Plant Materials
Cassava cultivars (Manihot esculenta Crantz) Arg7 and Ku50, and W14 (Manihot esculenta ssp. Flabellifolia ), a wild ancestor of cassava were used in this study. W14 was donated by CIAT, KU50 came from Argentina and ARG7 came from Royal Agricultural University of Thailand, all of materials were introduced and identified by tropical crops genetic resources institute of Chinese Academy of Tropical Agriculture Sciences (CATAS), and deposited as MS000581, MS000124 and MS000580 respectively in variety resource bed of tropical crops genetic resources institute, CATAS.
Arg7 and Ku50 are cultivated varieties with high storage root yield and about 30% starch content in its storage root; W14 expressed 2–5 fold lower storage root yield and less than 5% starch content in the storage root. Comparison of the biological indexes between cultivars and the wild ancestor indicated the underlying features of the modern cultivar. They were planted in green house at early March 2015 with enough plants. The biomass, microstructure, sampling for several experiments were treated in the early stage (60 DAPE) involves the forming and elongation of the cassava storage root, middle stage (180 DAPE) involves storage root expansion and rapid accumulation of starch and later stage (270 DAPE) involves storage root approaching top weight or the highest dense starch. All original data collected from three plants with an average value for each genotype.
CF and [14C]Suc tracing
CFDA labelling was performed as described previously [9, 16]. The CFDA solution was introduced into the stem near ground of cassava plants. A cotton thread immersed in a tube at one end and with the other end passing through the phloem zone of the stem base of the storage root. This method was selected due to its high-speed of transport and reliability. After 40 min, the plants were labelled with approximately 500 µl of 1 mg.ml− 1 CFDA aqueous solution (prepared from a stock solution in acetone). The tubes were packaged in silver paper to avoid dye loss and fluorescence quenching under sunlight, and the CF was allowed to translocate for 72 h (which was selected after a time gradient experiment) with approximately 1000–2000 µl of 1 mg.ml− 1 CFDA aqueous solution as the storage root development. The sink tissues underground was subsequently sectioned and examined for CF fluorescence using CLSM (FV1000, Olympus, Japan). Samples were scanned using an excitation wave of 350–600 nm, and the strongest fluorescence peak was observed at 488 nm excitated by CF,
the strongest autofluorescence of cassava root was appeared at 405 nm. We scanned every section using a 405 nm and 488 nm excitation wave to determine the distribution of autofluorescence and the green fluorescence of CF.
Sucrose was labelled with 14C isotope under experimental condition. The [14C]Suc was injected into the upper stem with the same method as CFDA. After 48 and 72 hours,the storage roots were sampled and made paraffin sections. Freehand sections were gently compressed and autoradiographed in cassettes using Kodak (XBT-1) at 4 °C for 30 d. The distribution of [14C]Suc in the storage roots was detected by [14C]-autoradiography.
Measurement of Plasmodesmal Density
Plasmodesmal density was measured as described by [40] as used in a previous study[17]. Five serial sections of two orientations (transverse and longitudinal) of ultrathin sections were prepared from Spurr-infiltrated samples; each group comprised sections located approximately 20 µm apart. From each group, six ultrathin sections were selected at random and placed on copper grids (100-mesh). Five scopes (each consisting of phloem and the surrounding PCs) were observed from each ultrathin section for transmission electron microscope (TEM). Plasmodesmata were counted at all cell interfaces (i.e. the interfaces between SEs and CCs, SEs and PCs, CCs and PCs and PCs and PCs; PCs include phloem parenchyma and xylem storage parenchyma cells) in each selected field. The results of the plasmodesmal counting are shown as the number of plasmodesmata per micron of specific cell/cell interface length per transverse section, which is referred to as plasmodesmal density (no. of plasmodesmata. µm− 1); half plasmodesmata were counted as one.
