Auxin signal transduction in Arabidopsis vein formation

Abstract

The plant signaling molecule auxin has been implicated in the control of a confounding multitude of diverse processes in plants, including leaf vascular patterning. In Arabidopsis leaves, expression of the HD-ZIP III gene ATHB8 is initiated in files of isodiametric subepidermal cells that will elongate into vein-forming procambium. We have recently shown that ATHB8 is transiently required for preprocambial development and procambium differentiation, and that permanence of the effects of loss of ATHB8 function on vein formation depends on the activity of the auxin response factor MONOPTEROS (MP). Further, we have shown that the onset of ATHB8 expression is directly and positively regulated by MP through an auxin-response element in the ATHB8 promoter, suggesting a molecular path by which auxin signals are translated into vein patterning inputs. Within broad fields of MP expression, however, only a subset of cells initiates expression of ATHB8. Here we discuss putative mechanisms by which wide domains of MP expression could activate ATHB8 transcription in single cell files.

The plant vascular system is composed of bundles of cell files that extend throughout the plant, transporting water, minerals and nutrients.¹ Leaf vascular bundles (‘veins’) emerge de novo from a morphologically homogenous population of ground cells as strands of elongated, cytoplasm-dense procambial cells that generate mature vascular cells.² Accumulating evidence suggests that polar transport of the plant signaling molecule auxin plays a major role in vein patterning.^3–8 In leaves, initially broad domains of expression of the auxin efflux carrier PIN-FORMED1 (PIN1) are restricted to narrow fields that specify positions of procambial strand formation.^6,8 Within wide PIN1 expression domains, expression of the class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) gene ATHB8 is initiated in single files of leaf ground cells that will elongate into procambial cells.^9–11 Onset of ATHB8 expression maintains its association with prospective procambial cells upon interference with vein patterning (reviewed in ref. 12), suggesting that inception of ATHB8 expression identifies a ‘preprocambial’ cell state that preludes to vein formation.^3,10,11

Here we briefly discuss the role of ATHB8 in leaf vascular development, the direct regulation of ATHB8 preprocambial expression by the transcription factor MONOPTEROS/AUXIN RESPONSE FACTOR5 (MP/ARF5), and possible models by which broad domains of MP expression may lead to transcription of ATHB8 in single cell files.

Functions of ATHB8 in Leaf Vascular Development

Mature vein patterns in null athb8 mutants do not conspicuously deviate from wild type;^13,14 however, marker analysis in developing athb8 leaves reveals transient abnormalities during early stages of leaf histogenesis,¹⁵ suggesting a role for ATHB8 in preprocambial development and procambium differentiation (Fig. 1A). Such defects are corrected during athb8 leaf development, suggesting either an ephemeral function of ATHB8 in vein formation or functional redundancy among members of the HD-ZIP III gene family. Analysis of the genetic interaction between mutations in ATHB8 and in MP—a direct upstream regulator of ATHB8¹⁵—provides insight into this matter. Absence of ATHB8 activity results in a small increase in the sensitivity of leaves to auxin transport inhibition; however, in a weak mp background, additional mutation of ATHB8 enhances the phenotypic severity of both untreated and auxin transport-inhibited leaves. This observation suggests that loss of ATHB8 function has long-lasting consequences on vein formation, but that the role of ATHB8 in this process is masked in the athb8 mutant by wild-type MP activity. The downregulation of expression of other members of the HD-ZIP III family in mp³ suggests that resolution of early vascular defects in athb8 leaves may depend on MP-regulated recruitment of HD-ZIP III gene redundant activities. All members of the HD-ZIP III family have been suggested to have roles in vascular development.¹⁴ However, unlike ATHB8, the other members have additionally been implicated in regulation of acquisition of leaf adaxial-abaxial polarity: a process that has repercussions on leaf morphogenesis and vein patterning.¹⁶ Therefore, understanding nonredundant and overlapping functions of HD-ZIP III genes in leaf vascular development will require careful separation of their specific roles in vein formation from vascular readouts of their activities in leaf adaxial-abaxial patterning.

Figure 1.

