KANADI and class III HD-Zip gene families regulate embryo patterning and modulate auxin flow during embryogenesis in Arabidopsis

Abstract

Embryo patterning in Arabidopsis thaliana is highly affected when KANADI or Class III HD-Zip genes are compromised. Triple loss-of-function kan1 kan2 kan4 embryos exhibit striking defects in the peripheral-central axis, developing lateral leaf-like organs from the hypocotyls, whereas loss of Class III HD-Zip gene activity results in a loss of bilateral symmetry. Loss of KANADI activity in a Class III HD-Zip mutant background mitigates the defects in bilateral symmetry, implying that the two gene families act antagonistically during embryonic pattern formation. Dynamic patterns of auxin concentration and flux contribute to embryo patterning. Polar cellular distribution of PIN-FORMED1 (PIN1) mediates auxin flow throughout embryogenesis and is required for establishment of the apical-basal axis and bilateral symmetry. Defects in the pattern of PIN1 expression are evident when members of either the KANADI or Class III HD-Zip gene families are compromised. Abnormal expression patterns of PIN1 in KANADI or Class III HD-Zip multiple mutants and the phenotype of plants in which members of both gene families are mutated suggest that pattern formation along the central-peripheral axis results from interplay between auxin and the KANADI and Class III HD-Zip transcription factors, whose defined spatial and temporal expression patterns may also be influenced by auxin.

Figure 1.

The Phenotype of kan1 kan2 kan4. (A) and (B) Fourteen-day-old wild-type (A) and kan1 kan2 kan4 (k124) (B) seedlings. Outgrowths develop on the abaxial side of kan1 kan2 kan4 cotyledons, and radialized leaf-like structures develop on the hypocotyl. (C) and (D) Cotyledon (cot; asterisk) and hypocotyl (hyp) outgrowths (arrow) can be observed already in a 2-d-old kan1 kan2 kan4 seedling (D) but are lacking in a 2-d-old wild-type seedling (C). (E) and (F) In a 6-d-old kan1 kan2 kan4 seedling (F), outgrowths form radialized structures (arrows) on the hypocotyl, and cotyledon outgrows (asterisk) continue to develop on the abaxial side. Compare with a 6-d-old wild-type seedling (E). (G) and (H) The epidermal cell structure that in the wild type is characterized by an alternating pattern of large and small cell rows (G) is disrupted in the kan1 kan2 kan4 hypocotyl and consists of uniform small cells (H). (I) to (K) Ten-day-old (I), 21-d-old (J), and 28-d-old (K) kan1 kan2 kan4 plants. The hypocotyl outgrowths elongate and form radialized leaf-like structures (white arrows) with an occasional trumpet-shaped leaf (black arrow). Ectopic meristems develop in the axils of the hypocotyl leaves, giving rise to leaves with polarity.

Figure 2.

The Embryo Phenotype of kan1 kan2 kan4. (A) and (B) Wild-type (A) and kan1 kan2 kan4 (k124) (B) early heart stage embryos. Periclinal cell divisions are already visible in the kan1 kan2 kan4 subepidermal layer (arrows). (C) and (D) Cotyledons are at a more obtuse angle and the region that will give rise to the SAM is broader in kan1 kan2 kan4 embryos (D) than in wild-type embryos (C). Arrows in (D) indicate abnormal periclinal cell divisions that will give rise to the cotyledon and hypocotyl outgrowths. (E) and (F) Torpedo stage kan1 kan2 kan4 embryo (F) with cotyledon (cot) and hypocotyl (hyp) outgrowths (arrows), which are lacking in the wild-type torpedo stage embryo (E).

Figure 3.

