Plant Materials
The cassava (Manihot esculenta Crantz) cultivars Arg7 and Ku50 and cassava (Manihot esculenta ssp. Flabellifolia) accession W14, a wild ancestor of cassava, were used in this study. W14 was donated by CIAT, KU50 originated from Argentina, and Arg7 was provided by the Royal Agricultural University of Thailand. All materials were introduced and identified by the Tropical Crop Genetic Resources Institute of the Chinese Academy of Tropical Agriculture Sciences (CATAS) and deposited as MS000581, MS000124 and MS000580, respectively, in the variety resource bed of the Tropical Crop Genetic Resources Institute, CATAS.
Arg7 and Ku50 are cultivars with high-yielding storage roots, with approximately 30% starch content in them. Compared with Arg7, W14 presents 2-5-fold lower storage root yields and less than 5% starch content in its storage roots. Comparison of the biological indexes between cultivars and the wild ancestor were made to determine the underlying features of the modern cultivar. The plants were planted in a greenhouse in early March 2015, with enough plants planted. With respect to the biomass, microstructure, and sampling performed for the different experiments, treatments were applied at the early stage (60 DAPE), at which point the cassava storage roots were forming and elongating, at the middle stage (180 DAPE), at which point the storage roots were expanding and rapidly accumulating starch, and the late stage (270 DAPE), at which point the storage roots were near their maximum weight or had the highest density of starch. All the original data were collected from three plants, with the average value taken for each genotype.
CF and [14C]Suc Tracing
CFDA labeling was performed as described previously [9, 16]. The CFDA solution was introduced into the stem near the base of cassava plants. A cotton thread was placed in a tube at one end, with the other end passing through the phloem zone of the stem base of the storage roots. This method was selected for its high speed of transport and reliability. After 40 min, the plants were labeled with approximately 500 μl of 1 mg.ml−1 CFDA aqueous solution (prepared from a stock solution in acetone). The tubes were wrapped in aluminum foil to avoid loss of dye and fluorescence quenching under sunlight, and the CF was allowed to translocate for 72 h (which was selected after a time gradient experiment); approximately 1000-2000 μl of 1 mg.ml−1 CFDA aqueous solution was used for the developing storage roots. The belowground sink tissues were subsequently sectioned and examined for CF fluorescence using CLSM (FV1000, Olympus, Japan). The samples were scanned using an excitation wavelength of 350–600 nm, and the strongest fluorescence peak was observed at 488 nm excited by CF,
The strongest autofluorescence of cassava roots was detected at 405 nm. We scanned every section using 405 nm and 488 nm excitation wavelengths to determine the distribution of autofluorescence and the green fluorescence of CF.
Sucrose was labeled with the 14C isotope under experimental conditions. The resulting [14C]Suc was injected into the upper stem according to the same method as that used for CFDA. After 48 and 72 hours, the storage roots were sampled, and paraffin sections were made. Manually made sections were gently compressed and autoradiographed in cassettes using a Kodak XBT-1 instrument 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 in [40] and 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 samples (each consisting of phloem and surrounding PCs) were observed from each ultrathin section for transmission electron microscopy (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 both phloem parenchyma and xylem storage parenchyma cells) in each selected field. The results of the plasmodesma 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. plasmodesmata.μm−1); half plasmodesmata were counted as one.
Histochemical Analysis and Ultrastructural Observations
Antibodies against CWI, SAI and SuSy were generated against polypeptides by 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 (at four different developmental stages): CWI-1, CWI-2 and CWI-5. The last two isoforms with the same amino acids have the partial sequence QPYRTSYHFQPPK, whereas the last one has the specific sequence DPKQRQVQNYAVPK. The isoform-specific partial amino acid sequence of SAI was QKGSEQTFPSRE; this sequence was generously provided by 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 roots. The sequence of SuSy, which was also highly expressed in cassava roots, was generously provided by Dr. Luiz (EMBRAPA Genetic Resources and Biotechnology, Brasilia, DF, Brazil); its GenBank number is AAV74405.1, and the specific sequence of this isoform is RRKESKDLEEXA (basic information about these genes is shown in Table 3
Ultrathin sections were essentially prepared 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) for 4 h at 4 °C. The penetration of the glutaraldehyde buffer was performed 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 that time 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, which was then infiltrated for 24 h at room temperature. Polymerization was conducted at 70 °C for 8 h. These sections (approximately 80 nm in thickness) were mounted on 100-mesh copper grids or were nickel coated with 0.25% Formvar film for ultrastructural observations using a JEM 2100 transmission electron microscope.
