synthesis. In addition, another

395

pathogenesis related protein, Thaumatin-like protein, only had specially increasing

396

abundance in the leaves of T510 cultivar. Thaumatin-like proteins belong to widely existing 18


38-39.

A

Page 19 of 55


397

pathogenesis-related proteins in many species, and participate in the forming of disulfide

398

linkages, which impart stability to the protein under stress conditions 58. Overall, these data

399

provide a new insight into the cross-tolerance mechanisms in sugar beet response to biotic

400

and abiotic stresses.

401

Photosynthesis related proteins

402

Photosynthesis processes are the basic activities in plants and are very sensitive to salt

403

stress. In this study, several light reaction related proteins showed significant change in

404

abundance during 280 mM NaCl stress in the two sugar beet genotypes. For example,

405

oxygen-evolving enhancer proteins 1 (OEE 1) and 2 (OEE 2) in both genotypes were found

406

to be down-regulated in response to salt stress. OEE1 plays a key role in sustaining the

407

water-oxidizing capability and stability of PSII, and OEE2 is involved in the assembly of the

408

PSII complex. It is also reported that OEE 2 can be easily removed from the PSII complex in

409

the presence of NaCl 41. In this study, lower levels of OEE indicated that the integrity and

410

function of PSII were affected in sugar beet under salt stress. In addition, proteins related to

411

the CO2 carboxylation and Calvin cycle changed abundances under salt stress in both

412

genotypes. Ribulose-1, 5-bisphosphatecarboxylase (RuBisCO) was up-regulated under salt

413

stress in the two genotypes. The increase in RuBisCO levels may partially offset the energy

414

reduction that accompanies salinity stress

415

enzyme related to the Calvin cycle, was shown to increase under stress only in the

416

salt-tolerant

417

phosphorylation of ribulose-5-phosphate to ribulose-1, 5-bisphosphate (RuBP) 43. Therefore,

418

the enhanced abundance of phosphoribulokinase in salt-tolerant T510 demonstrated that

genotype

T510.

42.

However, phosphoribulokinase, another

Phosphoribulokinase

participated

19


in

catalyzing

the


419

regeneration of RuBP in the salt-tolerant cultivar could be better maintained under salinity

420

stress.

421

Signaling transduction related proteins

422

When plants were exposed to a saline environment, diverse salt responsive signaling

423

pathways were activated to resist damage. Abscisic acid (ABA) signaling pathway plays a

424

vital role in plant response to abiotic stress as it controls the closing of stomata to limit water

425

loss under stress conditions. In this study, abscisic acid-stress-ripening protein 1 (ASR1),

426

which is involved in the transduction of ABA and sucrose signaling pathway, was only

427

decreased in leaves of the salt-sensitive cultivar S210. ASR proteins are small

428

hydrophilic proteins that will increase their expression levels led by abscisic acid and diverse

429

stresses

430

tobacco significantly increased drought and oxidative stresses tolerance, and SiASR1 can

431

regulate the transcript levels of some oxidation-related genes 45. The reducing level of ASR1

432

may therefore affect the salt-stress tolerance in S210 through inhibition of inducing several

433

oxidation-related genes, compared with T510.

44.

Overexpression of SiASR1, an ASR gene from foxtail millet (Setaria italic), in

434

Furthermore, jasmonic acid (JA) and methyl JA (MeJA) are widespread throughout many

435

species and involved in plant stress responses and defense. When a plant was treated with

436

methyl JA or MeJA, many kinds of jasmonate-induced proteins (JIPs), such as JIP60 and

437

JIP15, rapidly accumulated 46. JIP60 is first operative as a ribosome-dissociation factor, and

438

eIF4E domain of JIP60 plays a role in recruiting a subset of stress cellular messengers for

439

translation 47. Recently, it was also found that JIP60 may be a candidate QTL for biotic and

440

abiotic stress tolerance 48. In this study, JIP60 was identified as being down-regulated in the 20


Page 20 of 55

Page 21 of 55


441

leaves of S210 and unchanged in T510 under salt stress. On the contrary, another

442

jasmonate-induced protein homolog was increasing protein level in the roots of T510 and

443

unchanged in S210. Thus, the high abundance of JIPs in salt tolerant genotype could

444

efficiently regulate the signaling transduction process in plant response to salt stress.

445

Other proteins

446

Differential expression of cell wall related proteins during plant response to biotic or

447

abiotic stress suggests that these proteins may contribute to plant adaptation to

448

environmental stress. Glycine-rich proteins (GRPs) are major structural protein components

449

in the cell walls of many higher plants. It is also reported that that GRPs play a vital role in

450

a repair system in the process of the protoxylem stretching phase

451

biosynthesis of GRPs and their accumulation in vascular tissues are important parts of the

452

plant defense system

453

structural proteins in leaves of the tolerant cultivar may demonstrate some cell wall

454

structural rearrangements that can be important for the salt-stress tolerance mechanism. In

455

addition, increasing abundance of the Casparian strip membrane protein (CASP) was also

456

only identified in the roots of salt-tolerant T510 under 280 mM NaCl. CASP can regulate

457

plant tolerance through recruiting the lignin polymerization 51.

50.

49.

Furthermore,

In this study, increasing abundances of two glycine-rich cell wall

458

Early nodulin-like proteins (ENODLs) belong to chimeric arabinogalactan proteins related

459

to the phytocyanin family. In legumes, ENODLs are thought to play an important role in cell

460

differentiation and cell wall reorganization in the process of nodulation. Furthermore,

461

ENODL also participated in determining reproductive potential and stress resistance in

462

non-legumes

52.

Overexpression 21


of

BcBCP1,

a


Page 22 of 55

463

phytocyanin-related early nodulin-like gene, increased tolerance to osmotic stress in tobacco,

464

as indicated by the higher photosynthetic rates and antioxidant enzyme activities after

465

osmotic stress 53. Early nodulin-like protein and early nodulin-like protein 2, respectively,

466

increased the protein abundance in the leaves of salt-sensitive and salt-tolerant genotypes

467

under salt stress. Presumably, increasing the expression of several early nodulin-like proteins

468

is a common response in leaves to survive salt stress, although different sugar beet

469

genotypes regulated different kinds of early nodulin-like proteins. However, nodulin-26-like

470

protein had decreasing abundance in the roots of salt-sensitive S210 and unchanging in T510.

