Figure 1.
T-DNA constructs created to combine promoterless GUS reporter vectors with different configurations and promoters to express selectable markers.
(A) Group of vectors constructed in head to tail orientation with selectable marker upstream of the GUS reporter. The selectable marker cassette is comprised of hygromycin phosphotransferase gene (hptII, 1026 bp, in gray box) driven by CaMV 35S (848 bp, a1), Nos (307 bp, a2) or tCUP1 (519 bp, a3) promoters and terminated by the same CaMV 35S terminator (225 bp). The β-glucuronidase reporter gene (GUS, 2053 bp, in blue box) with nopaline synthase (nos) terminator (268 bp) at the 3′ end near the T-DNA right border (RB). (B) Group of vectors constructed in head to head orientation with reversed selectable marker upstream of the GUS reporter. The selectable marker gene is differentially expressed by CaMV 35S (b1), Nos (b2) or tCUP1 (b3) promoters placed near the start codon of GUS reporter. (C) Group of vectors constructed in head to head orientation similar to B series vectors except that a 254 bp sequence (triangle) derived from pCAMBIA1300 was inserted to separate the GUS reporter and its adjacent inverted promoter used for hptII gene expression. (D) Group of vectors constructed in head to tail orientation with GUS reporter upstream of the selectable marker. The GUS reporter gene is stacked with ATG near the RB and nos terminator close to CaMV 35S (d1), Nos (d2) or tCUP1 (d3) promoters used for hptII gene expression.
Figure 2.
GUS leaky expression in calli transformed by promoterless GUS reporter vectors containing different selectable marker promoters in different stacking configurations.
GUS leaky expression in representative callus transformed by promoterless GUS reporter vectors in (A–D) configurations combined with 35S (a1,b1,c1,d1), Nos (a2,b2,c2,d2) or tCUP1 (a3,b3,c3,d3) selectable marker promoters as indicated in Figure 1. Fifty Transformed calli per vector were stained after 3 weeks selection and calli with the average numbers of staining spots were pictured individually at the same magnification. Pictures show representative calli for at least three replicates. Bars: 1 mm.
Figure 3.
Comparison of GUS leaky expression in calli among vectors with different selectable marker promoters in different stacking configurations.
(A) Distribution histograms showing the numbers of spots of GUS leaky expression in calli transformed by group A (a1–a3), C (c1–c3) and D (d1–d3) vectors. Numbers of GUS spots per callus was set in the range from 0 to 80 and divided into 9 intervals. A total of 50 calli were counted for each vector. (B) Average number of GUS spots per callus derived from each vector. The bar represents mean±SE of spot numbers from 50 calli counted in (A). Different letters above the column indicate a statistically significant difference (p<0.05) between vectors with the same selectable marker promoter. (C) Relative GUS leaky percentage between group A (a1–a3) and group C (c1–c3) vectors after normalization by average spot numbers per callus of the respective group D (d1–d3) vectors. Different letters above the column indicate a statistically significant difference (p<0.05) between vectors with the same configuration. Statistical analysis was performed with GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA) by using one-way ANOVA with a Tukey’s multiple comparisons test.
Figure 4.
Construction of DR5::GUS reporter vectors on the basis of A and C configurations.
(A) The synthetic auxin responsive promoter DR5 (267 bp, yellow arrow) was inserted in group A vectors before the GUS reporter to make a1-DR5, a2-DR5 and a3-DR5. (B) A similar strategy was used to construct the DR5 promoter in group C vectors and named c1-DR5, c2-DR5 and c3-DR5 respectively.
Figure 5.
Specific and non-specific expression of DR5::GUS in adventitious roots of T0 transgenic plants.
(A, B) Non-specific expression of DR5::GUS in root tip and stele in both weak (A) and strong (B) staining lines. (C) Staining pattern with DR5::GUS expressed in root cap and zone of elongation. (D, E) Specific localization of DR5::GUS in columella cells only (D) or in columella cells and quiescent center (E). (F, G) Specific expression of DR5::GUS in root cap, quiescent center and protoxylem cells in the root meristem. Roots positioned in a protophloem plane, xylem cells expressing GUS are arranged in a uni-cellular plate (F) or roots positioned in a protoxylem plane, two arrays of protoxylem cells expressing GUS (G) are shown. Bars: A–C, 200 µm, D–G, 100 µm. Adventitious roots of 1-month-old T0 plants were collected and stained in 1 mM X-gluc for 1 h (A–E) or 0.5 mM X-gluc for 4 h (F, G). Positions of columella cell (green arrow), quiescent center (red arrow) and xylem cell (yellow arrow) are indicated. Detailed organization of rice root tissue and cell types can be found in references [56], [57].
Figure 6.
Summary of DR5::GUS staining patterns in primary roots of T1 transgenic plants.
(A) Varied expression patterns of DR5::GUS (a-i) in primary root tips of 8-day-old T1 transgenic seedlings grown in IRRI rice solution. The most intense GUS expression was mainly observed in columella (a), in columella and zone of elongation (b), in root cap (c), in root cap and basal meristematic zone (d), in root cap and protoxylem (e), in root cap, protoxylem and meristematic zone (f), in basal meristematic and elongation zones, absent in root cap and promeristem (g), evenly stained in root cap and meristematic zone (h), in root cap, stele and meristematic zone (i). Samples were stained for 30 min for (c)-(i) or 16 h for (a) and (b). Bar: 100 µm. (B) Images (a-i) shown on the right are magnifications of the corresponding images shown on the left (A) to provide detailed staining patterns in the apical root. Positions of columella cell (green arrow), quiescent center (red arrow) and xylem cell (yellow arrow) are indicated. Bar: 100 µm. (C) Three major staining patterns in lateral roots (a-c) from all the analyzed T1 transgenic seedlings transformed by six DR5::GUS vectors. The most intense GUS expression was clearly observed in lateral root cap (a), in lateral root cap and xylem of both primary and lateral roots (b), in the whole vascular bundle and less specifically in the lateral root cap (c). Bars: 100 µm. (D) Patterns within T1 transgenic seedlings of each vector were classified and presented as a heat map of percentage. Number of GUS positive T1 seedlings observed for each vector is indicated in brackets.
Figure 7.
Auxin responsiveness in DR5::GUS transgenic lines selected by vectors with hptII controlled by tCUP1 promoter.
Both a3-DR5 (A–D) and c3-DR5 (E–H) transgenic lines using the tCUP1 promoter for hptII gene expression were selected for the auxin responsive test. Representative images are shown for two independent lines each showing the DR5 promoter strongly induced in primary root tips (B, F) and lateral roots (D, H) after 1 µM NAA treatment for 24 h. In the mock treatment, specific but different DR5::GUS expression was observed between a3-DR5 and c3-DR5 lines in primary root tips (A, E) as well as in lateral roots (C, G). Bars: 100 µm.