Fig 1.
(A) Amino acid sequence alignment of plum DELLAs PslGAI (KU845589), PslRGLa/b (KU845592/KU845593), and PslRGAa/b (KU845590/KU845591) using ClustalX program. Conserved residues are shaded in black. Dark- and clear-grey shadings indicate similar residues in four and three out of five of the sequences, respectively. Conserved motifs are shown above the alignment columns. A putative nuclear localization signal (NLS) is indicated by black triangles. Conserved LXXLL motif is indicated by black circles. Asterix and open circle within DELLA/TVHYNP motifs highlight the substituted amino acid residues in PslRGAa/b that are essential for interaction with the GID1-like proteins and complex stabilization, respectively. The arrow indicates the site of the three amino acid residues insertion (Ser-Gly-Gly) in PslRGLb (B) Schematic representation of PslGAI, PslRGL and PslRGA proteins domain organization. The triangles in PslRGA are to highlight the location of distinct DELLA motifs. The GA-responsive DELLA domain [DELLA (D) and TVHYNP (T) motifs], the poly STV (S/T/V) motif, and the functional GRAS domain [LHR1, VHIID, LHR2, PFYRE and SAW motifs] are indicated. Number of base pairs (bp) and amino acids (aa) refer to full-length nucleotides and amino acid residues of the predicted sequences. (C) Phylogenetic relationships between PslDELLAs and Arabidopsis orthologous (AtGAI, AtRGA, AtRGL1, AtRGL2 and AtRGL3). The tree was constructed using MEGA5 software. The scale bar represents a number of amino acid substitutions per site, in which 1 cm is equal to 0.1 amino acid substitutions per site.
Fig 2.
Interaction capacity between PslDELLA (PslGAI, PslRGL and PslRGA) and GA-receptors (PslGID1b and PslGID1c) using Y2H and BiFC approaches.
Y2H assays (A) were performed using PslDELLA as prey in Y187 yeast strain and PslGID1 as bait in Y2HGold yeast strain. The mated yeast was grown in 96-well plates containing DDO/X/A medium in the presence or absence of 100 μM GA3. For in planta BiFC assay (B), PslDELLA sequences were fused with the C-terminus (CY) of YFP; PslGID1 were fused with the N-terminus (NY) of YFP. Different combinations of NY and CY constructs were transiently co-expressed in GA-treated tobacco protoplasts. NLS-mCherry was included in each transfection to highlight the location of the nucleus. YFP fluorescence is yellow; the merged image is a digital merge of bright field and fluorescent images to illustrate the interaction location; bars = 10 μm. All experiments were repeated at least three times.
Fig 3.
Stabilization of GA–PslGID1–PslDELLA complexes is dependent on the type of bioactive GA, mediating the reaction.
Y2H interaction experiments of PslGAI, PslRGL and PslRGA with PslGID1b and PslGID1c on selective medium containing 100 μM GA1, GA3 and GA4. Selective medium without bioactive GA was used as control. Other details as in Fig 2.
Fig 4.
Substitutions in the DELLA domain of PslRGA protein prevent the GA-dependent interaction between PslRGA and PslGID1-like proteins.
(A) Alignment of amino acid sequences of the GA-sensitive PslRGL, GA-insensitive PslRGA and their mutated versions PslRGL.MU and PslRGA.MU, highlighting the changes in DELLA and TVHYNP motifs generated by a site-directed mutagenesis approach. (B) Subcellular localization of full-length ORFs of PslRGL, PslRGA and their mutated derivatives fused to the GFP tag. All constructs were transiently transformed for the assay into N. tabacum protoplasts. NLS-mCherry was included in each transfection to indicate the location of the nucleus. GFP fluorescence is shown as green; the merged image is a digital merge of bright field and fluorescent images to illustrate the protein compartments. Bars = 10 μm. Interaction properties of PslRGL.MU and PslRGA.MU proteins with PslGID1s using Y2H (C) and BiFC (D) approaches. Corresponding native proteins were included as controls. All experiments were repeated a minimum of three independent times. Other details are as in Fig 2.