Histochemical Analysis and Ultrastructural Observation
Antibodies against CWI, SAI and SuSy were generated against polypeptides by the ComWin Biotech Co., Ltd. (China). The gene sequences of CWI were obtained from Phytozome V12.1 (http://www. Phytozome.com), and we selected three isoforms of this enzyme that were expressed in cassava roots (four developmental stages), they were CWI-1, CWI-2 and CWI-5. The former two isoforms with the same amino acids have the partial sequence QPYRTSYHFQPPK, whereas the latter has the specific sequence DPKQRQVQNYAVPK. The isoform specific amino acid partial sequence of SAI was QKGSEQTFPSRE, which was generously provided by Dr. Zhang Peng (Institute of Plant Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) and was proven to be highly and specifically expressed in cassava root. The sequence of SuSy, which was also highly expressed in cassava root, was generously provided by Dr. Luiz (EMBRAPA genetic resources and biotechnology, Brasilia, DF, Brazil). Its Genebank number is AAV74405.1, and the specific sequence of this isoform is RRKESKDLEEXA (basic information of these genes is shown in Table 3).
Table 3
Basic information of the cassava genes of sucrose synthase, cell wall invertase and sucrose transporters
Gene | JGI ID v6.1 | Location | Transcript (bp) | CDS (bp) | Protein (aa) |
SuSy1 | Manes.03G044400 | Chr03:3583270..3588587 reverse | 2752 | 2421 | 806 |
SuSy2 | Manes.03G198900 | Chr03:28057541..28064463 reverse | 2598* | 2241 | 746 |
SuSy3 | Manes.01G221900 | Chr01:30850003..30857026 reverse | 2867 | 2436 | 811 |
SuSy4 | Manes.16G090600 | Chr16:24754130..24759053 forward | 2750 | 2421 | 806 |
SuSy5 | Manes.02G081500 | Chr02:6080529..6085146 reverse | 2881 | 2526 | 841 |
SuSy6 | Manes.14G107800 | Chr14:8748090..8752577 forward | 3026 | 2739 | 912 |
SuSy7 | Manes.01G123800 | Chr01:24140021..24144607 reverse | 2783 | 2526 | 841 |
CWI1 | Manes.03G049200.1 | Chr03:4223184..4226988 forward | 2401 | 1779 | 592 |
CWI2 | Manes.08G027200.1 | Chr08:2431570..2433947 forward | 1851 | 1719 | 572 |
CWI3 | Manes.11G025400.1 | Chr11:2236114..2240122 reverse | 2079 | 1767 | 588 |
CWI4 | Manes.04G140500.1 | Chr04:26727894..26729547 forward | 1369 | 1278 | 425 |
CWI5 | Manes.008G027200.1 | Chr08:2431570..2433947 forward | 1851 | 1719 | 572 |
CWI6 | Manes.0009G053500.1 | Chr09:7064243..7068843 reverse | 1719 | 1719 | 572 |
SUT1-1 | Manes.18G099400.1 | Chr18:8603890..8607266 forward | 2193 | 1785 | 594 |
SUT1-2 | Manes.02G190300.1 | Chr02:15510673..15513298 forward | 2189 | 1542 | 513 |
SUT4-1 | Manes.18G054200.1 | Chr18:4548075..4559586 forward | 3720 | 1497 | 498 |
SUT4-2 | Manes.05G186600.1 | Chr05:25724747..25733136 forward | 3144 | 1491 | 496 |
SUT2-2 | Manes.05G099000.1 | Chr05:8333636..8347861 reverse | 3028 | 1827 | 608 |
Ultrathin sections were prepared essentially as follows: the storage roots were cut into small cubes (approximately 2 mm3) that were immediately fixed with 4% (v/v) glutaraldehyde in 100 mM precooled phosphate buffer (pH 7.4) over 4 h at 4 °C. The penetration of the glutaraldehyde buffer was improved manually by using a syringe. After an extensive rinse with the precooled phosphate buffer (pH 7.4), the tissue cubes were postfixed in 1% (w/v) OsO4 for 1–2 h and shaken several times during this period. Following another extensive rinse with the same buffer, the samples were dehydrated through a graded ethanol series (50–100%). Propylene oxide was used to displace the spur epoxy resin and then infiltrated it for 24 h at room temperature. Polymerisation was conducted at 70 °C for 8 h. These sections (approximately 80 nm in thickness) were mounted on 100-mesh copper grids or nickel coated with 0.25% Formvar film for ultrastructural observation using a JEM 2100 TEM.