471

Another study indicated that most of the ENODL gene family showed spatially specific

472

expression in Arabidopsis, and different ENODL genes may perform diverse functions

473

Therefore, differently from the previous two early nodulin-like proteins, the level of

474

nodulin-26-like protein may be involved in determining salt stress tolerance in sugar beet

475

root.

52.

476

Overview of the response and tolerant mechanism of salt tolerance in T510 tolerant

477

cultivar

478

Based on results from this study and published literature, an overview of the mechanism

479

in salt-stress tolerant cultivar T510 is proposed (Figure 8). In T510 leaves (Figure 8a), some

480

key proteins involved in stress tolerance integrated into several pathways, including protein

481

modification, amino acid metabolism, TCA cycle, photosystem II, stress hormone signal

482

transduction and ROS scavenging. Here, we found that the key proteins mainly participated

483

in carbohydrate metabolism, protein modification, amino acid metabolism, TCA cycle, cell

484

wall synthesis and ROS scavenging in T510 roots (Figure 8b). Although the root and leaf of 22


Page 23 of 55


485

salt-tolerant beets have some of the same signaling pathways involved in resisting salt stress,

486

they may trigger different proteins in the same metabolic pathway to alleviate the damage

487

caused by salt stress. For example, ATP-citrate synthase, involving in TCA cycle, was only

488

increased in the roots of T510 compared to T510 leaves. Our data thus prove that sugar beet

489

roots and leaves have different mechanisms to cope with salt stress. Future studies focusing

490

on characterizing the biological significance of these key proteins will be highly valuable in

491

designing molecular breeding or engineering programs for enhancing sugar beet tolerance

492

and yield.

493

Supporting information：

494

Table S1. List of the QRT-PCR primer.

495

Table S2. Peptide information of differentially expressed proteins identified by mass spectrometry.

496

Notes

497 498

The authors declare no conflict of interest. Funding

499

We are grateful for the financial support from the National Natural Science Foundation of

500

China Project (31701487), National Sugar Industry Technology System of China

501

(CARS-170209), Basic Research Work Program of Heilongjiang Provincial Higher

502

Education Institutions (KJCXYB201706) and Youth Innovative Talents Training Program of

503

Heilongjiang Regular Universities (KJCXYB2017031).

504 505 506 23



Page 24 of 55

507

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15, 922-931.

656

(51) Roppolo, D,; Boeckmann, B,; Pfister, A,; Boutet, E,; Rubio, M. C,; Dénervaud-Tendon,

657

V,; Vermeer

658

Functional and evolutionary analysis of

659

protein family. Plant Physiol. 2014, 165, 1709-1722.

JE, Gheyselinck

J, Xenarios the casparian

30


strip

I, Geldner

N.

membrane

domain

Page 31 of 55


660

(52) Mashiguchi, K,; Asami, T,; Suzuki, Y. Genome -wide identification, structure and ex

661

pression studies and mutant collection of 22 early nodulin-like protein genes in Arabido

662

psis. Biosci Biotechnol Biochem. 2009, 73, 2452-2459.

663

(53) Wu, H,; Shen, Y,; Hu, Y,; Tan, S,; Lin, Z. A phytocyanin-related early nodulin-like

664

gene, BcBCP1, cloned from Boea crassifolia enhances osmotic tolerance in trans

665

genic tobacco. J Plant Physiol. 2011, 168, 935-943.

666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 31



682

Figure legends

683

Figure 1. Morphological and physiological changes in sugar beet (T510 and S210) under

684

control (0 Mm NaCl) and salt stress (280 mM NaCl) conditions. Ten-days-old sugar beet

685

seedlings were treated with 280 mM NaCl for 7 days. Growth performance (a), dry weight (b),

686

total leaf area (c), leaf relative water content (d) and chlorophyll (a+b) content (e) were

687

measured in sugar beet seedlings. Asterisk (*) indicates significantly different at P < 0.05.

688

Three biological replicates were performed.

689

Figure 2. Effects of salinity stress on photosynthesis of leaves and proline or free amino acids

690

level of root in two sugar beet genotypes. Chlorophyll (a+b) content (a) and Net

691

photosynthetic rate (b) were measured in the leaves of sugar beet seedlings, and proline (c)

692

and free amino acids (d) content were detected in the roots of sugar beet seedlings.

693

Figure 3. Effects of salinity stress on Na+, K+ and Cl − content in two sugar beet genotypes.

694

Leaves Na+ (a), K+ (b) , Cl − (c) content and Na+/K+ ratio(d) of sugar beet treated with 0 mM

695

and 280 mM NaCl. Asterisk (*) indicates significantly different at P < 0.05. Three biological

696

replicates were performed.

697

Figure 4. Effects of salinity stress on N and P content in two sugar beet genotypes. N (a) and

698

P (b) contents in leaves of sugar beet treated with 0 mM and 280 mM NaCl. of NaCl for 7

699

days. Asterisk (*) indicates significantly different at P < 0.05. Three biological replicates

700

were performed.

701

Figure 5. Effects of salinity stress on antioxidant enzyme system in two sugar beet genotypes.

702

Leaves malondialdehyde (MDA) content (a), relative electrical conductivity (b), superoxide

703

dismutase (SOD) (c) and ascorbate peroxidase (APX) (d) of sugar beet treated with 0 mM and 32


Page 32 of 55

Page 33 of 55


704

280 mM NaCl. Asterisk (*) indicates significantly different at P < 0.05. Three biological

705

replicates were performed.

706

Figure 6. Functional classification of the identified differentially accumulated proteins in two

707

sugar beet genotypes treated with salinity stress. Differentially accumulated proteins in S210

708

leaf (a) and root (c); Differentially accumulated proteins in T510 leaf (b) and root (d).

