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Regulation Network of Sucrose Metabolism in Response to Trivalent and Hexavalent Chromium in Oryza sativa Yu Xi Feng, Xiao Zhang Yu, Ce-Hui Mo, and Chun Jiao Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01720 • Publication Date (Web): 14 Aug 2019 Downloaded from pubs.acs.org on August 16, 2019
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Journal of Agricultural and Food Chemistry
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Regulation Network of Sucrose Metabolism in Response to Trivalent and
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Hexavalent Chromium in Oryza sativa
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Yu-Xi Feng†, Xiao-Zhang Yu†,*, Ce-Hui Mo‡,*, Chun-Jiao Lu†
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6
†
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541004, People's Republic of China
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‡
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Materials, College of Life Science and Technology, Jinan University, Guangzhou 510632,
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College of Environmental Science & Engineering, Guilin University of Technology, Guilin
Guangdong Provincial Research Center for Environment Pollution Control and Remediation
People's Republic of China
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*Corresponding
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Xiao-Zhang Yu. Phone: +86 7735897016. E-mail:
[email protected] 14
Ce-Hui Mo. Phone: +86 2085223405. E-mail:
[email protected] authors
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Yu-Xi Feng, ORCID iD: 0000-0002-4917-7283
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Xiao-Zhang Yu, ORCID iD: 0000-0001-7846-5017
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Ce-Hui Mo, ORCID iD: 0000-0001-7904-0002
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Chun-Jiao Lu, ORCID iD: 0000-0002-4321-1532
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ABSTRACT: The presence of chromium (Cr) in cultivated field affects carbohydrate
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metabolism of rice (Oryza sativa L.) and weakens its productivity. Little is known about the
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molecular mechanism of sucrose metabolism underlying Cr stress response in rice plants. In
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the present study, the transcriptome map of sucrose metabolism in rice seedlings exposed to
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both trivalent and hexavalent chromium was investigated using Agilent 4X44K rice
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microarray analysis. Results indicated that Cr exposure (3-day) significantly (p < 0.05)
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improved sucrose accumulation, and altered the activities of sucrose synthetase, sucrose
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phosphate phosphatase, and amylosynthease in rice tissues. We identified 119 differentially
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regulated genes involved in 17 sucrose metabolizing enzymes; and found that gene responses
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in roots were significantly (p < 0.05) stronger than in shoots under both Cr(III) and Cr(VI)
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treatments. The network maps of gene regulation responsible for sucrose metabolism in rice
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plants provide a theoretical basis for further cultivating Cr-resistant rice cultivars through
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molecular genetic improvement.
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KEYWORDS: Oryza sativa, Cr speciation, sucrose metabolism; enzymes, transcriptomes
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Journal of Agricultural and Food Chemistry
1. INTRODUCTION
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Chromium (Cr), one of the most commonly used metal, is considered to be one of the top
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20 hazardous substances on the Superfund priority list.(1) It is widely applied in electroplating,
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manufacture of dyes, steel production, leather and wood preservation, etc.(1) Since the 1950s,
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Cr production has been increasing significantly, and its cumulative global production was
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approximately up to 105.4 million tons in 2000.(2) Cr contamination has become a global issue
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because of its high concentration in the environment media, for example soil, water, dusts and
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vegetations, resulting from various industrial and agricultural activities.(3) In natural
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conditions, Cr exists in several oxidation states (from -2 to +6), where trivalent Cr(III) and
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hexavalent Cr(VI) species are two relatively common and stable oxidation states in the
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environment.(1) Nutritionally, Cr(III) in small amount is an essential element that plays a
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positive role in lipid and sugar metabolism in human and animal.(4) However, Cr(VI) is
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regarded as highly toxic to human and animal, causing various of clinical diseases including
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carcinogenic and mutagenic effects.(1) Accumulation of Cr in plants, especially Cr(VI) can
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pose serious damage on plant growth and development through restraining nutrient balance
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and transpiration, degrading photosynthetic pigments, reducing mitochondrial electron
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transport, and activities of antioxidant enzymes(5). Cr(VI) can be reduced to Cr(III) possibly
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by the Fe3+ reductase enzyme in plants, but this process hardly happen, whereas Cr(III) cannot
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be oxidated to Cr(VI).(6) It should distinguish between Cr(III) and Cr(VI) in plant tissues
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because their phytotoxicity effects are very different.
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Sucrose, the primary end product of photosynthesis, provides some key information
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related to crop productivity such as a pivotal energy substance and a signal molecule
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mediating various physiological processes.(7) It serves as metabolite for plant cellular
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respiration and is a crucial osmolyte to maintain cell homeostasis.(8) Expression of genes
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related growth and stress of plants are regulated by sucrose, and plants respond to abiotic
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stress through the sucrose sensing mechanisms.(7) Partitioning of sucrose in plant cells is
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variable in response to many abiotic and biotic stimuli.(7) Previous studies have confirmed that
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environmental stresses, e.g. water/drought stress, heat/cold stress, and salt stress, can pose a
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significant effect on sucrose metabolism in plant tissues. However, research on the pathway
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of sucrose metabolism and its associated molecular mechanism under toxic metal stress is
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currently not available.