Anatomical Features of kan1 kan2 kan4. (A) and (B) Transverse sections through 12-d-old seedlings. Although in wild-type seedlings, young leaf primordia exhibit polarity in the abaxial–adaxial axis (A), kan1 kan2 kan4 (k124) leaf primordia lack polarity and are radialized (B). (C) and (D) Transverse sections through wild-type (C) and kan1 kan2 kan4 (D) hypocotyls. The vascular anatomy of the kan1 kan2 kan4 hypocotyl (D) is indistinguishable from that of the wild type (C) with a single phloem (ph) strand on either side of the primary xylem (xy). The kan1 kan2 kan4 ground tissue has more cell layers than the wild-type tissue and lacks the distinct endodermis (en), inner cortex (ic), outer cortex (oc), and epidermis (ep) cell layers seen in the wild type. Early in development (D), the hypocotyl outgrowth (arrow) resembles leaf primordia. (E) and (F) The leaf primordia develop into a radialized leaf lacking any signs of polarity, as seen in transverse (E) and longitudinal (F) sections. The vasculature differentiates initially at the distal end of the developing outgrowth (E). (G) to (I) Meristems develop in the axils of hypocotyl leaves. A young hypocotyl leaf with a meristem (m) developing at its base is seen in a transverse section (G). A second meristem is associated with another hypocotyl leaf that is not included in the section. As the hypocotyl leaves mature, ectopic meristems continue to develop at their axils and are observed in longitudinal sections through the base of mature hypocotyls ([H] and [I]). The ectopic meristems give rise to leaf primordia (lp).

Figure 4.

YABBY and the kan1 kan2 kan4 Cotyledon and Hypocotyl Outgrowths. (A) and (B) In kan1 kan2 kan4 fil yab3 yab5 (k124fily35) (B), outgrowths (arrows) forming on the hypocotyl are similar in shape to kan1 kan2 kan4 (k124) (A) outgrowths (arrows) but do not reach the full length of the kan1 kan2 kan4 outgrowths. Three narrow cotyledons (cot) develop in kan1 kan2 kan4 fil yab3 yab5 plants (B). The cotyledons lack the outgrowths (asterisks) observed on the abaxial side of kan1 kan2 kan4 cotyledons (A). (C) to (G) mRNA expression pattern of FIL in kan1 kan2 kan4 embryos. At the early (D) and late (E) heart stages, FIL expression in kan1 kan2 kan4 is similar to that in the wild type (C) and confined to the abaxial side of developing cotyledons. During the torpedo stage, FIL expression is seen throughout hypocotyl outgrowth primordia (arrow) (G) and on the abaxial side of the cotyledons, as in the wild type (F).

Figure 5.

PIN1 Expression and DR5 Auxin Response during Embryogenesis. (A) and (B) PIN1 is expressed throughout both wild-type (A) and kan1 kan2 kan4 (k124) (B) globular embryos. (C) and (D) In wild-type embryos (C), PIN1 is expressed during the transition stage at the sites of incipient cotyledons and developing vasculature, whereas in kan1 kan2 kan4 embryos (D), PIN1 expression can also be seen in the embryonic cells of the L1 layer that will give rise to the hypocotyl (arrows). (E) to (G) At the heart stage, PIN1 is excluded from the hypocotyl cells in the wild type (E) but continues to be expressed in these cells in kan1 kan2 kan4 embryos ([F] and [G]). A transverse section through the hypocotyl region of the kan1 kan2 kan4 embryo at the heart stage (G) exhibits ectopic expression of PIN1 in the L1 cells, whereas no changes in cell division are evident at this stage. (H) to (M) By the late heart (H) and later (L) stages, PIN1 expression in wild-type embryos is restricted to the SAM, vasculature, and tips of the cotyledons. In kan1 kan2 kan4, ectopic expression of PIN1 in the hypocotyl is maintained through the late heart ([I] and [J]) and later (M) stages, correlating with the aberrant phenotype. An abnormal polar arrangement of PIN1 in the hypocotyl cells with protein at the base of more apical cells and at the apex of more basal cells (K) leads to ectopic auxin maxima within the hypocotyl. (N) and (P) In phb phv rev embryos, PIN1 is expressed throughout the apical part of the embryo and in the provasculature cells from the globular through the heart stages. (Q) and (R) At later stages, PIN1 expression is lacking from the single radialized cotyledon (cot) and expressed only in the central and basal regions of the vasculature. (S) and (T) GFP expression driven by the artificial DR5 promoter can be seen in kan1 kan2 kan4 embryos (T) during the torpedo stage in the hypocotyl and cotyledon outgrowths (arrows), indicating areas of auxin accumulation not present in wild-type embryos (S).

