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. 2003 Oct;133(2):538-48.
doi: 10.1104/pp.103.026278. Epub 2003 Aug 21.

Altered life cycle in Arabidopsis plants expressing PsUGT1, a UDP-glucuronosyltransferase-encoding gene from pea

Ho-Hyung Woo  1 , Kym F Faull, Ann M Hirsch, Martha C Hawes
Affiliations

Altered life cycle in Arabidopsis plants expressing PsUGT1, a UDP-glucuronosyltransferase-encoding gene from pea

Ho-Hyung Woo et al. Plant Physiol. 2003 Oct.

Abstract

Alfalfa (Medicago sativa) and Arabidopsis were used as model systems to examine molecular mechanisms underlying developmental effects of a microsomal UDP-glucuronosyltransferase-encoding gene from pea (Pisum sativum; PsUGT1). Alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited delayed root emergence, reduced root growth, and increased lateral root development. The timing of root emergence in wild-type and antisense plants was correlated with the transient accumulation of auxin at the site of root emergence. Cell suspension cultures derived from the antisense alfalfa plants exhibited a delay in cell cycle from 24-h in the wild-type plants to 48-h in the antisense plants. PsUGT1::uidA was introduced into Arabidopsis to demonstrate that, as in alfalfa and pea, PsUGT1 expression occurs in regions of active cell division. This includes the root cap and root apical meristems, leaf primordia, tips of older leaves, and the transition zone between the hypocotyl and the root. Expression of PsUGT1::uidA colocalized with the expression of the auxin-responding reporter DR5::uidA. Co-expression of DR5::uidA in transgenic Arabidopsis lines expressing CaMV35S::PsUGT1 revealed that ectopic expression of CaMV35S::PsUGT1 is correlated with a change in endogenous auxin gradients in roots. Roots of ecotype Columbia expressing CaMV35S::PsUGT1 exhibited distinctive responses to exogenous naphthalene acetic acid. Completion of the life cycle occurred in 4 to 6 weeks compared with 6 to 7 weeks for wild-type Columbia. Inhibition of endogenous ethylene did not correct this early senescence phenotype.

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Figures

Figure 1.
Figure 1.
Correlation between time of emergence of adventitious roots and a transient increase in free IAA at the site of adventitious root emergence: Adventitious roots (arrow) emerge at d 7 from wild-type stem cuttings (WT; A) and at d 11 from stem cuttings of plants expressing CaMV35S::PsUGT1 antisense mRNA (AS; B). C, Emergence of roots from both antisense (dotted line) and wild-type plants (solid line) was correlated with a transient increase in free IAA at d 7 and 11, respectively. IAA levels were quantified using gas chromatography/mass spectrometry (GC/MS) analysis of tissue from the base of stem cuttings; values represent means and sds from three independent experiments.
Figure 2.
Figure 2.
Altered lateral root emergence and morphology in alfalfa plants expressing CaMV35S::PsUGT1 antisense mRNA. A, In wild-type adventitious roots, lateral roots emerged at an average number of 4.5 per centimeter; inset, in longitudinal section, root cells were elongated and arranged in parallel files; B, in roots of antisense plants, lateral roots emerged at an average number of 5.9 per centimeter; inset, in longitudinal section, the cells appear rounded and not elongated. Bars = 3 mm.
Figure 3.
Figure 3.
Increased duration of the cell cycle in alfalfa cell suspension cultures derived from plants expressing CaMV35S::PsUGT1 antisense mRNA. Durations of cell cycle between S phases were 24 and 48 h in cell suspension cultures of wild-type (white squares) and antisense plants (black circles), respectively. Peaks of DNA synthesis are denoted with arrows.
Figure 4.
Figure 4.
Expression of PsUGT1::uidA in Arabidopsis. Localized expression occurred in the outer tip of leaves (right arrows); leaf primordia (notched arrows); transition zone between hypocotyls and root (banded arrows); and the root apical and root cap meristems (left arrows). In the enlargement, the tissue was cleared to show the staining better. Bar = 3.5 mm.
Figure 5.
Figure 5.
Quantitative assessment of senescence based on loss of chlorophyll over time in leaves of wild-type plants (dotted line) and transgenic plants expressing PsUGT1 (solid line).
Figure 6.
Figure 6.
Altered auxin responsiveness in plants expressing CaMV35S::PsUGT1, demonstrated by the synthetic auxin-responsive reporter DR5::uidA. A, In wild-type Columbia 7 d after germination, expression of the DR5::uidA (top to bottom) is localized to the leaf primordium and the tip of the leaf, with no expression in roots except as sites of lateral root initiation and the primary root apex. B, Ectopic PsUGT1 expression lines 7 d after germination also exhibit expression of DR5::uidA (top to bottom) in the leaf primordium and the tip of the leaf, and there is increased expression at sites surrounding lateral root primordia and throughout the primary root apex.
Figure 7.
Figure 7.
Phenotype effects of NAA treatment implant expressing CaMV35S::PsUGT1. A, No effect from treatment of 10–8 m NAA in wild-type roots. B, Treatment of 10–8 m NAA caused root curling/waving in ectopic expression lines. C and E, Treatment of 10–7 m NAA inhibited root growth and enhanced lateral root development in wild-type plants. D and F, Treatment of 10–7 m NAA enhanced lateral root development and root hair growth in ectopic expression lines. Bars in A through D = 10 mm. Bars in E and F = 2.5 mm.
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