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Sucrose cannot be used directly in plants and must instead be cleaved into hexoses by
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sucrose-metabolizing enzymes(9). In particular, the key enzyme involved in sucrose anabolism
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is
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sucrose-6-phosphate + UDP ), which catalyzes the biosynthesis of sucrose in the cytosol of
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photosynthetic cells.(10) It is perceived as a primary control enzyme in sucrose synthesis.
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Notably, the key enzymes involved in sucrose degradation are invertase (INV, sucrose + H2O
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→ glucose + fructose) and sucrose synthetase (SS, sucrose + UDP ⇌ fructose +
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UDPglucose).(11) The first step in cleavage of sucrose is achieved by either INV or SS. INV
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catalyzes irreversible hydrolysis of sucrose to glucose and fructose, while SS is a cytosolic
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and reversible enzyme that cleaves sucrose to UDPglucose and fructose.(11) The regulation of
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these reactions and its consequences have become a crucial issue in responsive to external
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environmental signals such as heavy metal stress.
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sucrose
phosphate
synthase
(SPS,
UDPglucose
+
fructose-6-phosphate
⇌
Previous studies reported that high levels of toxic metals, e.g., cadmium (Cd), nickel (Ni),
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aluminium (Al), and copper (Cu) in soil can significantly perturb sucrose metabolism in
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growing plants, altering the levels of sucrose and the activity of key enzymes in their
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metabolic pathways.(8),(12),(13),(14) Prado et al. (2017) demonstrated that Cr(VI) affect
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accumulation pattern of sucrose, sucrose-related enzyme activities, and carbon allocation
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toward leaf development in floating (fronds) and submerged (lacinias) leaves of S. minima
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plants.(15) Nonetheless, the previous studies were only conducted at biochemical level, few
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was involved in the expression of sucrose metabolizing enzymes genes at transcriptional level
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under toxic metal stress. The variation in the molecular information of biochemistry trait
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among these enzymes remains unknown. Therefore, more comprehensive studies are needed
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to identify the regulation mechanism of sucrose metabolism in plant tissue under toxic metal
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stress.
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To achieve this purpose, we performed the following work: (1) measure the response to
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sucrose content and sucrose metabolizing-enzymes at different rice tissues in the presence of
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Cr(III) and Cr(VI); (2) investigate the differentially expression genes (DEG) encoded with
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sucrose metabolizing enzymes in rice seedlings exposed to both Cr species using microarray
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analysis and qPCR; (3) plot the network maps of gene regulation responsible for sucrose
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metabolism in different parts of rice exposed to two Cr species.
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2. MATERIALS AND METHODS
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2.1 Plant Cultivation and Cr Treatment. Rice (Oryza sativa L. cv. XZX 45) seedlings were
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cultivated in sandy soils inside the artificial climate chamber with controlled temperature of
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25 ± 0.5°C at relative humidity of 60 ± 2%. The modified 8692 nutrient solution was prepared
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in 1.0L of water conditions (KNO3 285.2 mg/L, MgCl2·H2O 12 mg/L, CaCl2 18 mg/L,
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MgSO4 15 mg/L, KH2PO4 33.46 mg/L, NaHCO3 150 mg/L, H3BO3 0.1855 mg/L, MnCl2
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0.415 mg/L, Fe-EDTA 4.83 mg/L, NaMoO4 7 mg/L, CuSO4 6.25 mg/L, ZnSO4 2.99 mg/L,
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CoCl2 1.5 mg/L). After growing for 16 days, rice seedlings were collected and rinsed with
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distilled water and incubated in a pretreated solution containing 1 mM CaCl2 + 2 mM
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MES-Tris buffer (pH = 6.0) for 4 h to remove the Cr ions from the cell surface.(5) Exposure
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tests of rice seedlings were conducted in accordance to our previous study with minor
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modification.(16) Briefly, ten pre-treated seedlings of similar size were selected and exposed to
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a 50 mL erlenmeyer flask spiked with 50 mL Cr(VI) (0, 2.0, 8.0, and 16.0 mg/L) or Cr(III)
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solution (0, 12.0, 24.0, and 40.0 mg/L). These concentrations of these Cr solutions were
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determined based on three different effective concentrations (EC20, EC50 and EC75) which
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were derived from the 20%, 50% and 75% relative growth inhibition rates of young seedlings,
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respectively.(17) Each treatment was performed with four independent replicates. Potassium
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chromate (Cr(VI), CAS: 7789-00-6) and chromium nitrate (Cr(III), CAS: 7789-02-8) of
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guaranteed reagent was used in this study. To minimize evaporation of water and prevent
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algal growth, erlenmeyer flasks were covered with aluminum foil. All the young seedlings
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were placed in the plant growth chamber for a 3-day exposure.