709

Figure 7. Relative gene expression for salt-stressed proteins in two sugar beet genotypes

710

examined using RT-qPCR. mRNA and protein expression levels of ten candidate proteins i.e.,

711

abscisic acid-stress-ripening-inducible 1(ASR1), thioredoxin-1 (Thx1), late embryogenesis

712

abundant protein 47 (LEA47), v-type proton ATPase subunit G6 (ATP6V1G6) and

713

auxin-binding protein19a ( ABP19a) in two genotypes sugar beet leaves (a, b), and

714

cytochrome c oxidase subunit 6a (COX6A), jasmonate-induced protein homolog (JIPH),

715

malate dehydrogenase (MDH), glutathione synthetase (GSS) and

716

localized protein23 (AHL23) in two sugar beet genotypes roots (c, d) are shown.

717

Figure 8. Schematic presentation of the potential mechanism of salt stress tolerance in T510

718

tolerant cultivar. Differentially accumulated or different levels of changes between the two

719

cultivars in leaf (a) and root (b) were indicated. Red and blue highlighted proteins indicate

720

only increase or decreased in salt tolerant cultivar T510 under salt stress, compared with salt

721

sensitive cultivar S210. The green highlighted proteins indicate only decreased in salt

722

sensitive cultivar S210 under salt stress.

723 724

33


AT-hook motif nuclear


Page 34 of 55

Table 1 Identification of differential expression proteins (DEPs) from control and salt stress treated samples of S210 (salt sensitive) and T510(salt tolerant) in leaves. Unique

Fold Change

Peptides

(Salt/control)

11.6

2

0.56

0.03

4.84

9.036

2

0.58

0.03

6.06

7.71

24.79

2

0.60

0.022

19.01

6.33

3.4

4.86

2

1.69

0.01

A0A0J8B8Q3

36.84

6.61

13.9

12.12

4

1.58

0.001

Cytochrome C

A0A0J8BJ03

12.10

9.32

30.35

24.12

3

1.44

0.04

S7

Acid phosphatase 1

A0A0J8BL25

30.36

8.94

7.38

5.62

2

1.62

0.027

S8

Hexokinase

A0A0J8BT88

53.78

5.9

8.43

9.38

3

1.34

0.043

Number

Sequence coverage

Protein name

Uniprot ID

MW ( kDa)

PI

S1

Probable methyltransferase 26

A0A0J8D1I2

87.9

5.35

9.9

S2

Carbamoyl-phosphate synthase large chain

A0A0J8EAX1

130.11

5.53

S3

Malate dehydrogenase

A0A0J8BJ08

41.16

S4

Purple acid phosphatase

A0A0J8B5H2

S5

4-hydroxy-tetrahydrodipicolinate reductase 2

S6

(%)

Score

P-value

Metabolism DEPs in S210

S9

Protein phosphatase 2C

A0A0J8CA29

48.42

7.4

7.73

6.33

2

1.37

0.02

S10

Aldose 1-epimerase

A0A0J8CDX8

37.31

6.52

17.10

10.33

3

1.31

0.04

S11

Adenosylhomocysteinase

A0A0J8CM32

53.42

6.19

32.44

41.82

11

1.43

0.047

S12

H+ ATPase

A0A0J8CEG9

105.47

6.52

5.92

18.51

2

1.44

0.03

Probable prolyl 4-hydroxylase 6

A0A0J8FMV1

35.31

7.23

6.98

4.65

2

1.30

0.004

A0A0J8BKW8

93.50

4.59

16.29

23.09

8

1.31

0.040

DEPs in T510 T1 T2

Serine/threonine-protein phosphatase 6 regulatory subunit 3

T3

Glycoside hydrolase, family 10

A0A0J8BI66

123.83

6.54

3.33

4.24

2

1.31

0.038

T4

Probable beta-D-xylosidase 5

A0A0J8BQN3

87.30

8.66

11.01

22.31

5

1.34

0.017

T5

Cysteine protease XCP2

A0A0J8BNR8

38.98

5.8

15.76

29.95

5

1.35

0.0061

T6

Chorismate synthase

A0A0J8D2L6

111.04

6.15

4.02

2.74

3

1.41

0.015

34


Page 35 of 55


T7

Probable protein phosphatase 2C 62 isoform X1

A0A0J8B454

105.39

4.06

3.72

7.03

2

1.54

0.037

T8

V-type proton ATPase subunit G

A0A0J8BEX4

12.23

5.58

74.45

36.43

9

1.77

0.002

T9

Polyphenol oxidase, chloroplastic

A0A0J8BT15

67.57

7.99

14.19

100.82

4

1.82

0.018

T10

Cysteine proteinase inhibitor

A0A0J8EMH9

12.75

9.2

49.12

29.45

10

2.02

0.018

T11

S-adenosylmethionine synthase

A0A0J8CM32

53.43

6.19

6. 77

77.79

2

2.22

0.029

S13

Thioredoxin 9

A0A0J8BM14

24.30

5.54

10.13

5.82

2

1.33

0.029

S14

14-3-3 protein 2 isoform X2

A0A0J8C465

29.24

4.75

18.14

11.79

2

1.77

0.04

A0A0J8CJS6

53.04

9.32

9.90

10.28

2

1.60

0.11

Stress and defense DEPs in S210

S15

Heavy metal-associated isoprenylated plant protein (HIPP)