Sections prepared from paraffin-embedded tissues were used for histological structure observations. The small cubes (2–3 cm3) of cassava storage root was immediately fixed with formalin–acetic acid–alcohol (FAA) fixation for 36 h at room temperature and dehydrated through a series of graded ethanol (80–100%). The following transparent was processed by n-butyl alcohol with three times and embedding using paraffin for 12 h at 60 °C-65 °C.
Immunogold Labelling
Immunogold labelling was conducted as described by [16]. Briefly, ultrathin sections were prepared as described above except they were fixed with 4% glutaraldehyde. The sections were first incubated with rabbit antiserum specific for SAI, CWI or SUT prepared as described above and then incubated with a secondary antibody (goat anti-rabbit IgG antibody conjugated to 10 nm gold). Finally, the sections were double-stained with uranyl acetate and alkaline lead citrate and examined with a JEM-2100 TEM. The specificity and reliability of the immunogold-labelling experiments were verified using two negative controls. In the first negative control, the antiserum was omitted to test for the potential nonspecific labelling by the goat anti-rabbit IgG antibody-gold conjugate. In the second negative control, the rabbit preimmune serum was used instead of the rabbit antiserum prior to immunogold labelling to determine the specificity of the anti-serum. At least three replicates of the control experiments were performed for each sample.
Extraction of mRNA, Determination of Its Levels and Enzymatic Activity and Western Blotting Analysis
Total RNA from cassava roots was extracted using RNAplant plus Reagent (TIANGEN, China). Primers for SuSy, CWI and SAI were designed based on the sequences described above, and amplicons were detected using a real-time quantitative PCR cycler (Rotor-Gene6000, QIAGEN, German). β-actin was used as the reference gene.
Enzyme extraction and assays of SAI or CWI activity were performed as described previously [41]. Briefly, the extraction buffer A medium was composed of 150 mM Tris-HCl (pH 8.0), 2 mM ethylenediamine tetra acetic acid, 10 mM MgCl2, 0.2% (v/v)-mercaptoethanol, 0.1 mM phenylmethyl sulfonyl fluoride, 1 mM benzamidine, 10 mM ascorbic acid and 3% (w/v) polyvinylpolypyrrolidone. The slurry was passed through four layers of cheesecloth. The filtrate was centrifuged at 16,000 g for 20 min, and the supernatant was used for the SAI assays. The residue, which was used to prepare CWI, was rinsed with the same buffer without polyvinylpolypyrrolidone until the effluent was free of protein. From this material, CWI was extracted in buffer A supplemented with 0.5 M NaCl with gentle shaking for 24 h. After centrifugation, the supernatant was used for the CWI enzymatic assays. All extraction procedures were performed at 4 °C. SAI activity was assayed using soluble and insoluble fractions, and each assay contain 0.3 ml of 100 mM sodium acetate buffer (pH 4.8), 0.1 ml of 100 mM sucrose and 0.1 ml of enzyme sample as described by [42].
The enzymatic extraction and assays of SuSy activity were performed as described by [43]. Briefly, frozen root tissues were ground in 5 ml of medium containing 50 mM HEPES buffer (pH 7.0), 10 mM 2-mercaptoethanol, 2% polyvinylpolypyrrolidone, 1% polyvinylpyrrolidone, 1 mM EDTA and 10 mM MgCl2. Next, 0.1 ml of this desalted extract was added to 0.9 ml of reaction medium composed of 25 mM HEPES-NaOH buffer (pH 6.5), 125 mM sucrose, 15 mM MgCl2 and 2 mM UDP. The enzymatic activity in the direction of the sucrose cleavage was assayed at 28 °C in the presence of sucrose. The reducing sugars produced were assayed using the 3, 5-dinitrosalicylic acid-based method described by [44].