Figure 6.

The Phenotype of kan1 kan2 kan4 phb phv rev. (A) A 14-d-old kan1 kan2 kan4 phb phv rev seedling (k124 phb phv rev) has two cotyledons (cot) and ectopic leaf-like outgrowths developing from the hypocotyl (arrows), similar to kan1 kan2 kan4 seedlings (cf. with Figure 4A, same age). In addition, in kan1 kan2 kan4 phb phv rev seedlings, a single radial central leaf (cl) develops in the position normally occupied by the SAM. The central leaf exhibits outgrowths on its distal end and is radialized toward its proximal end. (B) to (D) The central leaf substituting for the SAM in kan1 kan2 kan4 phb phv rev is initiated during embryogenesis, with subepidermal periclinal cell divisions giving rise to a leaf primordium (C) instead of anticlinal cell divisions, as in wild-type SAMs (D). Arrows in (B) indicate areas of periclinal cell divisions. (E) The vascular bundle at the proximal end of the central leaf is radialized with patches of phloem (arrows) surrounding a ring of xylem (xy) and parenchyma cells located at the center. (F) to (I) Ectopic meristems (m) develop around the base of the central leaf, as seen in longitudinal sections through the central leaf ([F] to [H]; [G] is a closeup of [F]), and give rise to leaf primordia (lp) (I).

Figure 7.

A Model for Auxin Flow and the Establishment of Bilateral Symmetry during Embryo Patterning. (A) and (B) In wild-type embryos at the transition stage (A), changes in auxin flow occur, with the reversal of auxin flow from the SAM central region outward toward the periphery of the embryo (red arrows). At the same time, the earlier flow toward the tip of the incipient cotyledons and down through the center of the globular embryo continues (black arrows). The reversal of auxin flow leads to auxin maxima at the incipient cotyledon tips and to cotyledon primordia development, as manifested in a heart-shaped embryo. During the heart stage (B), a second reversal of auxin flow from the cotyledon primordia toward the SAM region is proposed to occur (blue arrows). However, the effects of auxin maxima are repressed by genes expressed in the central domain, where the SAM will develop, such as Class III HD-Zip genes, with only the peripheral domain being responsive to auxin maxima. (C) and (D) In phb phv rev embryos during the transition stage (C), auxin flows from the basal part of the embryo upward in the epidermal cells, toward the apical tip, and down through the center of the embryo (black arrows). The reversal of auxin flow from the SAM region to the incipient cotyledons does not occur, because of ectopic KANADI expression in that region; thus, only a single auxin maxima is formed at the apex of the embryo during the transition stage, leading to an embryo bearing a single cotyledon primordium (D). (E) and (F) In kan1 kan2 kan4 embryos during the transition stage (E), auxin flows upward to the apical part of the embryo (black arrows). Apical reversal of the flow from the SAM region to the incipient cotyledons (red arrows) and back from the cotyledons to the SAM (blue arrows) takes place at the transition stage, similar to that in wild-type embryos. However, at the heart stage (F), an abnormal reversal of auxin flow occurs in the abaxial region of the cotyledon and hypocotyl cells (red arrows), resulting in bidirectional auxin flow. Auxin accumulation in cells located at the abaxial side of the cotyledons and hypocotyl promotes outgrowths and leaf primordia initiation, respectively. (G) and (H) In kan1 kan2 kan4 phb phv rev embryos, apical reversal of auxin flow at the transition stage (G) occurs normally (red arrows), as a result of KANADI loss of function in this background, leading to the establishment of a bilateral symmetry. At the heart stage (H), the second reversal of auxin flow back toward the meristem once the incipient cotyledons are established also occurs (blue arrows), similar to that in the wild type. This reversal of auxin creates an auxin maximum in the position normally occupied by the SAM in the wild type. However, because PHB, PHV, and REV are needed for SAM establishment, the ectopic auxin maximum induces an ectopic central leaf instead of a SAM. Loss of KANADI activity in this background leads to the reversal of flow on the abaxial side of the cotyledons and the hypocotyl (red arrows), resulting in auxin maxima and the formation of outgrowths and leaf primordia in the cotyledons and hypocotyl, respectively.