S16

Small heat shock protein, chloroplastic

A0A0J8CPW5

20.08

8.16

62.16

56.13

11

1.31

0.021

S17

Heat shock 70 kDa protein 6, chloroplastic

A0A0J8D2Y9

47.26

5.45

17.09

28.98

2

1.35

0.01

S18

B12D-like protein

A0A0J8D2L5

10.00

8.97

19.11

10.23

2

1.35

0.046

S19

Monodehydroascorbate reductase 5

A0A0J8F7Z1

53.66

7.59

9.64

9.08

2

1.53

0.03

S20

DNA damage-repair/toleration protein

A0A0J8FCQ4

25.76

5.27

15.76

8.93

2

1.36

0.041

DRT102 DEPs in T510 T12

Thioredoxin-1 isoform X1

A0A0J8BDD0

20.07

8.59

32.26

64.32

8

1.37

0.044

T13

Thaumatin-like protein 1b

A0A0J8BJP1

33.73

4.64

8.79

16.93

2

1.38

0.029

T14

Late embryogenesis abundant protein 47

A0A0J8C8Z8

14.84

4.63

61.11

37.33

6

1.59

0.012

A0A0J8D7Q4

17.45

4.86

42.86

19.31

5

1.68

0.013

A0A0J8D2Y9

47.26

5.45

17.09

38.82

2

1.91

0.020

Late embryogenesis abundant protein

T15 T16

Lea14-A-like Heat shock 70 kDa protein 6 chloroplastic

Protein synthesis

35



Page 36 of 55

DEPs in S210 S21

40S ribosomal protein

A0A0J8BI12

15.1

5.95

23.2

23.6

3

0.59

0.029

S22

40S ribosomal protein S20-2

A0A0J8B5L7

13.58

9.58

19.9

24.1

2

1.71

0.04

S23

60S ribosomal protein L28-1

A0A0J8BNV1

16.75

10.58

16.77

16.77

3

1.48

0.018

S24

60S acidic ribosomal protein P2A

A0A0J8EUG9

11.31

4.55

46.84

30.75

4

1.37

0.02

S25

50S ribosomal protein 5 alpha, chloroplastic

A0A0J8CMB5

15.36

11.47

10.76

7.61

2

1.49

0.049

A0A0J8B783

66.59

5.49

8.35

12.1

2

0.51

0.026

Eukaryotic translation initiation factor 3

S26

subunit

S27

Eukaryotic translation initiation factor 4B3

A0A0J8BN48

44.99

5.71

33.41

42.47

11

0.68

0.044

S28

Ribosome inactivating protein

A0A0J8BRJ6

15.1

4.86

28.75

391.91

2

0.29

0.009

S29

Ribosome-inactivating protein

A0A0J8CDM0

15.02

4.68

90.5

307.86

2

0.62

0.039

S30

Polyadenylate-binding protein

A0A0J8BIC9

72.65

7.11

35.68

90.85

20

1.42

0.035

A0A0J8B783

66.59

5.49

8.35

12.1

2

0.52

0.026

A0A0J8B5L7

13.58

9.58

19.9

24.1

2

1.72

0.04

A0A0J8BQ23

82.56

6.14

7.02

9.09

5

0.61

0.027

A0A0J8E607

65.01

6.55

4.21

7.25

2

2.01

0.04

DEPs in T510 Eukaryotic translation initiation factor 3

T17

subunit

T18

40S ribosomal protein S20-2

Photosynthesis DEPs in S210 S31 S32

4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IsPG) Probable GTP diphosphokinase CRSH, chloroplastic

S33

Ribulose 1,5-bisphosphate carboxylase

A0A023ZPS4

52.49

6.68

11.2

307.8

2

1.43

0.046

S34

Oxygen-evolving enhancer protein 1

A0A0K9RJ24

26.6

6.34

11.1

30.45

2

0.34

0.049

S35

Oxygen-evolving enhancer protein 2

A0A0K9RJ56

27.8

6.96

9.81

57.89

2

0.40

0.003

DEPs in T510 (salt tolerant genotypes)

36


Page 37 of 55


T19 T20

Thylakoid lumenal protein, chloroplastic Outer envelope protein 80, chloroplastic isoform X1

A0A0J8CKE1

28.13

8.53

48.11

91.61

14

1.35

0.044

A0A0J8FF47

74.52

7.27

6.90

6.90

2

1.39

0.010

T21

Thylakoid lumen 18.3 kDa protein

A0A0J8C3W9

13.12

5.07

35.86

9.87

2

1.83

0.046

T22

Ribulose 1,5-bisphosphate carboxylase

A0A023ZPS4

52.49

6.68

11.2

32.5

2

1.44

0.0032

T23

oxygen-evolving enhancer protein 2

A0A0K9RJ24

26.6

6.34

11.1

41.35

2

0.44

0.049

T24

oxygen-evolving enhancer protein 1

A0A0K9RJ56

27.8

6.96

9.81

56.18

2

0.45

0.004

T25

Phosphoribulo kinase

A0A0J8CH28

45.20

6.28

10.6

105.56

2

2.09

0.0038

A0A0J8BRZ7

112.46

6.46

2.05

2.25

2

0.69

0.048

A0A0J8BLJ1

53.81

6.05

40.74

48.58

10

1.31

0.027

A0A0J8D703

29.50

7.09

15.79

12.52

2

1.41

0.050

A0A0J8CXJ1

46.07

5.31

14.28

14.35

2

1.34

0.0003

A0A0J8CQK5

31.37

5.57

28.11

23.75

8

1.41

0.017

A0A0J8BSB4

117.45

5.41

2.33

5.54

2

1.62

0.011

A0A0J8C9N4

12.75

9.2

20

11.35

7

2.26

0.027

Transcription related protein DEPs in S210 S36 S37 S38

RNA polymerase II CTD phosphatase like 1 GATA zinc finger domain-containing protein 10-like Transcription factor bHLH

DEPs in T510 (salt tolerant genotypes) T26 T27 T28 T29

SWI/SNF chromatin-remodeling complex subunit SNF5-like Transcription factor Pur-alpha 1 Zinc finger BED domain-containing protein DAYSLEEPER isoform X2 Histone H1

