Construction of plant transformation vectors
Previously, the promoter fragment PEPC7-P4 of the alfalfa nodule-enhanced phosphoenolpyruvate carboxylase gene PEPC-7 (L39371) consisting of nucleotides −592 to 86 relative to the transcription start site was cloned and inserted into the XbaI and SmaI restriction sites in the plant expression vector pBI101.1  producing the PEPC7-P4::GUS chimeric reporter gene . Sequencing the PEPC7-P4 promoter during this study revealed a 56 bp direct repeat that was not reported in the original gene sequence submission. A sequence correction has been submitted to GenBank for the PEPC-7 gene. In an earlier study, the cDNA from a nodule-enhanced sucrose synthase gene (MsSUS1; AF049487) was isolated and cloned into pBluescript . For this study, the gusA sequence in the PEPC7-P4::GUS construct was replaced with an XmaI-SacI fragment containing the MsSUS1 cDNA to produce the PEPC7-P4:: MsSUS1 expression vector. The nucleotide sequences of the cloned DNA fragments were verified by sequencing at the University of Minnesota BioMedical Genomics Center.
Plant transformation and selection of transformed lines
Alfalfa (cultivar Regen SY) was transformed with the PEPC7-P4::GUS or the PEPC7-P4:: MsSUS1 construct by Agrobacterium tumefaciens-mediated transfer as previously described . Transformed plants were selected by kanamycin resistance and confirmed to be transgenic by the presence of nptII by PCR assays as described previously . The transformants selected to be used as transgenic control plants (M22, M35) contained the PEPC7-P4::GUS construct but lacked GUS expression as tested by histochemical staining. The transformants containing the PEPC7-P4:: MsSUS1 construct selected for further evaluation (M17, M18) had the lowest MsSUS1 expression as measured by qRT-PCR. The presence of the PEPC7-P4:: MsSUS1 construct in MsSUS1 down-regulated plants was confirmed by PCR using primers in the promoter and MsSUS1 coding sequence.
Plant material and culture conditions
Selected primary transformants were propagated clonally by stem cuttings and grown in the greenhouse. Primary transformants were used due to the severe inbreeding depression in alfalfa. For most experiments, plants were grown in a sand:soil mixture (2:1,v/v), one plant per cone-tainer (Stuewe & Sons, Tangent, OR; 7 cm width, 35 cm depth). Plants were grown in a randomized complete block with three or four replicates. Plants were watered weekly with quarter-strength Hoagland’s nutrient solution containing 25 ppm N . For qRT-PCR, immunbloting, and enzyme assay experiments, 10 stems were harvested from each replicate. ES internodes (apical four to five internodes) and PES internodes (seventh or eighth internode from the stem apex) were harvested from flowering plants as previously described . Stem material from each replicate (approximately 1 g fresh weight) was combined, frozen in liquid nitrogen, and stored at −80 °C until assayed. For experiments comparing growth of transformants, plants were grown in lime-amended sand and were watered with half-strength Hoagland’s nutrient solution containing 100 ppm N. Every fourth day, plants received only water. Plants were harvested at the time of flowering. Dry weight of roots and shoots were measured after drying at 60 °C. For examining expression of PEPC7-P4::GUS in roots and nodules, plants were grown in quartz sand. Plants were inoculated with Sinorhizobium meliloti (Nitragin®, Novozymes, Davis, CA) and watered daily with half-strength Hoagland’s nutrient solution without N.
Quantitative reverse-transcriptase PCR (qRT-PCR)
RNA was isolated from ES and PES samples using the RNeasy Plant Mini kit (Qiagen, Valencia, CA). Following DNase I treatment with the DNA-free kit (Ambion Inc., Austin, TX), first strand cDNA for each sample was made from 2 μg total RNA using Superscript II RT (Invitrogen, Carlsbad, CA) following the manufacturer’s recommendations and diluted 10-fold before use in PCR. Gene-specific primers (Additional file 3) were designed based on MsSUS isoform sequences retrieved from GenBank and alfalfa RNA-seq data (Additional file 1; http://plantgrn.noble.org/AGED/). qRT-PCR was performed using the iTaq Universal SYBR Green Supermix (BioRad, Hercules, CA) in 12.5 μL reactions containing 4 pmol of each forward and reverse primer and 2.5 or 3.0 μL of template cDNA. Samples from three biological replicates were run in triplicate on a StepOnePlusTM Real-Time PCR System (Applied Biosystems, Grand Island, NY) following the manufacturer’s recommendations. The PCR conditions were as follows: 30 s of pre-denaturation at 95 °C, 40 cycles of 3 s at 95 °C and 30 s at 60 °C, followed by steps for melting curve generation (15 s at 95 °C, 60 °C, 95 °C). The StepOne software (Applied Biosystems) was used for data collection. Disassociation curves for each amplicon were examined to confirm presence of a single amplicon. Melting curves showed that only one SUS transcript was measured demonstrating that the primers were specific for transcripts of each isoform. Relative transcript accumulation for each sample was obtained using the comparative Ct method  using the Ct value of the alfalfa actin gene (JQ028730.1) for sample normalization.
