Figure 10.

In vivo phosphorylation of LIP5 under abiotic stresses. A, In vivo phosphorylation of LIP5 under heat stress. Transgenic lip5-1/35S::LIP5^WT and lip5-1/35S::LIP5^6A plants with similar levels of myc-LIP5 transcripts were treated in a 45°C chamber, and leaf samples were collected at the indicated time points. The levels of myc-LIP5 transcripts were analyzed by RNA blotting using a ³²P-labeled myc tag DNA fragment as probe (top). Ethidium bromide staining of ribosomal RNA (rRNA) is shown for the assessment of equal loading. Total proteins were also isolated and separated on a regular SDS-PAGE (middle) gel and a Phos-tag SDS-PAGE (bottom) gel. The myc-LIP5^WT and myc-LIP5^6A proteins were detected with an anti-myc monoclonal antibody. Rubisco staining of the regular SDS-PAGE gel was used to assess equal protein loading. B, In vivo phosphorylation of LIP5 in salt stress. Seedlings of transgenic lip5-1/myc-LIP5^WT and lip5-1/myc-LIP5^6A lines containing similar levels of myc-LIP5 transcripts were treated with 150 mm NaCl. Samples were collected at the indicated hours for total RNA and protein isolation. RNA blotting, regular immunoblotting, and Phos-tag immunoblotting were performed as in A.