Protein folding and degradation DEPs in S210 S39

Trypsin inhibitor III

A0A0J8CA79

11.32

7.64

17.14

5.26

2

1.73

0.0008

S40

Small ubiquitin-related modifier

A0A0J8CL60

10.88

5.1

40.62

17.7

5

1.34

0.0045

37



S41

Ubiquitin-conjugating enzyme E2 22

A0A0J8CMD1

29.50

T30

Ubiquitin-conjugating enzyme E2 27

A0A0J8CAU9

T31

SH3 domain-containing protein C23A1.17

A0A0J8BGP0

T32

Polyubiquitin 9

T33 T34

Page 38 of 55

8.97

22.82

2.85

2

1.61

0.005

21.18

4.88

25.10

54.99

10.95

39.20

10.65

2

0.64

0.038

72.51

16

1.61

0.015

A0A0J8BFX3

11.99

4.91

46.15

18.87

4

1.32

0.048

S-phase kinase-associated protein 1

A0A0J8C6Z1

18.12

4.7

31.01

25.98

4

1.50

0.026

Polyubiquitin-like

A0A0J8D7Y7

42.67

7.08

77.23

339.17

4

1.96

0.020

60 kDa jasmonate-induced protein

A0A0J8B3E1

15.1

4.68

71.7

190.85

7

0.49

0.0046

A0A0J8D8Q9

12.44

7.24

40

11.42

3

0.67

0.03

A0A0J8BU18

197.03

5.71

13.07

70.19

18

0.67

0.04

A0A0J8CT65

10.86

6.74

67.68

44.29

6

1.44

0.032

A0A0J8BV83

86.50

4.48

19.72

14.94

5

1.49

0.033

DEPs in T510

Signaling transduction DEPs in S210 S42

Abscisic acid-stress-ripening-inducible 1

S43

protein Tetratricopeptide repeat (TPR)-like

S44

superfamily protein

DEPs in T510 (salt tolerant genotypes) T35

Probable steroid-binding protein 3 Serine/threonine-protein phosphatase 6

T36

regulatory subunit 3

T37

Receptor-like protein kinase HERK 1

A0A0J8EQP9

91.64

5.82

3.21

5.43

2

1.58

0.019

T38

Auxin-binding protein ABP19a-like

A0A0J8CT76

26.91

4.31

16.27

40.76

2

1.84

0.009

A0A0J8CHM3

16.84

5.39

18.33

4.56

2

0.69

0.04

SPX-MFS (Major Facility Superfamily) family A0A0J8CJB9

79.2

6.2

11.9

23.08

2

0.64

0.02

11.49

8

28.44

171.30

11

1.8

0.0008

Transport related protein DEPs in S210 S45 S46 S47

Phosphatidylglycerol/phosphatidylinositol transfer protein Non-specific lipid-transfer protein

A0A0J8C2Q8

38


Page 39 of 55


DEPs in T510 T39

Non-specific lipid transfer protein GPI-anchored 2

A0A0J8BU12

18.98

5.48

6.42

13.50

3

1.72

0.019

T40

Lipid transfer protein

A0A0J8BCS5

11.84

8.69

19.09

8.45

2

1.81

0.040

T41

Non-specific lipid-transfer protein

A0A0J8BJX5

12.03

8.32

10.93

8.44

2

2.27

0.023

A0A0J8BGP0

54.9

10.95

40.5

82.4

17

1.31

0.03

Others DEPs in S210 S48

Src homology 3 (SH3) domain containing protein C23A1.17

S49

Binding partner of ACD11 1

A0A0J8CSD9

28.14

6.15

23.48

16.64

4

1.46

0.029

S50

Early nodulin-like protein

A0A0J8D5T8

25.89

7.42

9.96

10.14

2

1.57

0.002

S51

Cell wall protein IFF6-like isoform X1

A0A0J8DXL5

47.29

6.58

11.21

12.15

3

1.44

0.049

S52

ATP-dependent, DEAD-box RNA helicase

A0A0J8E6F9

67.38

7.93

9.40

24.98

2

1.51

0.03

S53

15-kDa selenoprotein (Sep15)

A0A0J8FHW5

17.94

4.92

12.33

12.05

2

1.32

0.04

S54

Nuclear pore complex protein NUP98A

A0A0J8FJ79

103.38

8.56

10.07

15.59

2

1.37

0.036

T42

C2 domain-containing protein At1g53590

A0A0J8BA84

80.55

6.33

9.0673

8.20

2

1.35

0.039

T43

Nuclear pore complex protein NUP1

A0A0J8BSG2

78.67

9.26

3.54

2.56

2

1.46

0.047

T44

Early nodulin-like protein 2

A0A0J8CHF1

45.74

7.15

9.35

22.96

4

1.56

0.027

T45

Blue copper protein

A0A0J8FFN4

27.69

4.6

6.98

7.56

2

1.66

0.023

T46

Glycine-rich cell wall structural protein 1.8

A0A0J8D5G7

32.33

6.06

58.69

72.07

13

1.80

0.042

PEBP (phosphatidylethanolamine-binding

A0A0J8F906

19.13

5.05

51.74

95.50

10

1.86

0.008

A0A0J8CSJ7

32.11

10.52

48.40

45.73

7

1.87

0.013

DEPs in T510

T47 T48

protein) family Glycine-rich cell wall structural protein

Unknown DEPs in S210

39



Page 40 of 55

S55

Uncharacterized protein

A0A0J8C2M0

15.98

9.38

20.83

8.95

3

1.33

0.005

S56


A0A0J8FF16

58.09

5.99

15.70

13.76

5

1.31

0.025

T49


A0A0J8BJH3

135.36

5.47

20.77

58.49

17

1.40

0.023

T50


A0A0J8BWR8

56.88

5.21

9.23

5.24

2

1.97

0.005

DEPs in T510

40


Page 41 of 55


Table 2 Identification of differential expression proteins (DEPs) from control and salt stress treated samples of S210 (salt sensitive) and T510 (salt tolerant) in roots. Number

Sequence coverage

Unique

Fold Change

Peptides

(Salt/control)

7.97

3

0.46

0.006

8.97

11.96

3

0.57

0.040

6.06

7.71

24.79

2

0.62

0.036

34.22

8.00

52.68

71.19

4

0.63

0.033

Protein name

Uniprot ID

MW (Da)