SUS enzyme assay
ES and PES internode samples (0.3 g) were ground with a mortar and pestle in 3.0 mL extraction buffer [100 mM MES, pH 6.8, 15 % (v/v) ethylene glycol, 2 % (v/v) β-mercaptoethanol, 60 mg polyvinylpolypyrrolidone (PVPP), 30 μL of 0.1 M phenylmethanesulfonylfluoride (PMSF), and 30 μL protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO)]. The homogenate was centrifuged (16,800 x g, 25 min) at 4 °C. The supernatant was applied to a desalting column (PD minitrap G-25, GE Healthcare, Buckinghamshire, UK) that had been equilibrated in extraction buffer. The eluent was used for the assays. Enzyme activity was assayed in a 1 mL reaction mixture containing 50 mM HEPES, pH 7.4, 2 mM magnesium acetate, 5 mM dithiothreitol (DTT), 2 mM EGTA, 50 mM sucrose, 1 mM potassium pyrophosphate, 1 mM UDP, 1 mM NAD, 0.02 mM D-glucose-1,6-diphosphate, and 1 unit each of phosphoglucomutase, uridine-5′-diphosphoglucose pyrophosphorylase, and glucose 6-phosphate dehydrogenase (from Leuconostoc mesenteroides; Sigma-Aldrich). Enzyme activity was monitored by measuring absorbance at 340 nm (24 °C) using a Thermo Scientific Genesys 6 spectrophotometer (Thermo Electron Corp., Madison, WI).
Invertase enzyme assays
The acid invertase assay procedure was adapted from Sergeeva et al. . Frozen tissue (0.4 g) from ES and PES internodes was ground in a mortar and pestle with liquid nitrogen then homogenized in 2 mL extraction buffer [50 mM HEPES · KOH, pH 7.4, 5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 5 mM DTT, 10 % glycerol, 40 mg PVPP, 20 μL 0.1 M PMSF, 20 μL protease inhibitor cocktail for plant cell and tissue extracts (Sigma-Aldrich)]. The homogenate was centrifuged (16,800 x g, 1 min, 4 °C) and the supernatant was transferred to a fresh tube on ice for assay of soluble (vacuolar) acid invertase. The pellet was washed three times with 1 mL of extraction buffer minus PVPP and DTT. Washing involved homogenizing the pellet followed by centrifugation (16,800 x g, 4 °C). Centrifugation lasted 1 min for the first two washes and 5 min for the final wash. The washed pellet was resuspended in extraction buffer consisting of 20 mM MES · KOH, pH 6.0, 1 M NaCl and incubated overnight at 4 °C. The next day the suspension was centrifuged (16,800 x g, 20 min, 4 °C) and the supernatant was transferred to a fresh tube on ice for assay of the cell wall bound acid invertase. Both cell wall and soluble acid invertase were measured using a two-step assay involving glucose formation (reaction A) and subsequent spectrophotometric assay of glucose involving NAD reduction (reaction B). For assay of soluble acid invertase, the reaction A medium contained 150 μL plant extract, 200 μL of 150 mM citrate-phosphate buffer, pH 4.5, and 50 μL of 400 mM sucrose. The assay was conducted at 30 °C and stopped after 10 min by placing tubes in a boiling water bath for 4 min. The tubes were centrifuged at room temperature to pellet precipitated proteins. Controls were run without added sucrose and used for background subtraction in the final calculations. The reaction B assay cuvette contained 450 μl of 2X HEPES assay buffer (100 mM HEPES, pH 7.4, 4 mM Mg acetate, 10 mM DTT, 4 mM EGTA), 100 μL substrate mix (10 mM NAD and 5 mM ATP in sterile water) and boiled reaction A (25 μL and 100 μL for ES and PES extracts, respectively) and water (375 μL and 300 μL for ES or PES tissue extracts, respectively). The reaction was run to completion (3 min) and background absorbance was measured at 340 nm using a Thermo Scientific Genesys 6 spectrophotometer. Next, 50 μL of an enzyme mix [1 unit each of hexokinase and glucose 6-phosphate dehydrogenase (from L. mesenteroides; Sigma-Aldrich) in 2x HEPES assay buffer] was mixed into each assay cuvette and incubated for 15 min at room temperature. Absorbance readings were taken on each reaction and the change in absorbance was used to calculate glucose content. For assay of cell wall acid invertase, the assay protocol was the same as for the soluble acid invertase assay except that 50 μL of cell wall extract was used for reaction A conducted at pH 4.8 and 200 μl of reaction A was used for the NAD reduction assay (reaction B).
For alkaline and neutral invertase assays, tissue was extracted in the buffer used for extracting acid invertase except PVPP was not included. The extract was centrifuged (16,800 x g, 25 min, 4 °C). Activities of both enzymes were measured in a two-step assay as described above for acid invertase except that the buffers of reaction A consisted of 100 mM HEPES-NaOH (pH 7.5) or 100 mM sodium borate (pH 9.4) for measuring neutral and alkaline invertase activity, respectively. Reaction A contained either 100 μL ES or 50 μL PES extract for measuring neutral invertase and 100 μL ES or PES extract for measuring alkaline invertase. For the neutral invertase assay of ES and PES extracts, reaction B contained 200 μL boiled reaction A. For the assay of alkaline invertase, reaction B contained 200 μL or 100 μL boiled reaction A for ES and PES extracts, respectively. Alkaline and neutral invertase assays were also run in the presence of Tris to check for known Tris inhibition.
