Auxin response factors mediate Arabidopsis organ asymmetry via modulation of KANADI activity

Abstract

Members of the KANADI gene family in Arabidopsis thaliana regulate abaxial identity and laminar growth of lateral organs. Promoter APETALA3-mediated ectopic expression of KANADI restricts petal expansion and was used in a genetic screen for factors involved in KANADI-mediated signaling. Through this screen, mutations in ETTIN (ETT; also known as Auxin Response Factor3 [ARF3]) were isolated as second site suppressors and found to ameliorate ectopic KANADI activity throughout the plant as well. Mutant phenotypes of ett are restricted to flowers; however, double mutants with a closely related gene ARF4 exhibit transformation of abaxial tissues into adaxial ones in all aerial parts, resembling mutations in KANADI. Accordingly, the common RNA expression domain of both ARFs was found to be on the abaxial side of all lateral organs. Truncated, negatively acting gene products of strong ett alleles map to an ARF-specific, N-terminal domain of ETT. Such gene products strongly enhance abaxial tissue loss only when ARF activities are compromised. As KANADI is not required for either ETT or ARF4 transcription, and their overexpression cannot rescue kanadi mutants, cooperative activity is implied. ARF proteins are pivotal in mediating auxin responses; thus, we present a model linking transient local auxin gradients and gradual partitioning of lateral organs along the abaxial/adaxial axis.

Figure 1.

ETT Mediates Ectopic KAN Activity. Arabidopsis wild-type petals are laterally expanded and composed of epidermal cells that exhibit distinct abaxial/adaxial morphology (A). Ectopic expression of KAN1 using the floral-specific AP3 promoter results in radial petals ([B] and [C]). AP3:KAN1 ett-11 plants have laterally expanded petals despite ectopic KAN activity (D). In the wild type, abaxial petal cells are relatively cubic with wavy cuticular ridges, while adaxial ones are conical with undiluted cuticular ridges ([E] and [F]). Distal epidermal petal cells of AP3:KAN1 are cubic all around (G), while the distal abaxial epidermal petal cells of ett-11 are intermediate between abaxial and adaxial wild-type petal cells (H). (I) shows a comparison of abaxial 7th leaf surface of wild-type (a) and kan1-2 kan2-1/+ (b), ett-1 kan1-2 kan2-1/+ (c), and kan1-2 kan2-1 double mutants (d). Novel phenotype of small blade outgrowths appears on the abaxial leaf surface of the ett-1 kan1-2 kan2-1/+ leaves, which resemble kan1-2 kan2-1double mutants. ett-1 mutants are indistinguishable from the wild type (J) before flowering. Ectopic expression of KAN2 through the ANT promoter results in narrow cotyledons, radial leaves, and growth arrest (K), while in ett-1 ANT≫KAN2 plants (L), all lateral organs form and expand, albeit with altered morphology. p, petal; rp, radial petal; ab, abaxial; ad, adaxial. Bars = 10 μm in (E) to (H).

Figure 2.

ett arf4 Double Mutants Resemble Loss of KAN Phenotypes. Plants mutated at either ETT or ARF4 are indistinguishable from the wild type at the vegetative stage (A); however ett-1 arf4-1 double mutants exhibit severe morphological alterations (B): leaves are narrow and curled upwards and have ectopic blade outgrowths on their abaxial side only. Similar narrow leaves with abaxial outgrowth formed by kan1-2 kan2-1 mutants are shown (C). At the base of ett-1 arf4-1 leaves, ectopic stipules (asterisks) can be found (D). The abaxial blade outgrowths of these leaves emerge in expanding leaf primordia and are nearly radial with unexpanded cells all around (E). Abaxial epidermal cells are extremely small, similar in size to guard cells (F). The temporal pattern of cell divisions is monitored by CYC1B:GUS reporter activity in the wild type (G). In ett-1 arf4-1 leaves of comparable age, it displays prolonged activity (H). A single vascular bundle connects the ett-1 arf4-1 blade outgrowth to the leaf main bundles (I). Unlike the wild type (J), the ett-1 gynoecium is composed of a short valve and basally expanded style (K). Similar alterations are found in plants with partially compromised activity of the redundant KAN genes as in kan1-2 kan2-1/+ (L). All floral organs of ett-1 arf4-1 are misshapen (M). The gynoecium of ett-1 arf4-1 (N) is short and nearly devoid of valve tissue, and ovules are distally positioned both adaxially and abaxially. bl, blade; blo, blade outgrowths; gc, guard cell; vb, vascular bundle; v, valve; st, style. Bars = 50 μm in (D) and (E), 10 μm in (F), and 200 μm in (N).

Figure 3.

Unique and Common Functional Activities of ETT and ARF4. (A) A summary of complementation tests of ett-1, ett-1 arf4-1, and kan1 kan2 mutant backgrounds with ectopic ETT and ARF4 constructs. (B) to (F) The typical gynoecium morphology of ett-1 af4-1 plants complemented by the various ectopic ETT and ARF4 constructs.