PI

S1

2-hydroxyisoflavanone dehydratase

A0A0J8BRL7

34.29

5.63

10.58

S2

GDSL esterase/lipase 7-like

A0A0J8C0I2

40.77

5.64

S3

Malate dehydrogenase

A0A0J8BJ08

41.16

Glucan endo-1,3-beta-glucosidase isoform X2 A0A0J8CRC0

(%)

Score

P-value

Metabolism DEPs in S210

S4 S5

Sucrose synthase

A0A0J8ESB8

93.68

6.37

5.35

14.98

3

0.65

0.02

S6

UDP-sulfoquinovose synthase

A0A0J8CW47

53.56

8.21

18.88

26.02

7

1.34

0.027

S7

Arginase 1, mitochondrial

A0A0J8EAJ7

37.12

6.30

28.99

26.06

5

1.34

0.042

S8

Citrate synthase

A0A0J8BI86

71.01

9.14

8.68

23.02

2

1.35

0.009

ATP-citrate synthase alpha chain protein 1

A0A0J8EVJ0

46.67

5.48

8.75

6.21

2

1.39

0.031

A0A0J8CPW2

48.62

5.66

48.42

75.63

17

1.35

0.016

S9 S10

Guanosine nucleotide diphosphate dissociation inhibitor

S11

Carboxylesterase 5

A0A0J8CQ53

34.21

5.38

20.45

13.46

5

1.36

0.033

S12

Chorismate synthase

A0A0J8FNJ1

46.83

7.9

16.93

18.05

5

1.40

0.019

S13

Allene oxide synthase-like

A0A0J8BK97

54.97

7.09

5.10

8.00

2

1.42

0.034

A0A0J8C156

43.83

8.18

31.65

30.71

10

1.43

0.0005

S14

Pyruvate dehydrogenase E1 component subunit alpha

S15

ATP synthase subunit

A0A0J8FKE6

62.60

7.03

13.04

30.99

5

1.55

0.008

S16

Ferredoxin nitrite reductase

A0A0J8CM16

67.21

7.03

25.29

44.58

13

1.30

0.047

Xyloglucan endotransglucosylase/hydrolase

A0A0J8EH61

33.04

9.17

26.21

23.99

5

0.63

0.010

DEPs in T510 T1

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T2

Xyloglucan endotransglucosylase/hydrolase

A0A0J8B630

34.75

5.43

18.67

14.83

5

0.66

0.015

T3

ATP synthase subunit epsilon, mitochondrial

A0A0J8CI46

7.807

9.25

27.14

4.17

2

0.65

0.007

T4

Glucan endo-1,3-beta-glucosidase-like

A0A0J8FFF8

67.50

5.95

36.83

71.40

2

0.65

0.006

T5

Cysteine synthase

A0A0J8D7W0

39.68

7.43

46.74

32.66

12

0.67

0.009

T6

Glycosyl hydrolase family protein isoform 1

A0A0J8C819

71.66

6.38

5.74

7.54

2

0.67

0.014

T7

Purple acid phosphatase

A0A0J8EUF0

49.61

7.06

5.04

8.93

2

1.31

0.004

A0A0J8C9W8

11.68

8.19

22.62

7.49

2

1.32

0.05

A0A0J8CN69

38.00

6.11

47.11

57.89

13

1.33

0.014

A0A0J8C836

38.91

6.54

16.09

28.89

5

1.33

0.033

A0A0J8E1Y7

84.84

5.5

2.63

7.5

2

1.34

0.030

Cytochrome c oxidase subunit 6a,

T8

mitochondrial

T9

Probable aldo-keto reductase 2 Probable 2-oxoglutarate-dependent

T10 T11

dioxygenase At3g49630 Pyrophosphate-energized membrane proton pump 3

T12

Glycylpeptide N-tetradecanoyltransferase

A0A0J8FLE4

49.58

6.33

25.46

28.30

9

1.42

0.043

T13

Fructose-bisphosphate aldolase

A0A0J8CPN6

43.36

7.36

41.06

42.32

6

1.44

0.032

T14

S-adenosylhomocysteine synthase

A0A0J8CM32

53.43

6.19

30.18

67.79

17

1.47

0.029

T15

Protein glycosyltransferase subunit 1

A0A0J8CUT8

53.72

8

5.53

7.29

2

1.63

0.045

T16

Polygalacturonase inhibitor

A0A0J8FHI9

37.54

8.65

37.91

30.52

10

1.71

0.049

T17

Polygalacturonase inhibitor

A0A0J8CPB7

37.53

8.76

23.21

17.16

6

1.31

0.041

T18

Glutathione synthetase

A0A0J8FCL3

56.48

8.19

5.39

9.40

2

2.71

0.046

T19

tRNA(adenine(34)) deaminase

A0A0J8C4C7

168.19

7.56

1.66

2.33

2

1.64

0.040

S17

Dirigent protein 1-like

A0A0J8BEV5

20.47

8.07

58.60

36.64

7

0.52

0.019

S18

Dirigent protein 23

A0A0J8EEN5

20.83

8.97

19.79

8.65

2

0.68

0.039

S19

L-ascorbate oxidase homolog

A0A0J8CBM0

61.21

9.01

29.15

57.95

13