Summary and perspectives. (A) Molecular events in early vein formation. In Arabidopsis leaf primordia, MP binds a TGTCT G sequence motif in the ATHB8 promoter and initiates ATHB8 transcription (wavy line). ATHB8, in turn, controls preprocambial development and procambium differentiation. These two activities could represent completely separated and self-sufficient functions of ATHB8 in leaf vascular development; they could overlap to various extents; or they could simply correspond to different readouts of the same regulatory potential. While the schematic nature of the relations presented here should be emphasized, the diagram illustrates how MP-dependent global auxin signaling inputs are molecularly partitioned into defined patterning events in leaf vascular development. TC, transcription complex. (B) Mechanisms for restriction of ATHB8 transcription to a subpopulation of MP-expressing cells. At least three formally different scenarios can be envisaged. According to the first (left), expression of ATHB8 (ring) in a subset of the cells expressing MP (circle) would depend on the presence of an activating signal (plus sign) in preprocambial cells. This cue may not depend on the transcriptional regulatory properties of MP and does not necessarily implicate sequence-specific DNA binding activities, but could reflect, for example, different chromatin configurations at the ATHB8 promoter in different cells.²⁴,²⁵ In the second scenario (central), confined activation of ATHB8 expression within wide fields of MP expression would rely upon removal of an inhibiting signal (minus sign) in preprocambial cells, such as transcription-independent degradation of repressors of the Aux/IAA family.²³ Finally, according to the third scenario, MP protein would be present in an instructive gradient in leaf primordia with maximum expression in cells that initiate ATHB8 transcription. Because the ATHB8 preprocambial element is expected to be suboptimal for MP binding,²⁶ activation of ATHB8 expression would only occur where MP levels are sufficiently high.

Regulation of ATHB8 Preprocambial Expression

Both auxin responsiveness and correct initiation of ATHB8 expression depend on a TGTCTG element in the ATHB8 promoter¹⁵ (Fig. 1A). This motif closely resembles the canonical auxin response element TGTCTC necessary for auxin inducible gene expression.^17,18 The TGTCTG sequence in the ATHB8 promoter is an in vivo binding site for MP/ARF5¹⁵ (Fig. 1A), and onset of ATHB8 expression is extremely delayed in a strong mp background.¹⁵ In addition to their reduced organ vascularization, mp mutants are characterized by severe auxin insensitivity and defects both in the formation of the apical-basal axis of the embryo and in the patterned outgrowth of post-embryonic lateral organs, suggesting a broader role for MP in auxin-mediated axial patterning. ^3,19–22 MP/ARF5 belongs to the Auxin Response Factor (ARF) family of transcription factors and is one of five ARFs identified as transcriptional activators (reviewed in ref. 23). The five activating ARFs are characterized by a high degree of functional redundancy, raising the possibility that any of them could regulate transcription of ATHB8. However, the inability of a null athb8 mutation to enhance vascular patterning defects in mature leaves of a strong mp mutant¹⁵ suggests that nonredundant functions of ATHB8 in vein formation are primarily dependent on MP, and that the contribution of other ARFs to regulation of ATHB8 activity in vein ontogeny is probably minor.

Like other members of the HD-ZIP III family, ATHB8 undergoes post-transcriptional regulation by microRNA activity.¹⁶ However, this level of control does not seem to affect initiation of ATHB8 expression in leaves,¹⁵ suggesting that ATHB8 preprocambial expression is mainly dependent on MP activity.

Stage-Specific Expression Dynamics in Early Vein Development

Similar to PIN1, MP expression is initially detected in broad domains that eventually narrow to sites of vein formation,^8,20 while ATHB8 expression is initiated in single files of isodiametric ground cells within wide MP expression domains.¹⁵ As the TGTCTG element in the ATHB8 promoter is a direct target of MP activity, why is ATHB8 transcription not promoted in all MP-expressing cells? At least three formally different possibilities are conceivable (Fig. 1B). The first is that MP is not sufficient to activate ATHB8 transcription and requires the simultaneous presence of a preprocambial-specific coactivating signal. The second is that MP activity depends on the specific removal of an inhibiting cue in preprocambial cells. Finally, the third is that MP protein is present in an instructive gradient in leaf primordia, with maximal expression in cells that will activate ATHB8 transcription. It should be emphasized that these scenarios do not necessarily exclude one another, but that they could all, to varying extents, co-exist. In the future, it will be interesting to understand how ATHB8 expression is initiated in single cell files within broad domains of MP expression. Nevertheless, identifying a role for ATHB8 in vein formation and its direct regulation by MP provides a molecular mechanism through which general auxin signal transduction controls leaf vascular development.

Addendum to: Donner TJ, Sherr I, Scarpella E. Regulation of preprocambial cell state acquisition by auxin signaling in Arabidopsis leaves. Development. 2009;136:3235–3246. doi: 10.1242/dev.037028.