However, excessive autophagic responses could become hazardous and harmful

d transplanted as needed. Plants were grown for anywhere from one to six months depending on the experiment. We used a ‘leaf plastochron index’ system to assure that stems were at the same developmental stage for any given test or manipulation. We defined ‘the apex’ as Auxin Transport during Woody Stem Development the tight cluster of leaves above the first internode that could be clearly identified with the unaided eye. The leaf that subtended this internode was approximately 1.5 cm long with the basal one-third of the leaf margin still curled. Under our growing conditions, saplings 518303-20-3 maintained between 100 to 125 leaves beneath the apex before they began to abscise and had an outer stem diameter of about 1.5 cm at a position 100 nodes beneath the apex. Auxin response in PtaDR5 lines All 14 PtaDR5 lines were tested for an auxin response by incubating plant tissue in half-strength MS liquid growth media containing 30 mM IAA at 22uC for 4212 hrs following brief vacuum infiltration. Whole in vitro grown plantlets and stem and root segments from both in vitro and greenhouse grown plants were tested and the auxin response was compared against matched controls incubated in the same media without IAA. In order to test for an auxin response to endogenous IAA, lanolin containing 50 mM NPA was applied in a 0.5-cm-wide ring around the epidermis of stems 0.4 to 1 cm in diameter, covered with foil for 2 weeks, and harvested above and below the application site. Control plants were treated with DMSO in lanolin. GUS assays were performed 18339876 on fresh and fixed tissue following Jefferson et al with a 428 hr incubation at 37 uC in X-Gluc solution containing 2 mM potassium ferrocyanide and 2 mM potassium ferricyanide. For all tissues examined, ice-cold-acetone-fixed and LN2-plunge-frozen tissue was tested to check for wounding artifacts. Acetone fixation greatly reduced but did not eliminate the signal; LN2 freezing did not reduce the signal relative to fresh tissue but did significantly disrupt soft tissues. Unless otherwise stated, images are from fresh tissue in which localization of GUS was verified with matched LN2-frozen tissue. Tissue was cleared in 70% EtOH to remove chlorophyll. Endogenous GUS expression was characterized in three PtaDR5 lines in more detail during active growth and the onset of dormancy. Expression of GUS was chosen over GFP as a reporter for all experiments because stem tissues generally needed to be sectioned, fixed and cleared, whereas viewing GFP requires live whole mounts. The GFP signal was also weak relative to the background autofluorescence typical of secondary vascular tissue. Dormancy was induced over 12 weeks following transfer to soil. Plants were grown under 8 hr days at 15 uC for four weeks, then 10 uC for eight weeks, at which time plants had set bud and dropped all of their leaves. Dormant plants were compared against actively growing plants with a comparable number of internodes. In both cases fresh tissue was incubated in X-Gluc as described above, fixed in 4% paraformaldehyde for 24 hrs and cleared in 70% EtOH. Tissue browning 23551948 due to phenolic oxidation was particularly problematic in dormant apices and required additional clearing in 0.5% sodium hypochlorite, which proved more effective than traditional clearing methods using chloral hydrate. Soft tissues were embedded in 5% agarose blocks and sectioned at 50 to 100 mm on a vibratome. Woody stem segments were sectioned at 24 mm on a sliding microtome. assays base