0.65

0.037

Stress and defense DEPs in S210

42


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S20

Peroxidase

A0A0J8BZT4

34.91

8.47

40.80

56.76

10

0.55

0.023

S21

Germin-like protein 2-1

A0A0J8D8V3

26.73

5.2

26.77

18.56

2

1.34

0.018

S22

Thaumatin-like protein

A0A0J8CPA1

26.37

8.34

10.44

5.78

2

1.35

0.048

S23

Thioredoxin M3

A0A0J8BK72

19.63

8.18

15.91

10.98

3

1.37

0.029

T20

Acidic endochitinase

A0A0J8CVL8

33.72

8.98

11.55

11.28

2

0.60

0.040

T21

Chitinase-like protein 1

A0A0J8CG05

35.62

6.83

11.18

7.77

2

0.61

0.033

T22

Dirigent protein 10

A0A0J8EQN6

35.75

5.11

6.57

7.61

2

0.64

0.012

A0A0J8D0P4

40.40

8.03

27.76

17.04

7

1.30

0.015

DEPs in T510

T23

L-ascorbate peroxidase 6, chloroplastic isoform X2

T24

Peroxidase

A0A0J8CK75

34.28

5.62

9.35

4.98

2

1.35

0.024

T25

Peroxidase

A0A0J8FT54

34.91

8.87

36.84

69.02

12

1.76

0.026

T26

Universal stress protein PHOS32

A0A0J8CRN2

18.17

7.11

46.34

29.83

7

1.49

0.027

S24

60S ribosomal protein L31

A0A0J8CQ15

13.88

8.8

9.67

8.99

2

1.38

0.019

S25

40S ribosomal protein S26

A0A0J8FLJ9

14.38

10.76

10.03

9.59

2

1.82

0.004

T27

60S acidic ribosomal protein P2A

A0A0J8EUG9

11.31

4.55

46.85

78.54

6

0.64

0.005

T28

Ribosome-inactivating proteins

A0A0J8BRN0

35.75

5.86

39.49

86.45

20

0.69

0.009

T29

Protein translation factor SUI1 homolog

A0A0J8BER8

12.60

8.78

19.47

9.57

2

1.41

0.044

A0A0J8BYC6

29.98

5.17

30.47

31.31

6

0.67

0.007

UBX domain-containing protein 1 isoform X2 A0A0J8CE48

25.93

6.96

19.21

9.73

2

1.43

0.029

25.50

10.58

25.49

9.06

2

1.45

0.033

Protein synthesis DEPs in S210

DEPs in T510

Transcription related protein DEPs in S210 S26 S27 S28

RNA-binding protein CP29B Histone H2A

A0A0J8BZC2

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DEPs in T510 T30 T31 T32

GRF1-interacting factor 2 isoform X1 DNA-directed RNA polymerases II, IV and V subunit 3-like Small nuclear ribonucleoprotein-associated protein B

A0A0J8D779

24.27

6.24

19.73

2.60

2

1.36

0.042

A0A0J8BMG1

118.26

5.78

9.87

7.81

4

1.37

0.049

A0A0J8D1F7

30.47

10.76

7.64

4.54

3

1.38

0.040

Protein folding and degradation DEPs in S210 S29

Small heat shock protein

A0A0J8E1T2

19.59

8.02

57.87

75.31

8

1.36

0.020

S30

E3 ubiquitin-protein ligase RNF25

A0A0J8BRE6

48.92

5.64

4.69

9.49

2

1.36

0.039

S31

F-box protein At3g08750

A0A0J8BRV6

40.08

5.33

5.20

9.09

2

1.41

0.003

DEPs in T510 T33

E3 ubiquitin-protein ligase RNF25

A0A0J8BRE6

48.92

5.64

4.69

9.49

2

1.36

0.039

T34

Protein disulfide isomerase-like 2-3

A0A0J8BM84

47.93

5.85

26.71

23.44

11

1.39

0.003

Signaling transduction DEPs in S210 S32

Auxin-binding protein ABP19a

A0A0J8CM37

23.39

5.8

26.44

143.94

2

0.59

0.002

S33

Auxin-binding protein ABP19a

A0A0J8CT70

22.40

5.47

45.67

331.53

2

0.63

0.018

S34

Auxin-binding protein ABP19b

A0A0J8B5D5

22.42

7.50

38.46

228.12

5

0.65

0.026

A0A0J8CWA4

134.15

6.14

5.08

9.80

2

1.60

0.005

A0A0J8BJN4

34.68

6.51

5.95

8.67

2

0.43

0.049

A0A0J8C7Y5

70.54

8.48

37.73

117.91

25

0.69

0.019

A0A0J8C915

38.80

7.97

12.61

13.00

4

1.30

0.029

S35

LRR receptor-like serine/threonine-protein kinase GSO2

DEPs in T510 T35 T36 T37

Cysteine-rich receptor-like protein kinase 39 LRR receptor-like serine/threonine-protein kinase FLS2 GDSL esterase/lipase At2g23540