Figure 4.

In Situ RNA Distribution of ARF4 and ETT in Vegetative and Reproductive Tissues. Twelve-day-old seedlings of wild type ([A], [B], [D], and [E]), kan1-2 kan2-1 ([C] and [F]), and ett-1 (G) probed with antisense digoxigenin-labeled RNA for ETT ([A] to [C]) and ARF4 ([D] to [G]). m, meristem; s, stipules; blo, leaf blade outgrowth; vb, vascular bundle; ph, phloem; im, inflorescence meristem; fm, flower meristem; cb, cryptic bract; sp, sepal; st, stamen; g, gynoecium. (A) ETT transcript is detected at low levels throughout the SAM and at much higher levels in leaf anlagen. Expression marks the sites of provascular differentiation both in the shoot and leaves. (B) In the transverse section, a gradual restriction from throughout leaf primordia to the adaxial marginal domain of leaves (arrowheads), stipules, and vascular bundles is evident. (C) In kan1-2 kan2-1 background, ETT expression initiates normally, but further expression is restricted to the abaxial leaf side where high levels of ETT mRNA demarcate the initiation sites of abaxial blade outgrowths. (D) and (E) ARF4 is expressed abaxially in the proximal part of wild-type leaf primordia starting at P0 to P4, and its expression is gradually restricted to a narrow band of cells connecting the vascular bundle with the leaf lateral margins (arrows) and the phloem. (F) and (G) An expression pattern of ARF4 similar to the wild type is detected in kan1-2 kan2-1 vegetative apices (F) with slight expansion to more distal leaf domains (G). No alteration relative to wild-type ARF4 transcript distribution was found in ett-1 apices. (H) and (I) ETT mRNA is detected throughout the inflorescence meristem and the sites of provascular differentiation, but much higher mRNA levels are found in initiating flower meristems, which is later confined to the cryptic bract and abaxial domain of sepals, stamens, and gynoecium. (J) to (L) Low ARF4 expression is detected in inflorescence and flower meristems. Later, it resolves to cryptic bract primordia and throughout all initiating floral organ primordia. (M) At later stages, expression of ARF4 is restricted to the phloem as seen in a transverse section of a stage 10 flower pedicel.

Figure 5.

Strong ett Alleles Result from Truncated Gene Products with Negative Effects. (A) Structure of the ETT gene and position of lesions in the different ett alleles (exons shown as boxes, and introns are marked by lines). T-DNA insertion positions and the premature stop codon in ett-11 are indicated by arrows. Regions indicated by A and B correspond to TAS3 trans-acting siRNA (ta-siRNA) target sites (Allen et al., 2005). The black lines denote the extent of the ETT-N150 and ETT-N333 constructs. (B) ett-13 gynoecia have an elongated gynophore topped by valves of unequal length as indicated by arrows. (C) ett-114 gynoecium has the typical strong ett phenotype with apical cell types shifted basally. (D) In ett-13 arf4-1 plants, leaves are slightly up-curled with hardly any abaxial outgrowths. (E) ett-13 arf4-1 flowers have normal numbers of organs (one petal removed), albeit narrower. (F) Occasionally, nearly radial petals or stamens missing locules are formed (asterisk). The gynoecium has an extended gynophore with very reduced or complete loss of valve tissue, style expanded basally, and limited distal placenta. (G) and (H) Close to null nature of ett-13 is evident by lack of RNA signal in the shoot (G), compared with the strong and more equal expression between meristem and primordia detected in ett-1 (H). (I) and (J) Missexpression of ETT-N150 (I) and ETT-N333 (J) lead to a significant enhancement of the ett-13 gynoecium defects, phenocopying the strong ett-1 allele. Bars = 2 mm in (B), (C), (I), and (J). DBD, DNA binding domain; MR, middle region; v, valve; s, stamen; st, style; gy, gynophore; p, petal; rp, radial petal.

Figure 6.

Proposed Role for Auxin in Mediating Abaxial/Adaxial Partitioning of Organ Primordia. Initiating organ primordia cells coexpress abaxial and adaxial factors. The partitioning of lateral organs into the abaxial KAN-expressing domain and the adaxial PHB-like–expressing domain is gradual and evolves by mutual antagonism between the two types of factors and external morphogenic input. With the rapid expansion of the growing primordium, auxin concentrations form a slight gradient via asymmetric auxin influx carrier distribution and due to conversion from being a sink to a new source of auxin synthesis (Reinhardt et al., 2003). This gradient of auxin is translated into differential action of specific subsets of ARFs (ETT, ARF4, and others), enabling KAN to override PHB-like activities at the abaxial domain. Subsequently, gradients of these ARFs help differentially translate auxin presence to maintain abaxial fate, leading to stable abaxial/adaxial partitioning.