Fig 5.
Steady-state transcript levels of PslGAI, PslRGL and PslRGA mRNAs assessed by qPCR during EG plum fruit development, including flowers (FL), fruit set (FI), and the 4 different stages of fruit development (S1-S4).
Results represent data from three biological and three technical replicates. Standard curves were used to calculate the number of target gene molecules per sample. These were then normalized relative to PslAct expression. Error bars represent SD. The y-axis refers to the mean molecules of the target gene per reaction/mean molecules of PslAct. The x-axis in each figure represents the developmental stage as indicated by the number of days after bloom (DAB). The expression of the three genes during fruit ripening was over-exposed to visualize the changes in transcription levels.
Fig 6.
(A) PslDELLA transgene levels and (B) the accumulation of the GA-metabolism mRNAs in WT and the different transgenic events overexpressing PslGAI (L.1), PslRGL (L.9) and PslRGA (L.6) genes. Transcripts accumulation was determined using qPCR on three biological and three technical replicates. Standard curves were used to calculate the numbers of target gene molecules per sample, which were then normalized relative to AtAct expression. ND means non-detectable. (C) Aerial portions of 45-day-old WT and the different transgenic mutant plants grow under standard conditions; bars = 10 cm.
Fig 7.
(A) Representative 15-day-old seedlings primary roots of WT, PslGAI, PslRGL and PslRGA genotypes. Plants were grown on MS medium in the presence or absence of GA3 (50 μM); bar = 10 mm. (B) Differential response of WT, PslGAI, PslRGL and PslRGA root growth to GA treatment. Root length measurements are the means (±SD) of 24 seedlings.
Fig 8.
Representative 50-day-old aerial portions of WT (A), PslGAI (B), PslRGL (C), PslRGA (D) and PslRGA.MU (E) plants grow under standard conditions with or without GA3 (100 μM) treatment; bars = 10 cm. (F) Differential response of WT, PslGAI−, PslRGL−, PslGAI− and PslRGA.MU−plant growth to GA treatment. Plant height measurements are the means (±SD) of 24 plants.
Table 1.
Vegetative and reproductive growth characteristics of WT Arabidopsis plants expressing plum PslGAI, PslRGL, PslRGA, PslRGA.MU genes in the presence or absence of GA as shown in Figs 6 and 8.
Fig 9.
Close-up views of WT, PslGAI−, PslRGL−, PslRGA−, and PslRGA.MU−flowers (A) and siliques (B) from plants grow under standard conditions with or without GA3 (100 μM) treatment.
Sepals and petals were removed to reveal the anthers and pistil; bars = 10 mm.
Fig 10.
PslRGA is not subjected to GA-induced degradation.
GFP fluorescence of primary 5-day-old Arabidopsis seedling roots, expressing (A) PslGAI−GFP, (B) PslRGL−GFP, (C) PslRGA−GFP, and (D) PslRGA.MU−GFP. Fluorescence were monitored with confocal laser scanning microscopy after treatment with water (mock) or GA3 (100 μM) for 60, 120 and 240 minutes.
Fig 11.
Modifications in PslRGL and PslRGA DELLA domain alter their GA-dependent responses.
Confocal microscopic images of GFP fluorescence in tobacco BY-2 cells transiently expressed PslRGL−GFP (A), PslRGA−GFP (B), PslRGL.MU−GFP (C) and PslRGA.MU−GFP (D) chimeric proteins. BY-2 cells were treated with GA3 (100 μM), paclobutrazol (10 μM) and a joint treatment of PAC and GA. Non-treated cells were used as control. NLS-mCherry was included in each transfection to indicate the location of the nucleus. GFP fluorescence is shown as green; the merged image is a digital merge of bright field and fluorescent images to illustrate the protein compartments. All experiments were repeated a minimum of three independent times; bars = 10 μm.