The Arabidopsis auxin-inducible gene ARGOS controls lateral organ size

Abstract

During plant development, the final size of an organ is regulated and determined by various developmental signals; however, the molecular mechanisms by which these signals are transduced and the mediators involved are largely unknown. Here, we show that ARGOS, a novel Arabidopsis gene that is highly induced by auxin, is involved in organ size control. Transgenic plants expressing sense or antisense ARGOS cDNA display enlarged or reduced aerial organs, respectively. The alteration in organ size is attributable mainly to changes in cell number and the duration of organ growth. Ectopic expression of ARGOS prolongs the expression of AINTEGUMENTA (ANT) and CycD3;1 as well as the neoplastic activity of leaf cells. Moreover, organ enlargement in plants overexpressing ARGOS can be blocked by the loss of function of ANT, implying that ARGOS functions upstream of ANT to affect the meristematic competence of organ cells. The induction of ARGOS by auxin is attenuated or abolished in auxin-resistant1 (axr1), and overexpression of ARGOS partially restores axr1 organ development. These results suggest that ARGOS may transduce auxin signals downstream of AXR1 to regulate cell proliferation and organ growth through ANT during organogenesis.

Figure 1.

Sequences and Expression of ARGOS. (A) Nucleotide and predicted amino acid sequences of ARGOS. The Leu-rich region is shaded. (B) Induction of ARGOS by auxin. Ten-day-old seedlings of Arabidopsis ecotype Columbia grown vertically on MS medium were sprayed with 5 μM NAA. Roots and aerial parts were harvested at the times indicated and processed for RNA gel blot analysis. (C) Organ-specific expression of ARGOS. Inflorescence stems (St), leaves (L), flowers (F), and siliques (Si) were taken from 6-week-old plants grown in a growth chamber. Roots (R) and young rosette leaves (YL) were taken from 2-week-old seedlings grown vertically on MS medium. (D) to (F) ARGOS-GUS expression in a 12-day-old seedling (D), flower (E), and young silique (F). The inset in (D) shows the GUS expression pattern in the root pericycle and root tip. Bars = 5 mm.

Figure 2.

Phenotypic and Molecular Characterization of ARGOS Transgenic Plants. (A) Thirty-day-old transgenic plants carrying 35S-anti-ARGOS (left), vector control (middle), and 35S-ARGOS (right) grown in a growth chamber at 23°C under a 16-h-light/8-h-dark photoperiod. Bar = 1 cm. (B) Expression analyses of ARGOS in transgenic plants. A vector control line (CK1-4) and two independent lines of 35S-anti-ARGOS (A3-5 and A13-3) and 35S-ARGOS (S1-1 and S6-4) were used to analyze transgene and endogenous gene expression. The RNA gel blot was probed with the ARGOS coding region to monitor transgene expression, with an anti-ARGOS RNA to detect the overexpression of ARGOS (ARGOS-OE), and with a 5′ nontranslated region of ARGOS for endogenous ARGOS expression (ARGOS). (C) Leaf fresh weight of 6-week-old plants. At least 10 plants from two independent lines were measured in vector control, 35S-anti-ARGOS, and 35S-ARGOS plants (n = 10). (D) Morphology (top) and dimensions (bottom) of 5-week-old fifth leaves. For the top panel, bar = 5 mm; for the bottom panel, n = 10. (E) Phenotypes of flowers, inflorescence stems, and siliques of 35S-anti-ARGOS, vector control, and 35S-ARGOS plants (from left to right). Bars = 5 mm.

Figure 3.

Anatomical Analysis of Fifth Leaves in Transgenic ARGOS Plants. (A) Adaxial epidermal pavement cells of fully expanded fifth leaves of 35S-anti-ARGOS (left), vector control (middle), and 35S-ARGOS (right) plants. Bars = 100 μm. (B) Transverse sections of leaf blades of 35S-anti-ARGOS (top), vector control (middle), and 35S-ARGOS (bottom) plants. Bars = 100 μm. (C) Dimensions of palisade cells. X, Y, and Z axes correspond to the directions of leaf width, length, and thickness, respectively. At least 40 cells of each line were measured with a microscope. (D) Number of palisade cells and total number of mesophyll cells in X and Y axes of leaves. Four leaves of each line were sectioned, and the cells were counted in the middle of each leaf in the X axis and ∼1 mm from the midvein in the Y axis.

Figure 4.

Effect of ARGOS on Growth and Cell Meristematic Competence. (A) Eight-week-old vector control (left) and 35S-ARGOS (right) plants grown in a growth chamber. Bar = 1 cm. (B) Growth kinetics of the fifth leaves in 35S-anti-ARGOS, vector control (CK), and 35S-ARGOS plants. After emergence, leaf length was measured every 3 days (n = 10). (C) CycB1-GUS activity in 16-day-old seedlings of CK (left) and 35S-ARGOS (right) plants. Bars = 5 mm. (D) ANT and CycD3;1 transcript levels in juvenile and fully expanded rosette leaves of vector control (CK) and ARGOS transgenic plants. (E) and (F) Neoplasia in leaf explants of ARGOS transgenic plants. Bars = 5 mm. (E) Leaf explants from 4-week-old transgenic plants were cultured on hormone-free B5 medium, and photographs were taken at 10 days after excision. Note the callus formation in 35S-ARGOS (bottom) but not in the vector control (top). (F) Callus growth in leaf explants of vector control (top), 35S-ARGOS (middle), and 35S-anti-ARGOS (bottom) plants. The explants were cultured on B5 medium containing 4.5 μM 2,4-D and 0.5 μM kinetin and photographed at 40 days without changing the medium.

Figure 5.

Loss of Function of ANT Blocks Organ Enlargement in 35S-ARGOS Transgenic Plants. (A) Morphology of 4-week-old plants (top) and inflorescences (bottom) of 35S-ARGOS/ANTANT or 35S-ARGOS/ANTant-1 (35S-ARGOS/ANT_) and 35S-ARGOS/ant-1ant-1 plants in line L2-4. Bars = 10 mm. (B) Endogenous ANT mRNA and transgenic ARGOS mRNA levels in the two types of plants shown in (A).

Figure 6.

ARGOS Acts Downstream of AXR1. (A) ARGOS expression in wild-type (WT), axr1-3, and axr1-12 plants with and without auxin treatment. RNA was extracted from 12-day-old seedlings treated with 5 μM NAA (+) or water (−) for 3 h. Numbers above the lanes refer to ARGOS expression levels relative to 28S rRNA levels. (B) Three-week-old plants of wild-type Columbia [WT (Col.)], axr1-3, transgenic axr1-3 carrying an empty vector, and transgenic axr1-3 carrying a 35S-ARGOS transgene. Bar = 1 cm. (C) Dimensions of fifth leaves of 5-week-old wild-type (1), axr1-3 (2), axr1-3/vector (3), and axr1-3/35S-ARGOS (4) plants The corresponding fresh weights (mg) are as follows: (1) 29.8 ± 4.1; (2) 15.5 ± 5.2; (3) 16.1 ± 3.8 ; and (4) 32.8 ± 5.2. At least 12 leaves from each genotype were measured. (D) Endogenous ARGOS mRNA and transgenic ARGOS (ARGOS-OE) mRNA levels in the wild type (lane 1), axr1-3 (lane 2), axr1-3/vector (lane 3), and axr1-3/35S-ARGOS (lane 4).

Figure 7.

A Proposed Model for ARGOS Function in Plant Organ Size Control. The model was adapted from Mizukami and Fischer (2000).