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. 2018 Mar 7;30(3):620–637. doi: 10.1105/tpc.17.00840

Figure 1.

Figure 1.

Identification and Expression Analysis of the ARF8 Splice Variant ARF8.4.

(A) Schematic diagram of the splicing variants At5g37020.1, At5g37020.2, and At5g37020.4 encoding ARF8.1, ARF8.2, and ARF8.4, respectively. Rectangles (white, translated; gray, untranslated) represent exons, and black lines represent introns. Asterisks indicate the premature stop codon and the sequence at the splicing site in ARF8.2 and ARF8.4, and the black box indicates intron 8 retention in ARF8.4.

(B) Contribution of intron retention and premature stop codon to ARF8 protein domains. Thirty-eight amino acids in the terminal region of the PB1 domain are deleted in ARF8.2 and ARF8.4, and 28 amino acids are inserted in the ARF8.4 MR.

(C) Schematic diagram of the regions of interest (from exon 8 to 3′ untranslated region) of ARF8.1, ARF8.2, and ARF8.4 transcripts. Primers used for RT-PCR 1, RT-PCR 2, and qRT-PCR are shown. F, forward; R, reverse.

(D) RT-PCR analysis of ARF8.2, ARF8.1, and ARF8.4. Twenty-eight to 35 cycles were used to detect the abundance of each splice variant in wild-type flower buds at stages 9 and 10 using the following primers: 1F and 2R (amplicon size 1540 bp), 1F and 3R (amplicon size 1544 bp), 4F and 3R (amplicon size 1607 bp) for ARF8.1, ARF8.2, and ARF8.4, respectively (upper panel RT-PCR 1). Thirty-five and 40 cycles were used to detect the abundance of ARF8.4 (amplicon size 345 bp) compared with ARF8.1+ARF8.2 (amplicon size 261 bp) in wild-type stamens at stages 9 and 10, using 11F and 11R primers (lower panel RT-PCR 2). M, DNA markers.

(E) Quantitative analysis by qRT-PCR of ARF8.4 transcript in various organs, using 4F and 5R primers, showing negligible expression in organs other than inflorescences (value set to 1). ARF8.4 cDNA levels are relative to actin cDNA. Values are means ± se of nine data points obtained from three biological replicates that were each analyzed in triplicate. Biological replicates were obtained by pooling organs isolated from five independently grown plants or 7-d-old seedlings.

(F) Comparative analysis by qRT-PCR of ARF8.4 and ARF8.1+ARF8.2 transcript levels in wild-type stamens at different developmental stages, using 4F and 5R, and 1F and 5R primers, respectively. ARF8.1+ARF8.2 or ARF8.4 cDNA levels are relative to actin cDNA. Values are means ± se of nine data points obtained from three biological replicates that were each analyzed in triplicate. Biological replicates were obtained by pooling stamens from flowers at different developmental stages isolated from five independently grown plants.