Plant organ size control: AINTEGUMENTA regulates growth and cell numbers during organogenesis

Abstract

The control of cell proliferation during organogenesis plays an important role in initiation, growth, and acquisition of the intrinsic size of organs in higher plants. To understand the developmental mechanism that controls intrinsic organ size by regulating the number and extent of cell division during organogenesis, we examined the function of the Arabidopsis regulatory gene AINTEGUMENATA (ANT). Previous observations revealed that ANT regulates cell division in integuments during ovule development and is necessary for floral organ growth. Here we show that ANT controls plant organ cell number and organ size throughout shoot development. Loss of ANT function reduces the size of all lateral shoot organs by decreasing cell number. Conversely, gain of ANT function, via ectopic expression of a 35S::ANT transgene, enlarges embryonic and all shoot organs without altering superficial morphology by increasing cell number in both Arabidopsis and tobacco plants. This hyperplasia results from an extended period of cell proliferation and organ growth. Furthermore, cells ectopically expressing ANT in fully differentiated organs exhibit neoplastic activity by producing calli and adventitious roots and shoots. Based on these results, we propose that ANT regulates cell proliferation and organ growth by maintaining the meristematic competence of cells during organogenesis.

Figure 1

The loss-of-function ant-1 allele reduces mature organ size and cell number. (A) Fully grown seventh-rosette leaves from an ant-1 mutant (Left) and wild-type plant (Right). (B) Width and length of mature rosette leaves and petals. Wild-type (n = 12) and ant-1 (n = 12) mature third-rosette leaves, and wild-type (n = 58) and ant-1 (n = 41) mature petals (stage 15) were analyzed. Bars on columns show SD. (C and D) Epidermal cells (×150) from the abaxial, distal portion of mature (stage 15) ant-1 (C) and wild-type petals (D).

Figure 2

Gain-of-function 35S::ANT transgene expression causes multiple organ hyperplasia. (A–H) Arabidopsis. (I and J) tobacco. A, C, and E–G show plants or organs of 35S-vector-only control (Left or Upper) and 35SANT (Right or Lower) transformants grown under the long-day conditions. Specimens in each panel were photographed together (A, C, G, and H), or are shown at the same magnification (E and F). (A) Whole plants after bolting. (B) Leaf mass. The average from four independent 35S::ANT and control transgenic plants is shown. Bars indicate SD. (C) Mature flowers. (D) Mass of 10 flowers each from four independent 35S::ANT (AYM 18, 19) and control (AYM 27) T₁ transgenic plants. (E) Mature anthers. The 35S::ANT anther fails to dehisce. (F) An enlarged ovule from the weak 35S::ANT transformant AYM19.4.17 with abnormal growth of nucellus cells. Embryo sac (asterisk) and nucellus cells (arrowhead) are indicated. (G) A fruit obtained after hand-pollination of an AYM19.4.17 pistil with wild-type pollen and an autonomously self-pollinated control fruit. (H) ant-1 (left), control (middle), and 35S::ANT (right) plants grown under short-day (8-h light) conditions. (I) Km^R R₁ tobacco seeds obtained from hand-pollination. (J) Two-day-old tobacco seedlings.

Figure 3

Loss- or gain-of-ANT-function influences the extent of cell proliferation during organogenesis. (A–C) Fully differentiated cells (×135) with centripetal ridges characteristic of the adaxial epidermis of mature petals at stage 15. (D–F) Undifferentiated epidermal cells (×450) from the adaxial, distal portion of mid-stage 9 petals (Insets, ×45). (A and D) ant-1 petal. (B and E) Control (wild type). (C and F) 35S::ANT. (G) Comparison in petal area, cell size, cell number per unit area, and numbers per petal. Percentages of results from ant-1 and 35S::ANT petals to those from control petals are shown. Bars indicate SD. The analysis was performed with the abaxial, distal portion of petals by using more than 20 flowers.

Figure 4

Gain-of-ANT-function prolongs meristematic potential in developing and mature organs. (A) Growth of leaves in control and 35S::ANT seedlings. Length of leaves from six vector-only controls and six 35S::ANT plants was measured at 16, 19, 21, and 26 days after germination (DAG). (B) Effect of ANT activity on CycD3 RNA level in line AYM19.4.7. (C) Ectopic ANT expression causes abnormal outgrowth of cells, or neoplasia. Nine-week-old control and 35S::ANT leaves were excised and placed on an MS-agar plate without phytohormones and were photographed two weeks after excision. Calli (arrowhead) and adventitious roots (arrows) were formed around the cut surface of the 35S::ANT leaf. Inset shows an enlargement of the mass of calli in the 35S::ANT leaf. (D) A stem of a 19-week-old, 35S::ANT plant shows clusters of green growing calli (arrowheads).

Figure 5

A model of ANT function in plant organ size control.