Induction of UGT74E2 by H₂O₂ and Environmental Stresses.
(A) Rapid induction of the UGT74E2 transcript by high-light treatment in catalase-deficient (cat2) and wild-type background (Vanderauwera et al., 2005) as determined by quantitative PCR. Error bars are se (n = 3).
(B) Left side: Induction of UGT74E2 protein levels in 14-d-old wild-type seedlings exposed for 5 d to increased concentrations of exogenous H₂O₂ as determined by protein immunoblots. Right side: UGT74E2 transcript induction was quantified in pUGT74E2:LUC seedlings treated with 1 and 3 mM H₂O₂ for 5 d. Error bars are se (n = 3).
(C) Stress-related expression patterns of the Arabidopsis group L glycosyltransferases. Log2-transformed relative expression values of the group L glycosyltransferases in publicly available microarray data on biotic and abiotic stress treatments (Genevestigator) (Zimmermann et al., 2004). Magenta, green, and black indicate upregulation, downregulation, and no change versus control experiments, respectively.
(D) Histochemical GUS staining of pUGT74E2:GUS seedlings under control conditions (1 to 4 and 9 to 12) or exposed for 5 h to 50 μM MV (5 to 8) or 500 mM PEG (13 to 16). On 12-d-old seedlings (1 to 8), pUGT74E2:GUS expression is visible in the distal tip and base of leaf primordia (1 and 2), mesophyll of cotyledons (1), first formed lateral root primordia in the hypocotyl-root junction (4), and root tip (1 and 3). On 16-d-old seedlings (9 to 16), GUS staining is prominent in young leaf primordia and developing leaf tips and stipules (9 and 10), in hydathodes of developed leaves and on margins of petioles and bases (12), and in roots tips and lateral root primordia (11). MV or PEG stress increased pUGT74E2:GUS expression in these tissues (5 to 8 and 13 to 16). Bars = 2 mm in panels 1, 2, 5, 6, 9, and 13, 0.5 mm in panels 3, 7, 10, 12, 14, and 16, and 0.1 mm in panels 4, 8, 11, and 15.

 Identification of UGT74E2 as an IBA Glucosyltransferase.
(A) Relative conversion rates of different plant hormones to their glucosylated form by recombinant UGT74E2. The naturally occurring auxin IBA is the preferred substrate. Error bars are se (n = 2). JA, jasmonic acid.
(B) Separation by liquid chromatography of buffered samples containing UDP-Glc and IBA without (left) and with (right) addition of recombinant UGT74E2. Peak identity was determined by mass spectrometry. A significant proportion of IBA was glucosylated only in the presence of UGT74E2.
(C) Separation by liquid chromatography of buffered samples containing UDP-Glc and IAA (top) or IBA (bottom) in short-term incubation assays (10 min). No IAA-Glc could be detected, whereas a significant proportion of IBA was glucosylated.

 Modulation of Arabidopsis Architecture by Overexpression of UGT74E2.
(A) UGT74E2 mRNA levels in wild-type and two UGT74E2OE plants assessed by real-time RT-PCR. Error bars are se (n = 3).
(B) UGT74E2 protein levels in wild-type and UGT74E2OE plants assessed by protein gel blot with leaves from 1-month-old plants. For each lane, 200 μg of soluble protein was loaded.
(C) Rosette shapes of 3-, 4-, and 5-week-old (top to bottom) wild-type and UGT74E2OE plants growing under long-day (top, middle) or short-day (bottom) regimes, respectively. Note the intense green color of UGT74E2OE plants and the compressed rosette structure.
(D) Mature UGT74E2OE plants that were shorter and displayed a higher level of shoot branching than the wild type. Diagram illustrates the concept of inflorescence node order, in which 1 is the primary inflorescence and 2 to 4 are the subsequent orders of axillary nodes.
(E) Higher order of inflorescence branching of UGT74E2OE plants compared with wild-type plants (P < 0.001, n = 9 to 16).

 Increased Tolerance of UGT74E2OE Plants against Osmotic Stress.
(A) One-month-old wild-type and overexpressing transgenic seedlings grown on MS agar plates and transferred to plates supplemented with 150 mM NaCl. Survival was assessed 8 d after transfer. Overexpressing lines showed significantly increased survival compared with wild-type plants (P < 0.05, Student’s t test). Measurements were made on two independent replicates of 20 seedlings each. Error bars indicate se.
(B) Plants of wild-type Col-0 and two independent UGT74E2OE lines grown under a controlled watering regime for 3.5 weeks followed by watering for 3 weeks with 500 mM NaCl.
(C) Plants of wild-type Col-0 and two independent UGT74E2OE lines grown under a controlled watering regime for 3.5 weeks and deprived from further watering for 12 d. On day 13, plants were rewatered and observed for recovery on day 14.
(D) Plants were grown with 1.5 g water/g dry soil for 3.5 weeks, after which both groups were not watered for 9 d. The total weight (pot + soil + plant) was recorded at different times. During the dehydration period, the water economy of the UGT74E2OE plants was significantly better (P < 0.05, Student’s t test) than that of the wild-type plants already after 3 d. Error bars indicate se (n = 6).

 ABA Homeostasis and Response in Transgenic Lines.
(A) Relative abundance of ABA and ABA-Glc per unit fresh weight of wild-type and UGT74E2OE plants quantified by HPLC-MS under control and dehydrated conditions. Data are based on three biological replicates (means ± se).
(B) Percentage of emerged radicles for wild-type and UGT74E2OE lines scored 3 d after seed imbibition in the absence (control) or presence of 2 μM ABA, 10 μM IBA, 10 μM IBA-Glc, 0.5 μM IAA, or a combination of each auxin with ABA at the same concentration as mentioned before. Data are means ± se and are based on three biological replicates of 50 seeds each and P < 0.05.
(C) Percentage of emerged green cotyledons 8 d after imbibition in wild-type and transgenic lines grown as described in (B). Data are means ± se and are based on three replicates of 50 seeds each and P < 0.05.