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2.2 Cr Analysis. After a 3-day exposure, rice seedlings were collected and rinsed with
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distilled water and divided into shoot tissues and root tissues. The remaining procedure was
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described in our previous work.(16) Dry seedling materials were digested with HNO3-HClO4
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(v/v, 4:1) solution. The concentrations of total Cr in different tissues of rice seedlings were
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measured using Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES,
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PerkinElmer Optima 700 DV). The detection limit, determined as mean blank plus three times
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the standard deviation of ten blanks, was 0.07 μg Cr/L.
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2.3 Determination of Sucrose. Rice tissues were dried immediately at 70°C in an oven for 48
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hours and the contents of sucrose were determined colorimetrically. Roots and shoots were
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homogenized with 3 mL 80% (v/v) using a cold pestle and mortar. After heating the
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homogenate at 80°C water bath for 10 minutes, the supernatant was removed by
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centrifugation at 10,000 × g for 10 minutes. The extraction process was carried out for three
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times with 3 mL 80% (v/v) ethanol and centrifuged as described above. Then, 10 mg
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activated carbon was added to supernatant for decoloring followed by addition of 0.1 M
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NaOH solution to 0.9 mL supernatant. After cooling to room temperature, 3 mL 10 M HCl
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and 1 mL 0.1% resorcinol were added to the supernatant at 80°C water bath (0.1g resorcinol
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with 100 mL 95% ethanol). After that, sucrose contents were determined using ultraviolet
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spectrophotometer at 480 nm.(18)
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2.4 Determination of Sucrose Metabolizing Enzymes Activity. Sucrose phosphate
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phosphatase (SPS), sucrose synthetase (SS), and amylosynthease (SSS) were extracted from
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rice roots and shoots. All fresh samples were frozen in liquid nitrogen with buffer solution,
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and the supernatants were collected using centrifugation with 3,000 × g for 20 minutes. More
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specifically, to analyze the activities of SPS and SS, frozen fresh tissues were ground in liquid
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nitrogen with 3 mL HEPES-NaOH buffer (pH = 7.5) containing 15 mM MgCl2, 25 mM
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fructose 6-phosphate, 25 mM glucose 6-phosphate, and 25 mM UDP-glucose.(19) To
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determine the activities of SSS, frozen fresh tissues were ground in liquid nitrogen with 3 mL
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extraction buffer containing 10 mM pH 8.0 Tricine-NaOH butter, 8 mM MgCl2, 2 mM EDTA,
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50 mM mercaptoethanol, 12.5% glycerinum, 5% PVP-K40.(20) The extract was used for
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quantifying SPS, SS, and SSS activities using the ELISA Kit purchased from Nanjing
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Jiancheng Biology Engineering Institute (P.R. China).
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2.5 Microarray Analysis. After 3-day exposure, rice seedlings were separated into roots and
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shoots. Agilent 4X44K Rice Microarrays which contain more than oligonucleotide 44,000
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probes were applied in this analysis. The extraction of RNA and remaining procedure of
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microarray analysis were described in our earlier work.(17) The threshold values for selecting
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differentially expression genes (DEGs) were set as the P-value < 0.05. Meanwhile, the
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expression change ratio between non-treated and treated tissues was either < 0.5 or > 2.0.
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2.6 Identification of Genes Involved in Sources Metabolism in Rice. Identification of
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genes involved in sources metabolism in rice was performed via BLAST-P search in rice
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database RGAP, CRTC and RAP-DB based on their respective sequences as queries from the
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Arabidopsis database TAIR. After removing redundant and query hits, 119 genes were
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identified
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http://www.ricedata.cn/gene/index.htm;
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http://www.arabidopsis.org/, v10.0).
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2.7 qRT-PCR Analysis. Real-time quantitative PCR (qRT-PCR) was used to verify the
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expression levels of sucrose metabolism enzymes after Cr exposure. The total RNA (from
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different plant tissues of rice seedlings) was extracted using Trizol (Invitrogen, Carlsbad, CA,
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USA) and was used to conduct the qRT-PCR analysis. The remaining procedures are similar
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to our previous study.(21) The cycling conditions were denaturation at 95°C for 10 seconds →
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annealing 58°C for 30 seconds → extension at 72°C for 32 seconds. This cycle was repeated
(RGAP,
http://rice.plantbiology.msu.edu/analyses_search_blast.shtml; RAP-DB,
http://rapdb.dna.affrc.go.jp/;
CRTC, TAIR,
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for 40 times. The qRT-PCR analysis was measured using the 7500 Fast Real-Time PCR
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system
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(glyceraldehyde-3-phosphate dehydrogenase, LOC_Os08g03290.1) would be selected as
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house-keeping gene.(17) The standard 2−ΔΔCT was used to measure the relative expression of
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each of the targeted genes. All these values referred to mean ± SD with four independent
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replicates. Gene locus identifier and primer sequences are listed in Table S1.
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2.8 Data Analysis. Variance (ANOVA) and Tukey’s multiple range tests were used to
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measure the statistical significance at the level of 0.01 or 0.05 between the non-treatments and
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treatments. Asterisk refers to the significant difference between treatment and control (p