PMC

GH3.3, GH3.5, and GH3.6 Can Conjugate to Amino Acids in Vitro.
Thin layer chromatography analysis of -amino acid conjugates synthesized by recombinant GST:GH3.3, GST:GH3.5, and GST:GH3.6 proteins. Substrates added to each enzyme reaction mixture are indicated below the image. Concentrations for and were 10 and 1 mM, respectively. conjugate standards, shown to the right, migrated with somewhat greater mobility because they were loaded on the plate in organic solvent rather than the enzyme reaction mixture. Free and migrated to near the top of the plate.

Adventitious Root Initiation: A Proposed Model for a Regulatory Pathway.
ARF6, ARF8, and ARF17 and their regulatory microRNAs interact in a complex network () and act upstream of GH3.3, GH3.5, and GH3.6. ARF6 and ARF8 are positive regulators of the three GH3s, whereas ARF17 is a negative regulator. GH3.3, GH3.5, and GH3.6 control each other’s expression through a pathway yet to be identified. The three GH3 genes control homeostasis. The -Ile level in the hypocotyl is determined by the level. The level of -Ile negatively regulates adventitious rooting through the activation of the COI1 signaling pathway. In conclusion, auxin controls adventitious root initiation through the activation of ARF6 and ARF8 proteins, leading to a downregulation of the inhibiting COI1 signaling pathway.

GH3.3, GH3.5, and GH3.6 Regulate Adventitious Root Initiation by Modulating Homeostasis.
(A) to (C) Endogenous free (A), free (B), and free (C) contents were measured in the hypocotyl of wild type ecotype Columbia (Col-0) and the gh3.3-1gh3.5-2gh3.6-1 triple mutant. Seedlings were first etiolated in the dark, until their hypocotyls were 6 mm long (T0), and then transferred to the light for 72 h (T72L). Error bars indicate sd of eight biological replicates. A one-way analysis of variance combined with the Bonferroni posttest indicated that the values indicated by asterisks are significantly different from wild-type Col-0 values and the values indicated by hash signs are significantly different from values observed at T0 (P < 0.05; n = 8). FW, fresh weight.
(D) and (E) Endogenous free (D) and -Ile (E) were measured in the hypocotyl of wild type Col-0 and the gh3.3-1gh3.5-2gh3.6-1 triple mutant, etiolated as in (A) (T0) or transferred to the light for 3 h (T3L) or 9 h (T9L). Error bars indicate sd of nine biological replicates. A one-way analysis of variance combined with the Bonferroni posttest indicated that the values indicated by asterisks are significantly different from wild-type Col-0 values (P < 0.05; n = 9).

Variation in the Number of Adventitious Roots in the Hypocotyl of arf Mutants and ARF-Overexpressing Lines Correlates with the Expression Levels of GH3.3, GH3.5, and GH3.6.
(A) Adventitious roots (AR) were counted on seedlings of arf mutant lines and ARF-overexpressing lines. Seedlings were first etiolated in the dark, until their hypocotyls were 6 mm long, and then transferred to the light for 7 d. Data from three independent biological replicates, each of at least 30 seedlings, were pooled and averaged. Error bars indicate se. A one-way analysis of variance combined with the Tukey’s multiple comparison posttest indicated that the values marked with asterisks were significantly different from wild-type values (P < 0.01; n > 90). In addition, those marked with hash signs were significantly different from values obtained from single mutants or the ARF17-OX1 line (P < 0.01; n > 90). Col-0, ecotype Columbia; Ws, ecotype Wassilewskija.
(B) The GH3.3, GH3.5, and GH3.6 transcript abundance was quantified in the hypocotyls of the different arf mutants or overexpressing lines, which had been etiolated and then transferred to the light for 72 h. Gene expression values are relative to expression in the wild type, for which the value is set to 1. Error bars indicate se obtained from three independent biological replicates. A one-way combined with the Dunnett’s comparison posttest indicated that the values marked with ns were not significantly different from wild-type values, whereas the others were significantly different (P < 0.05; n = 3).

GH3.3, GH3.5, and GH3.6 Genes Have Redundant Functions in the Regulation of Adventitious Rooting.
(A) Average number of adventitious roots (AR) in two independent knockout alleles for gh3.3, gh3.5, and gh3.6 single mutants, in the double mutants gh3.3-1gh3.5-2 and gh3.3-1gh3.6-1, and the triple mutant gh3.3-1gh3.5-2gh3.6-1. Seedlings were first etiolated in the dark, until their hypocotyls were 6 mm long, and then transferred to the light for 7 d. Data from three independent biological replicates, each of at least 30 seedlings, were pooled and averaged. Error bars indicate se. A one-way combined with the Tukey’s multiple comparison posttest indicated that the differences observed in the mutants versus the wild type (*), in gh3.6 versus gh3.3 or gh3.5 (#), and in the triple mutant versus the double mutants (#) are significant (P < 0.01; n > 90). Col-0, ecotype Columbia.
(B) The steady state expression levels of GH3.3, GH3.5, and GH3.6 were quantified by quantitative RT-PCR in the hypocotyls of the different gh3 single and double mutants first etiolated in the dark, until their hypocotyls were 6 mm long, and then transferred to the light for 72 h.
(C) The relative amount of the total GH3.3+GH3.5+GH3.6 mRNAs in the hypocotyl of single and double gh3 mutants grown as in (B) was quantified by quantitative RT-PCR.
For (B) and (C) Gene expression values are relative to the expression in the wild type, for which the value is set to 1. Error bars indicate se obtained from three independent biological replicates. A one-way combined with the Dunnett’s comparison posttest confirmed that the differences between the wild type and the mutants (*) are significant (P < 0.001, n = 3).

The COI1 Signaling Pathway Is Required for the Inhibition of Adventitious Rooting by Jasmonate.
(A) to (E) Quantification by quantitative RT-PCR of JAR1 (GH3.11) JAZ1, JAZ3, JAZ5, and COI1 transcripts in hypocotyls of wild-type etiolated seedlings (T0), after an additional 3 or 9 h in the dark (T3D or T9D), and after transfer to the light for 3 or 9 h (T3L or T9L). Values are relative to the expression level of APT1, which was used as a reference gene as described in Methods. Error bars indicate se obtained from three independent biological replicates. A one-way analysis of variance combined with the Tukey’s multiple comparison posttest confirmed that the differences between the wild type and the mutants (*) are significant (P < 0.05, n = 3). Col-0, ecotype Columbia.
(F) and (G) Average number of adventitious roots (AR) in several mutants altered in jasmonate biosynthesis or signaling (F) and in the -deficient mutant lines eds5-1 and eds5-2 (G). Data from three independent biological replicates, each of at least 30 seedlings, were pooled and averaged. Error bars indicate se. A one-way analysis of variance combined with the Tukey’s multiple comparison test showed that the values indicated by asterisks are significantly different from wild-type values (P < 0.01; n > 90); the Bonferroni posttest indicates that the value indicated by the plus sign is significantly different from that of the myc2 mutant and that indicated by the hash mark is significantly different from that of jar1-12 (P < 0.01; n > 90).
(H) Average number of adventitious roots in single arf6-3 and arf8-7 mutants and the ARF17-OX line and in the corresponding double mutants with coi1-16. Data from three independent biological replicates, each of at least 30 seedlings, were pooled and averaged. Error bars indicate se. A one-way analysis of variance combined with the Dunnett’s multiple comparison test showed that the values indicated by asterisks are significantly different from wild-type values (P < 0.05; n > 90).