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. 2016 Sep 28;28(10):2666–2682. doi: 10.1105/tpc.16.00612

Figure 4.

Figure 4.

AAD Genes Induced by MYB115 and MYB118.

(A) to (C) RT-qPCR analysis of transcript abundance in cDNA prepared from myb115-1, myb118-1, and myb115-1 myb118-1 mutant seeds harvested 10 DAA (A), from rosette leaves of Pro35Sdual:MYB118 lines (OE1 and OE2) (B), or from rosette leaves of Pro35Sdual:MYB115 lines (OE11 and OE19) (C). Values are the means and se of three to nine replicates performed on cDNA dilutions obtained from three ([B] and [C]) or four (A) independent mRNA extractions. Asterisks indicate significant difference from the wild type according to t test at ***P < 0.001, **P < 0.01, and *P < 0.05, respectively.

(D) Transactivation assay in leaves of N. benthamiana. Pro:uidA reporter constructs alone or in combination with a vector allowing the expression of MYB115, MYB118, or MYB107 (negative control) were coinfiltrated in young leaves of N. benthamiana with a vector allowing the expression of the p19 protein. Leaf discs were assayed for GUS activity 3 d after infiltration. Tissues were incubated 4 h in a buffer containing 2 mM each of potassium ferrocyanide and potassium ferricyanide. Representative discs (diameter = 0.8 cm) are presented.

(E) and (F) Pattern of activity of the ProAAD2:uidA (E) and ProAAD3:uidA (F) cassettes in developing seeds harvested 8, 10, or 12 DAA (main pictures, from left to right) and in early maturing embryos harvested 8, 10, or 12 DAA (secondary small pictures, from left to right). For histochemical detection of GUS activity, tissues were incubated overnight in a buffer containing 2 mM each of potassium ferrocyanide and potassium ferricyanide. Microscopy observations of seeds and excised embryos were performed using Nomarski optics. Bars = 100 µm.

(G) Analysis of relative mRNA accumulation of AAD2 and AAD3 was performed in developmental series of endosperm and embryo fractions. The results obtained are standardized to the EF1αA4 (EF) gene expression level. Values are the means and se of three to six replicates performed on cDNA dilutions obtained from three independent mRNA extractions. Endo., endosperm fraction.