Proteins were extracted according to Miron and Schaffer [45]. The extraction buffer consisted of 100 mM Tris-HCl (pH 8.9), 250 mM sucrose, 10 mM MgCl2, 5 mM vitamin C and 3.5% crosslinking polyvingypyrrolidone. (NH4)2SO4 was used to precipitate CWI, SAI and SuSy proteins, and protein concentrations were determined using the Bradford [46] method with bovine serum albumin as a standard. SDS-PAGE and immunoblotting assays were performed as described by Pan [41], with some modifications. After electrophoretic transfer from the polyacrylamide gels, the nitrocellulose membranes were blocked and incubated overnight at 4 °C in the antiserum specific to SAI (1:4000), CWI (1:2000) and SuSy (1:5000), which were diluted with Tris-buffered saline (TBS: 10 mM Tris-HCl, 150 mM NaCl) + 0.05% Tween20 + 3% bovine serum albumin (BSA). The membranes were then washed in TBS with Tween 20 (TBST: 10 mM Tris-HCl, 150 mM NaCl + 0.05% Tween20) for three times, incubated for 45 min at room temperature with goat anti-rabbit IgG-alkaline phosphatase conjugate, diluted 1000 times with TBST2 (50 mM Tris-Hcl, 150 mM NaCl + 0.1% Tween20 + 1% BSA) and then used as secondary antibody. After being washed three times in TBST2 and TBS, these membranes were stained with BCIP/NBT Kit (ComWin Biotech Co., Ltd, China). β-actin was used as a reference.
Collection and Analysis of Phloem Exudates
Phloem exudates were collected from the stems of 60-day-old seedlings as described by King and Zeevaart [47]. The stem was cut near the base, washed with ultrapure water, and inserted into a solution of 20 mM EDTA (pH 7.5). The detached plant was kept in darkness at 30 °C and 95% humidity for 1 to 2 h and then transferred to ultrapure water for collection for 4–5 h. Phloem exudates were lyophilised and stored at − 80 °C. The sucrose, glucose, fructose, sorbitol, stachyose and raffinose contents of the exudates were examined by using HPLC with evaporative light scattering detector (ELSD). The samples were analysed in 10 µl increments with water amide (xBridge 3.5 µm, 4.6 mm × 150 mm, USA); the mobile phase was 70% (v/v) acetonitrile and 0.1%(v/v) ammonium hydroxide; flow rate: 1 ml min− 1; temperature: 25 °C; drift tube temperature: 85 °C; nitrogen flow rate: 2.0 l min− 1; gain value: 2.
Transcriptome Sequencing and Annotation
Ten RNA libraries from developing leaves and storage roots of KU50, Arg7 and W14 plants were sequenced using the Illumina High-Seq 2000 system with approximately 100 bp reads. After pre-processing, the mRNA sequence reads from 10 samples were mapped to the draft genome sequences of AM560-2. Approximately 20,000 UniGenes were annotated. The genes involved in the development of the all RNA-seq reads of 10 samples were uploaded in NCBI SRA with the following accession numbers: SRX551093, SRX553797, SRX553799, SRX553800, SRX553801, SRX553802, SRX553803, SRX553804, SRX553805, and SRX553807.
Chemicals
Sucrose, glucose, fructose, sorbitol, stachyose, and raffinose were purchased from TCI (Japan). Protein ladder (Fermentas, Canada), SYBR Premix ExTaq (TaKaRa, Bio Inc., Japan), the ReverAid first-strand cDNA synthesis kit (Fermentas, Canada), and glutaraldehyde (TED, PELLA Inc., USA) were obtained from the indicated suppliers; all other chemicals were purchased from Sigma (USA).
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version
abbreviations:
CF, carboxyfluorescein; CC, companion cells; CFDA, Nonfluorescent 6(5) carboxyfluorescein diacetate; CLSM, Confocal laser scanning microscopy; CW, cell wall; CWI, cell wall acid invertase; DAPE, days after plant emergence; GFP, green fluorescent protein; HT, hexose transporter; NI, neutral invertase; SAI, Soluble acid invertase; SEL, size exclusion limit; SE, sieve element; SFR, secondary fibrous root; SuSy, sucrose synthase; SUT, sucrose transporters; PC, parenchyma cells; PD, plasmodesmata; PFR, primary fibrous root; PPH, primary phloem; PPUs, pore-plasmodesmata units; S, starch granule; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TBST, Tris-buffered saline with Tween 20; TEM, Transmission electron microscopy; UDPG, Uridine Diphosphate Glucose; V, vacuole; Suc, sucrose; Glc, glucose; Fru, fructose; CATAS, Chinese Academy of Tropical Agriculture Sciences