Polar auxin transport controls suspensor fate

Abstract

Polar auxin transport is critical for normal embryo development in angiosperms. It has been proposed that auxin accumulates dynamically at specific positions, which in early Arabidopsis embryos correlates with developmental decisions such as specification of the apical cell lineage, specification of the hypophysis, and differentiation of the two cotyledons. In conifers, pattern formation during embryo development is different, and includes a free nuclear stage, nondividing suspensor cells, presence of tube cells, lack of hypophysis and formation of a crown of cotyledons surrounding the shoot apical meristem. We have recently shown that polar auxin transport is important for normal embryo development also in conifers. Here we suggest a model where auxin is transported from the suspensor cells to the embryonal mass during early embryogeny in conifers. This transport is essential for the developmental decisions of the tube cells and the suspensor, and affects both the amount of programmed cell death and the embryo patterning.

In the model plant Arabidopsis thaliana auxin is transported, already from the first cell division of the zygote, from the basal cell to the apical cell, where it is involved in establishing the identity of the apical cell lineage. At the 32-cell stage the polar auxin transport is reversed, leading to an auxin accumulation in the uppermost suspensor cell, which occurs concomitantly with the specification of the hypophysis. During the heart stage auxin is transported towards the cotyledonary primordia, giving positional information about the cotyledon outgrowth.¹ Formation of the apical-basal embryonic pattern during early embryogeny in conifers is quite different from that in Arabidopsis and proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass, the embryonal tube cells and terminally differentiated nondividing suspensor cells.²

The somatic embryo system of Picea abies (Norway spruce) includes a stereotyped sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments.^3,4 We are using this as a model system for elucidating the regulation of embryo development in conifers.² Early somatic embryos differentiate from proembryonic masses (PEMs) after withdrawal of the plant growth regulators (PGRs) auxin and cytokinin (Fig. 1A and B). We have previously shown that the organisation of the apical-basal polarity in early embryos is dependent on a gradient of PCD from the embryonal tube cells committed to death, to the cell corpses at the basal end of the suspensor.^5–7 Dysregulation of the PCD leads to aberrant apical-basal patterning.

Figure 1.

Model for polar auxin transport control of early embryo patterning in conifers. (A) Embryogenic cultures proliferate as proembryonic masses (PEMs) in the presence of the plant growth regulators (PGRs) auxin and cytokinin. (B) Early embryos start to differentiate from PEMs after withdrawal of PGRs. Endogenous auxin is transported to the newly formed embryonal mass. (C) Early embryos are formed within two weeks in PGR-free medium. Early embryos have a distinct embryonal mass, tube cells and a suspensor. IAA is transported from the suspensor and the tube cells to the embryonal mass. (D) Fully matured cotyledonary embryos are formed after 5–6 weeks on maturation medium. (E) Treatment with NPA blocks the polar auxin transport to the embryonal mass, leading to an IAA accumulation in the suspensor cells, tube cells and perhaps also in the cells of the embryonal mass most adjacent to the tube cells. (F) Embryos with supernumerary suspensor cells are formed if polar auxin transport is inhibited only during the earliest stages of suspensor differentiation. (G) Embryos with meristematic cells in the suspensor are formed if polar auxin transport is inhibited during both differentiation and elongation of the suspensor. We assume that these abnormalities abort further development and maturation of viable embryos. em, embryonal mass; s, suspensor; tc, tube cells. Green arrows indicate polar auxin transport, T indicates blocked polar auxin transport, green shadings indicate auxin accumulation.

We recently showed that in embryogenic cultures of Norway spruce treated with the polar auxin transport inhibitor NPA, the number of cells undergoing PCD decreases. As a consequence the balance between the number of cells in the embryonal mass and the number of cells in the suspensor develop abnormally, and concomitantly the endogenous free IAA content increases almost two-fold.⁸

In order to visualise the IAA accumulation within the embryos we used a -318 bp deletion of the auxin-responsive IAA4/5 promoter from Pisum sativum (pea), previously characterized by Oeller et al.,⁹ and Ballas et al.,¹⁰ fused to the GUS reporter gene.¹¹ In tobacco (Nicotiana tabacum) the promoter is expressed in rapidly elongating hypocotyls,¹² (our unpublished observations) and strong induction by auxin is clear in elongating zones of both roots and hypocotyls in transgenic pIAA4/5-GUS Arabidopsis plants.¹¹ However, to our knowledge, expression of IAA4/5 has not been reported in embryonal shoot apical meristems. Hence, the pIAA4/5-GUS may preferentially be used as a biosensor of auxin activity in non-meristematic cells during spruce embryo development. During normal somatic embryo development in spruce, pIAA4/5-GUS activity is detected in PEMs, tube cells and suspensor cells, but not in the embryonal mass. Early embryos of Norway spruce that are treated with NPA show increased pIAA4/5-GUS activity in tube cells and suspensor cells (unpublished), well in line with the increment of free IAA levels.

Our results indicate that IAA under normal conditions is transported from the suspensor cells to the cells in the embryonal mass (Fig. 1B and C). NPA-treatment blocks this polar transport of endogenous IAA, which results in an accumulation of IAA and increased pIAA4/5-GUS activity in the suspensor cells, the tube cells, and perhaps also in the cells of the embryonal mass most adjacent to the tube cells (Fig. 1F and G). Blocked polar auxin transport during early differentiation of the suspensor stimulates abnormal cell divisions of the meristematic cells most adjacent to the tube cells or perhaps even of the tube cells themselves. Consequently, embryos with supernumerary tube and suspensor cells are formed (Fig. 1F). If the polar auxin transport is blocked for a longer time, i.e., during both differentiation and elongation of the suspensor, the auxin accumulation leads to maintenance of meristematic fate and a failure to undergo PCD (Fig. 1G).

It has been proposed that the fate of the suspensor cells is regulated by signals from the embryo proper which impede developmental potential and initiate PCD.¹³ In accordance, we assume that the abnormal embryo morphologies formed after NPA-treatment may result from adverse inhibitory signals from the embryonal mass.

Abbreviations

IAA

indole-3-acetic acid

NPA

1-N-naphtylphthalamic acid

PCD

programmed cell death

Addendum to: Larsson E, Sitbon F, Ljung K, von Arnold S. Inhibited polar auxin transport results in aberrant embryo development in Norway spruce. New Phytol. 2008;177:356–366. doi: 10.1111/j.1469-8137.2007.02289.x.