Sections prepared from paraffin-embedded tissues were used for histological structure observations. Small cubes (2-3 cm3) of cassava storage roots were immediately fixed in formalin–acetic acid–alcohol (FAA) solution for 36 h at room temperature and dehydrated through a series of graded ethanol (80%–100%). The subsequent material was processed by n-butyl alcohol three times and then embedded in paraffin for 12 h at 60-65 °C.
Immunogold Labeling
Immunogold labeling was conducted as described in [16]. Briefly, ultrathin sections were prepared as described above except that 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 together with secondary antibodies (goat anti-rabbit IgG antibodies conjugated to 10 nm gold). Finally, the sections were double-stained with uranyl acetate and alkaline lead citrate and subsequently examined with a JEM-2100 transmission electron microscope. The specificity and reliability of the immunogold-labeling experiments were verified using two negative controls. In the first negative control, the antiserum was omitted to test for potential nonspecific labeling by a goat anti-rabbit IgG antibody-gold conjugate. In the second negative control, rabbit preimmune serum was used instead of rabbit antiserum prior to immunogold labeling to determine the specificity of the antiserum. At least three replicates of the control experiments were included for each sample.
Extraction of mRNA, Determination of mRNA Levels and Enzyme 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-Gene 6000, QIAGEN, German). β-Actin was used as a 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 ethylenediaminetetraacetic 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 subsequently centrifuged at 16,000 g for 20 min, after which the supernatant was used for 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 enzyme assays. All extraction procedures were performed at 4 °C. The SAI activity was assayed using soluble and insoluble fractions, and each assay consisted of 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 in [42].
The enzyme extractions and assays of SuSy activity were performed as described in [43]. Briefly, frozen root tissues were ground in 5 ml of media containing 50 mM HEPES buffer (pH 7.0), 10 mM 2-mercaptoethanol, 2% polyvinylpolypyrrolidone, 1% polyvinylpyrrolidone, 1 mM EDTA and 10 mM MgCl2. Afterward, 0.1 ml of the desalted extract was added to 0.9 ml of reaction media composed of 25 mM HEPES-NaOH buffer (pH 6.5), 125 mM sucrose, 15 mM MgCl2 and 2 mM UDP. The enzyme activity in the direction of sucrose cleavage was assayed at 28 °C in the presence of sucrose. The reducing sugars produced were assayed using the 3,5-dinitrosalicylic acid-base method described in [44].
Proteins were extracted according to the methods of 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 polyvinylpyrrolidone. (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 used as a standard. SDS-PAGE and immunoblotting assays were performed as described by Pan [41], with slight modifications. After electrophoretic transfer from the polyacrylamide gels, the nitrocellulose membranes were blocked and incubated overnight at 4 °C in 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% Tween 20 +3% bovine serum albumin (BSA). The membranes were then washed in TBS together with Tween 20 (TBST; 10 mM Tris-HCl, 150 mM NaCl +0.05% Tween 20) three times, incubated for 45 min at room temperature together with goat anti-rabbit IgG-alkaline phosphatase conjugate, diluted 1000-fold with TBST2 (50 mM Tris-HCl, 150 mM NaCl +0.1% Tween 20 +1% BSA) and then treated with secondary antibodies. After being washed three times in TBST2 and TBS, these membranes were stained with a BCIP/NBT Kit (ComWin Biotech Co., Ltd., China). β-Actin was used as a reference gene.
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 lyophilized and stored at −80 °C. The sucrose, glucose, fructose, sorbitol, stachyose and raffinose contents of the exudates were evaluated by using HPLC with an evaporative light-scattering detector (ELSD). The samples were analyzed in 10 μl increments with water amide (xBridge 3.5 µm, 4.6 mm × 150 mm, USA); the mobile phase consisted of 70% (v/v) acetonitrile and 0.1% (v/v) ammonium hydroxide; the flow rate was 1 ml min−1; the temperature was 25 °C; the drift tube temperature was 85 °C; the nitrogen flow rate was 2.0 l min−1; and the gain value was 2.
Transcriptome Sequencing and Annotation
Ten RNA libraries generated from developing leaves and storage roots of KU50, Arg7 and W14 plants were sequenced using an Illumina High-Seq 2000 system, with approximately 100 bp reads. After preprocessing, the mRNA sequence reads from 10 samples were mapped to the draft genome sequence of AM560-2. Approximately 20,000 unigenes were annotated. The genes involved in the development of all RNA-seq reads of the 10 samples were uploaded to the NCBI SRA under 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). The protein ladder (Fermentas, Canada), SYBR Premix ExTaq (TaKaRa, Bio Inc., Japan), ReverAid First-strand cDNA Synthesis Kit (Fermentas, Canada), and glutaraldehyde (TED, Pella, Inc., USA) were obtained from the indicated suppliers. All the other chemicals used were purchased from Sigma (USA).