Appendix S1. Supplementary experimental procedures:
cDNA library construction
First-strand cDNA synthesis was preformed on approximately 1 µg total RNA in one 20
µL cDNA reaction using SuperScript II RNase H- Reverse Transcriptase (18064-022,
Invitrogen) in combination with the SMART IV oligo from the Creator SMART cDNA
Library Construction Kit (634903, Clontech), using the Clontech provided first strand
buffer, DTT and dNTP, and a modified version of the CDS III/3΄ primer
(5΄ctagaggccgaggcggccgacatgttttgtttttttttcttttttttttvn3΄). The reverse transcriptase reaction
was further supplemented with 4 units of Protector RNase Inhibitor (03335399001,
Roche) per 20 µL cDNA reaction mixture. Reverse transcriptase reaction was preformed
at 42 °C for 1 hour.
The cDNA was subsequently amplified by PCR using the Advantage 2 Polymerase
Mix (639201, Clontech), 5΄ PCR primer form the Creator SMART cDNA Library
Construction Kit (634903, Clontech) and the modified CDS III/3΄ primer. For each 100
µL PCR reaction 2 µL cDNA reaction was used as template. A total of 600 µL PCR
reaction were subsequently preformed for bulk amplification of cDNA.
Amplified cDNA was treated with 360 µg Proteinase K, incubated 20 minutes at 45
°C, phenol-chloroform extracted and ethanol precipitated. The cDNA was digested with
SfiI and size-fractionated on ChromaSpin-400 Columns (636076, Clontech). Fractions
containing DNA species smaller than 100 base pairs were discarded. The remaining
fractions were ethanol precipitated and dissolved in TE yielding a total of 15 µg DNA.
Quantitative dot blot analysis using carbohydrate specific antibodies
Aliquots of 3 mg de-starched AIR was extracted with 200 µL 1 M or 4 M KOH with 1%
(w/v) NaBH4 over night at room temperature. To 160 µL of supernatant 59 µL of 8.7 M
acetic acid was added to reduce the pH. Approximately 2 mg purified 1 M KOH stem
carbohydrate preparations were dissolved directly in 0.8 M KOH to a final concentration
of 10 mg/mL for subsequent serial dilution. Serial dilutions were performed in 0.8 M
KOH in 96 well microtiter plates. Spotting was preformed onto nitrocellulose (162-0115,
BioRad) using a Biomek 2000 robot (Beckman) and a 96 well pin spotting tool. The
carbohydrate dot blots were then blocked with 5% dry milk (Spartan Stores, MI) in
DPBS for 30 minutes and incubated over night at room temperature with primary
antibody (LM10, LM11 or LM15) diluted 1:500 in DPBS. The membranes were then
washed three times in DPBS and incubated for one hour with rabbit anti-rat antibody
conjugated to horseradish peroxidase (P0450, Dako) diluted 1:1000 in DPBS. The
membranes were then washed three times in DPBS and developed by chemiluminescence
using the substrate SuperSignal West Pico (34083, Pierce). Emitted light was recorded in
a VersaDoc 4000MP (BioRad) and individual spots were quantified with the associated
Quantity One-4.6.5 software.
Crystalline cellulose analysis
After trifluoroacetic acid treatment of de-starched AIR the remaining residue was washed
with Updegraff reagent to strip of further hemicelluloses and amorphous glucan
(Updegraff, 1969). This remaining residue was then hydrolyzed with sulfuric acid
(Seaman hydrolysis; Selvendran and O’Neill, 1987) and the resulting monosaccharides
released quantified using the anthrone assay.
Lignin analysis
Acetyl bromide soluble lignin (ABSL) was determined essentially as described by
Fukushima et al. (1991). To samples of 2 mg de-starched AIR were added 250 μl acetyl
bromide solution (25% acetyl bromide in glacial acetic acid) and incubated at 50 °C for 3
hours with frequent mixing. Samples were subsequently centrifuged 15 min at 8,000 g
and 100 μl of the sample supernatant was added to tubes containing 400 μl 2 M NaOH
and 75 μl 0.5 M hydroxylamine hydrochloride and mixed thoroughly. Glacial acetic acid
was subsequently added to a final volume of 2 mL and absorbance at 280 nm was
determined. An extinction coefficient of 23.35 g-ILcm-1 for Arabidopsis Col-0 ABSL was
used for absolute quantification (Chang et al., 2008).
qRT-PCR analysis performed on Arabidopsis stems
Single stems from 5 weeks old Arabidopsis plants were harvested in liquid nitrogen,
homogenized and split in two aliquots. One aliquot was used to prepare AIR for
carbohydrate dot blot analysis while the other aliquot was used for extraction of total
RNA by TRIzol Reagent (15596-026, Invitrogen). The purified total RNA was DNase
treated with TURBO DNA-free (AM1907, Ambion) and given a final cleanup using the
RNeasy Mini Kit (74104, Qiagen).
cDNA synthesis was done using SuperScript III Reverse Transcriptase (18080-044,
Invitrogen) and qRT-PCR assays were run using Power SYBR Green Master Mix
(4367659, Applied Biosystems) and the predesigned qPCR primes QuantiTect Primer
Assays (At_CESA8_1_SG; At_IRX3_1_SG; At_FRA8_1_SG; At_IRX9_1_SG;
At_GUT2_1_SG; At_GUT1_1_SG; At_IRX14_1_SG; At_4CL_1_SG;
At_At5g67210_1_SG; Qiagen) and run on the ABI Prism 7900HT Sequence Detection
System (Applied Biosystems).