Assays were run in triplicate on three biological replicates. Statistical analyses were performed using the analysis of variance function of InStat (GraphPad, San Diego, CA) with Dunnett’s multiple comparison post-hoc test to compare down-regulated lines to the control line.
Protein extraction and immunoblotting
Proteins were extracted by grinding 0.4 g stem tissue with a mortar and pestle in liquid nitrogen followed by the addition of 2 mL extraction buffer (100 mM MES, pH 6.8, 15 % ethylene glycol, 2 % β-mercaptoethanol, 100 mM sucrose, and 20 μL of 0.1 M PMSF). Following centrifugation (16,000 x g, 20 min, 4 °C) the supernatant was transferred to a fresh tube and the protein was quantified using the Bio-Rad Protein Assay reagent (Bio-Rad, Hercules, CA). Proteins were separated on a 10 % SDS-PAGE gel  and transferred to nitrocellulose. SUS was detected with an antiserum produced against maize sucrose synthase 2 (SS2; ). Precision Plus Dual Protein standards (Bio-Rad) were used to estimate molecular weight. Bound antibodies were visualized with the SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Scientific, Rockford, IL).
Mass spectrometry of SUS proteins
Total soluble proteins of PES internodes from M22 (control) were extracted as described above and separated on a 10 % SDS-PAGE gel. A protein band corresponding to the MsSUS1 antibody-reactive band, determined by immunoblot analysis of an identical lane run on the gel, was excised and an in-gel tryptic digest was performed. The identity of MsSUS1 from M22 PES tissue was confirmed by MS/MS performed by the University of Minnesota Center for Mass Spectrometry and Proteomics. LC/MS samples were analyzed using Sequest (Thermo Fisher Scientific, San Jose, CA, version 27). Sequest was set up to search the non-redundant M. truncatula database modified to include the GenBank entry AF049487 for the MsSUS1 transcript. Scaffold (version 3.6.0, Proteome Software Inc., Portland, OR) was used to validate LC/MS based peptide and protein identifications. Peptide identifications were accepted if they were established at greater than 95 % probability as specified by the Peptide Prophet algorithm . Protein identifications were accepted if they could be established at greater than 99 % probability and contained at least two identified peptides. Protein probabilities were assigned by the Peptide Prophet algorithm .
Sucrose synthase and neutral invertase in situ enzyme assays
SUS and neutral invertase in situ enzyme assays were conducted by coupling enzyme activity to the reduction and precipitation of nitro-blue tetrazolium (NBT). Transverse sections of ES and PES samples (100–150 μm) were cut using a sledge microtome. The sections were immediately fixed by applying fixation medium to the knife and to each freshly prepared stem section. The fixation medium contained 1 % paraformaldehyde, 2 % (w/v) polyvinylpyrrolidone 40, and 5 mM DTT, pH 7.0. The tissue was fixed at room temperature for 30 min to 1.5 h. After fixation, sections were rinsed three times in water followed by a 3 h water wash at 4 °C. Cold water was refreshed once during the 3 h period. The sucrose synthase assay was performed as described by Wittich and Vreugdenhil  except that the concentration of sucrose was 100 mM and the incubation period was 1 h at room temperature. The assay medium contained: 100 mM sucrose, 15 mM UDP, 15 mM PPi, 150 mM NAD, 3 mM glucose-1,6-bisphosphate, 1 unit phosphoglucomutase, 1 unit glucose-6-phosphate dehydrogenase, 1 unit UDPG pyrophosphorylase, 50 mM HEPES-KOH, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.1 % bovine serum albumin (BSA), 5 mM MgCl2, and 0.07 % NBT. The neutral invertase in situ assay medium contained: 50 mM HEPES-KOH, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM NAD, 5 mM ATP, 100 mM sucrose, 0.1 % BSA, 0.03 % NBT, and 1 unit each of glucose-6-phosphate dehydrogenase, hexokinase, and phosphoglucoisomerase, 100 mM sucrose, 1 mM NAD, 5 mM ATP, and 0.03 % NBT. Assays were conducted at room temperature for 1 h for ES internodes and overnight for PES internodes. Negative controls were conducted without sucrose in the assay medium.
Histochemical GUS analysis
GUS activity was detected as described by Udhe-Stone et al. . Micrographs of stained stem sections were taken using a Nikon SMZ800 stereoscope attached to a Nikon DXM1200 digital camera.
Cell wall analysis
Ten stems (45 cm measured from the apex) were harvested at flowering and combined for each replicate. Stem cell wall sugar composition was determined using the Uppsala dietary fiber method  as previously described . Statistical analyses were performed using the analysis of variance function of R .
Availability of supporting data
The data sets supporting the results of this article are included within the article and its additional files.