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T38

Syntaxin-71

A0A0J8BYF9

30.33

5.5

8.21

11.09

2

1.31

0.009

T39

Jasmonate-induced protein homolog

A0A0J8B8K6

22.75

6.55

50.72

10.48

6

1.60

0.043

A0A0J8D346

11.18

7.33

20.59

5.45

2

1.68

0.033

A0A0J8BFX1

11.98

9.03

22.67

7.27

2

2.73

0.044

cAMP-regulated phosphoprotein 19-related

T40

protein

T41

Signal recognition particle 9 kDa protein

Transport related protein DEPs in S210 S36

Annexin

A0A0J8C5G7

35.85

5.48

10.75

14.00

3

1.45

0.023

S37

ADP/ATP carrier protein, mitochondrial

A0A0J8CL40

42.05

9.69

30.95

76.51

2

1.38

0.018

A0A0J8BC54

20.66

8.07

5.84

10.16

2

1.55

0.0004

S38

Non-specific lipid-transfer protein-like protein At5g64080 isoform X1

DEPs in T510 T42

Protein TIC110

A0A0J8BWB3

112.11

5.71

3.64

5.92

3

1.42

0.006

T43

Protein NRT1/ PTR FAMILY 1.2

A0A0J8BLG2

67.41

8.59

5.98

7.15

2

1.32

0.031

A0A0J8FEQ5

32.02

9.47

14.38

28.33

2

1.42

0.049

T44

Dicarboxylate/tricarboxylate transporter DTC, mitochondrial

Others DEPs in S210 S39

AT-hook motif nuclear-localized protein 22

A0A0J8CFB6

32.89

6.71

10.16

9.83

4

0.58

0.008

S40


A0A0J8F6U6

30.12

6.43

9.79

12.02

2

0.61

0.016

S41

Expansin-like A3

A0A0J8BP13

28.57

8.16

31.42

15.36

6

0.68

0.028

S42

Nodulin-26-like

A0A0J8C6D3

13.89

5.01

12.12

12.15

2

0.58

0.018

Glycine-rich cell wall structural protein 2-like A0A0J8BH85

S43

19.60

9.29

59.02

22.74

4

0.67

0.033

S44

Protein GOS9

A0A0J8CL92

17.73

8.82

67.27

71.84

12

1.43

0.016

S45

Putative exosome complex component rrp40

A0A0J8F4A9

26.03

8.53

14.02

8.51

2

1.44

0.040

DEPs in T510

45



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T45

Expansin-A4-like

A0A0J8CGK7

27.35

9.32

38.74

17.31

6

0.61

0.0005

T46

Lamin-like protein

A0A0J8FNB4

18.34

9.33

15.53

6.49

2

0.66

0.004

T47

Expansin-like A3

A0A0J8EE77

28.66

7.52

20.38

20.23

4

0.67

0.010

T48


A0A0J8F6U6

30.11

6.43

5.41

7.25

2

0.69

0.030

T49

Embryo-specific protein ATS3A-like

A0A0J8BUV5

21.09

7.34

14.05

5.30

2

1.38

0.028

T50

Signal peptide peptidase 2

A0A0J8CLI1

28.24

9.26

12.25

7.97

2

1.47

0.033

A0A0J8CAE8

35.35

8.75

7.02

9.35

2

1.51

0.042

Actin-depolymerizing factor 4

A0A0J8D098

15.93

5.44

13.67

5.40

2

2.03

0.014

S46


A0A0J8EJT3

41.22

9.11

35.23

395.10

19

0.64

0.006

S47


A0A0J8CHH9

47.19

9.13

34.47

64.11

12

0.69

0.027

T53


A0A0J8B7S7

36.57

5.5

6.29

4.55

2

0.53

0.018

T54


A0A0J8C7I3

18.41

4.68

43.90

58.51

10

0.62

0.042

T55


A0A0J8C885

17.72

4.94

32.05

44.48

9

0.64

0.023

T56


A0A0J8BNN1

13.91

7.12

32.81

11.51

4

0.68

0.023

T51 T52

Casparian strip membrane proteins (CASP) family

Unknown DEPs in S210

DEPs in T510

46


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Table of Contents (TOC) graphic

47



Figure 1. Morphological and physiological changes in sugar beet (T510 and S210) under control (0 Mm NaCl) and salt stress (280 mM NaCl) conditions. Ten-days-old sugar beet seedlings were treated with 280 mM NaCl for 7 days. Growth performance (a), dry weight (b), total leaf area (c), leaf relative water content (d) and chlorophyll (a+b) content (e) were measured in sugar beet seedlings. Asterisk (*) indicates significantly different at P < 0.05. Three biological replicates were performed. 167x207mm (300 x 300 DPI)


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Figure 2. Effects of salinity stress on photosynthesis of leaves and proline or free amino acids level of root in two sugar beet genotypes. Chlorophyll (a+b) content (a) and Net photosynthetic rate (b) were measured in the leaves of sugar beet seedlings, and proline (c) and free amino acids (d) content were detected in the roots of sugar beet seedlings. 182x144mm (300 x 300 DPI)



Figure 3. Effects of salinity stress on Na+, K+ and Cl− content in two sugar beet genotypes. Leaves Na+ (a), K+ (b) , Cl− (c) content and Na+/K+ ratio(d) of sugar beet treated with 0 mM and 280 mM NaCl. Asterisk (*) indicates significantly different at P < 0.05. Three biological replicates were performed. 166x130mm (300 x 300 DPI)


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Figure 4. Effects of salinity stress on N and P content in two sugar beet genotypes. N (a) and P (b) contents in leaves of sugar beet treated with 0 mM and 280 mM NaCl. of NaCl for 7 days. Asterisk (*) indicates significantly different at P < 0.05. Three biological replicates were performed. 74x133mm (300 x 300 DPI)



Figure 5. Effects of salinity stress on antioxidant enzyme system in two sugar beet genotypes. Leaves malondialdehyde (MDA) content (a), relative electrical conductivity (b), superoxide dismutase (SOD) (c) and ascorbate peroxidase (APX) (d) of sugar beet treated with 0 mM and 280 mM NaCl. Asterisk (*) indicates significantly different at P < 0.05. Three biological replicates were performed. 169x148mm (300 x 300 DPI)


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Figure 6. Functional classification of the identified differentially accumulated proteins in two sugar beet genotypes treated with salinity stress. Differentially accumulated proteins in S210 leaf (a) and root (c); Differentially accumulated proteins in T510 leaf (b) and root (d). 194x177mm (300 x 300 DPI)



Figure 7. Relative gene expression for salt-stressed proteins in two sugar beet genotypes examined using RT-qPCR. mRNA and protein expression levels of ten candidate proteins i.e., abscisic acid-stress-ripeninginducible 1(ASR1), thioredoxin-1 (Thx1), late embryogenesis abundant protein 47 (LEA47), v-type proton ATPase subunit G6 (ATP6V1G6) and auxin-binding protein19a ( ABP19a) in two genotypes sugar beet leaves (a, b), and cytochrome c oxidase subunit 6a (COX6A), jasmonate-induced protein homolog (JIPH), malate dehydrogenase (MDH), glutathione synthetase (GSS) and AT-hook motif nuclear localized protein23 (AHL23) in two sugar beet genotypes roots (c, d) are shown. 190x128mm (300 x 300 DPI)


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Figure 8. Schematic presentation of the potential mechanism of salt stress tolerance in T510 tolerant cultivar. Differentially accumulated or different levels of changes between the two cultivars in leaf (a) and root (b) were indicated. Red and blue highlighted proteins indicate only increase or decreased in salt tolerant cultivar T510 under salt stress, compared with salt sensitive cultivar S210. The green highlighted proteins indicate only decreased in salt sensitive cultivar S210 under salt stress 160x196mm (300 x 300 DPI)


Comparative Physiological and Proteomic Analysis of Two Sugar Beet

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