Supplementary Material
Supplementary Material and Methods
Plant Growth Conditions during Growth Cessation Experiments
Rooted cuttings of WT and aPttPHYA hybrid aspen lines were grown at constant temperature (18°C) and relative humidity (80%). During the experiment described in Supplemental Table 1, the main long day photoperiod was 10 h with irradiance about 200 mmol m-2 s-1 (Osram Powerstar HQI-T 400 W/D lamps, Osram, Germany). Day length extensions of 8 h were given using low-intensity light (20 mmol m-2 s-1) (Osram) before or after the main light photoperiod. In this experiment, between three and five plants of each of the seven transgenic lines and WT were examined for elongation every 3rd to 5th day starting when established. The light quality in the SD condition in Supplemental Table 2 (experiment 1) was the same as in the LD except of the five hours of extension. In this experiment, aPttPHYA lines -1, -2, -9, and WT (7 to 11 plants per line) were tested over a period of 76 days under LD and concluded with CDL conditions.
RNA Isolation and Gel Blot Analysis
Total RNA from the seven independent hybrid aspen aPttPHYA lines (-1, -2, -4, -5, -7, -8, and -9) transformed with the PttPHYA anti-sense construct and WT was extracted, loaded (either 48 mg or 38 mg, see legend of Supplemental Figure 2) onto formaldehyde agarose gels, blotted, and hybridized essentially as described by Eriksson et al. (2000).
As a template for ribo probes, a PCR fragment from the PttPHYA (position 2630 to 3047 bp), cloned between the T7 and T3 polymerase promoters of pT7/T3a-19, was used. Probes were synthesized using a Strip EZÔ RNA T3 kit (Ambion Inc, Austin, TX, USA), and transcription from the T7 promoter was carried out by substituting the T3 polymerase mix with T7 RNA polymerase and the RNAse inhibitor RNAGuard (Amersham Pharmacia Biotech, Uppsala, Sweden) at recommended concentrations. Between hybridizations, the Strip EZÔ RNA T3 kit (Ambion) was used to remove the probe.
Northern Blot
Plant Material and Sampling
To characterize the endogenous PttPHYA RNA expression, 11-week-old wild type plants were first grown under LD conditions (18 h light/6 h dark). Two plants were sampled and the remaining two were transferred to SD conditions (10 h light/14 h dark) and harvested after four days. All samples were harvested in the middle of the main photoperiod. Samples were collected from plants 1.7 – 1.9 m tall, from the apices and internodes 9, 27, and 68 (corresponding to early expanding, late expanding, and mature non-expanding tissues, respectively) together with adjacent leaves and petioles as well as actively growing roots. Tissues from two plants were harvested and pooled for each sample.
Poly A+ Extraction and Analysis
Total RNA extraction (as described above) was followed by poly (A)+ purification using oligo (dT)- cellulose type 7 (Amersham Pharmacia) according to the manufacturer’s recommendations, except that LiCl was substituted for NaCl. Eight mg of poly A+ RNA from each sample was loaded and blotted as described above. The blots were probed with purified alpha -[32P] dCTP (6000Ci/mmol; Amersham) labeled DNA fragments, which were hybridized, washed, and exposed to Phosphoimager screens as previously described (Eriksson et al., 2000). Northern blots were probed with a 1171 bp Eco RI/ Xba I PttPHYA fragment from its 3’-end, stripped with boiling hot 0.1 x SSPE in 0.1% (w/v) SDS, and re-probed with the ubiquitin-like ESTs, A046p07u (PttUBQ1), or A081p57u (PttUBQ2) (Sterky et al., 1998).
Supplementary References
Eriksson ME, Israelsson M, Olsson O and Moritz T (2000) Increased gibberellin biosynthesis in transgenic trees promotes growth, biomass production and xylem fiber length. Nat Biotechnol 18:784-788
Sterky, F., Regan, S., Karlsson, J., et al. (1998) Gene discovery in the wood-forming tissues of poplar: Analysis of 5,692 expressed sequenced tags. Proc. Natl. Acad. Sci. USA 95:13330-13335.
Supplementary Figure Legends
Supplemental Figure 1 aPttPHYA-7 line and WT shoots grown in tissue culture (A), Northern blot analysis of leaf tissues showing detected levels of PttPHYA transcript in WT and transformed lines (B).
(A) A bud set phenotype is seen in the former, with no internode elongation and an apical bud formed at the apex (arrow), while the WT is still growing showing leaf primordia at the apex and elongation (arrows).
(B) Percent of WT expression estimated in the plants is shown. Total RNA was loaded; 48 µg from all samples except line 7 and wild type plants indicated by*, for which 38 µg was loaded. The ethidium bromide staining shows ribosomal RNA intensity as a loading control.
Supplemental Figure 2 Northern blot analyses showing PttPHYA transcript levels in different WT tissues and different photoperiods. Eight µg of poly A+ RNA were loaded for each sample. (A) Expression under LD, left to right, in young roots (YR), mature non-expanding internodes (MI), late expanding internodes (LI), early expanding internodes (EI), and apices (A). (B) Expression under LD and SD in young roots (YR), late expanding internodes (LI), and apeices (A) is compared. For comparison, expression levels of the endogenous ubiquitin genes PttUBQ1 or PttUBQ2 are also shown.
Supplemental Figure 3 Growth of representative WT, aPttPHYA-1, and oatPHYAox lines grown 63 days under a 6 h light/6 h dark cycle.
Supplemental Figure 4 Expression levels of PttFKF1 in WT and aPttPHYA-1 line under continuous light. The subjective night length used for entrainment is shown in grey. The results are presented relative to the lowest value after normalization to 18S rRNA. Mean ± SE denotes technical replication of pooled leaf material from three plants.
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