HANABA TARANU is a GATA transcription factor that regulates shoot apical meristem and flower development in Arabidopsis

Abstract

We have isolated a new mutant, hanaba taranu (han), which affects both flower and shoot apical meristem (SAM) development in Arabidopsis thaliana. Mutants have fused sepals and reduced organ numbers in all four whorls, especially in the 2nd (petal) and 3rd (stamen) whorls. han meristems can become flatter or smaller than in the wild type. HAN encodes a GATA-3-like transcription factor with a single zinc finger domain. HAN is transcribed at the boundaries between the meristem and its newly initiated organ primordia and at the boundaries between different floral whorls. It is also expressed in vascular tissues, developing ovules and stamens, and in the embryo. han interacts strongly with clavata (clv) mutations (clv1, clv2, and clv3), resulting in highly fasciated SAMs, and we find that WUS expression is altered in han mutants from early embryogenesis. In addition, HAN is ectopically expressed both in clv1 and clv3 mutants. We propose that HAN is normally required for establishing organ boundaries in shoots and flowers and for controlling the number and position of WUS-expressing cells. Ectopic HAN expression causes growth retardation, aberrant cell division patterns, and loss of meristem activity, suggesting that HAN is involved in controlling cell proliferation and differentiation.

Figure 1.

han Mutant Flower and Floral Meristem Phenotypes. (A) A wild-type flower. (B) to (E) han-2 homozygotes. (F) to (H) han-1 homozygotes. (B) A han-2 flower. (C) The arrow points to the fusion of two stamens. (D) Filamentous structures in place of a petal or a stamen are indicated by the arrow and arrowhead, respectively. Sepals were removed. (E) Carpel defects include asymmetric silique valves (arrow) and extra carpel tissue positioned at the apical region of the silique (arrowhead). Siliques shown are not age-matched. (F) A han-1 inflorescence. (G) An early-arising han-1 flower. (H) A flower with sepals all fused into an open semicircle. (I) A wild-type stage 3 flower. Se, sepal; FM, floral meristem. (J) A han-1 stage 3 flower (because han floral meristem development is generally delayed, floral stage is determined based on sepal size). (K) A wild-type stage 6 flower. St, stamens. (L) A han-1 stage 6 flower. (M) A han-1 inflorescence at late stage. The black arrow points to a carpelloid sepal, and the white arrowhead points to a sepalloid carpel. (N) An enlarged view of the carpelloid sepal indicated in (M). Arrow points to stigmatic papillae at the edge of the sepal. (O) An enlarged view of the sepal (Se) and carpel (Ca) fusion structure in (M). (P) A wild-type SAM. (Q) A han-1 mutant SAM. Bars in (A) to (H) = 0.5 mm; bars in (I) to (Q) = 50 μm (unless indicated otherwise).

Figure 2.

Protein and Gene Structure. The HAN protein sequence deduced from the open reading frame of the full-length HAN cDNA. The position of the intron is indicated by the open triangle; a 14–amino acid domain that is highly conserved among some plant GATA factors is underlined twice; the zinc finger domain is underlined once. The mutation in han-1 results in deletion of the whole gene. The mutation in han-2 changes a Gly (GGC) to a Ser (AGC) at amino acid position 179 (asterisk). The mutation in han-4 results from a T-DNA insertion within the intron (arrow). The 5′ junction sequence is flanked by the Bar gene in the T-DNA, whereas the 3′ is flanked by the 4× 35S promoter in the T-DNA (near the right border).

Figure 3.

HAN Expression Pattern. (A) Budding axillary SAMs are shown in (a). Arrows point to the expression domains of HAN at the boundaries between the SAM and the adaxial sides of leaves. Lf, leaf. (b) Older axillary SAM. Arrows point to the expression domains of HAN at the boundaries between the SAM and the newly initiated organ primordia (pr, small arrowheads). Large arrowhead indicates the junction of the HAN expression domain in the SAM and its vascular expression in the stem. (c) Inflorescence SAM. Arrows point to two stripes of HAN expression at the boundaries between the SAM and newly initiated floral primordia. Small arrowheads indicate HAN expression in a stage 2 flower at the boundaries between the floral meristem and the soon-to-be-specified sepal primordia. Large arrowheads point to the junction of the HAN expression domain in the SAM and in the stem (note that the angle of this section is tilted toward the reader, and the domain indicated by the large arrow includes some of the expression of HAN between the SAM and a primordium pointing toward the reader). (d) Stage 5 flower. Arrow points to the connection arch of HAN expression in the floral meristem and its expression in the peduncle vascular tissue. Se, sepal. (e) Expression in the lateral and basal regions of carpel primordia in a stage 6 flower. (f) Early stage 12 ovary (cross section). HAN is expressed in initiating inner and outer integuments as well as in the vascular tissue (arrows). (g) Expression in integuments continues in the stage 13 ovary. Arrow points to expression in funiculus vascular tissue. (h) HAN expression in the stamens of a stage 8 flower. lo, locule. (i) HAN expression persists in the tapetum cell layer until it has degenerated and is absent in mature haploid pollen. t, tapetum. (j) Eight-cell stage embryo. HAN is expressed in all cells of the embryo proper. (k and l) Globular and transition stage embryos. HAN is expressed in the center files of cells. (m and n) Heart and torpedo stage embryos. HAN expression remains in the center cells destined to be provascular tissues. (o) Late torpedo stage. Bars = 10 μm. (B) Expression in SAM in serial longitudinal (a to f) and cross sections (g to j). Arrows point to expression at the boundaries between the SAM and new organ primordia as well as between organ primordia. Expression of HAN in the SAM merges with its expression in the vascular strands in the stem (arrowheads) as illustrated in (k). In (k), red lines represent the expression domains of HAN in the SAM, stem, and developing organ primordia (P1 and P2). Arrowhead corresponds to the regions indicated by large arrowheads in Figure 3A (b and c). P, primordium. Bars = 10 μm.

Figure 4.

Genetic Interactions of han and clv. (A) clv3-2 inflorescence, top view. (B) clv3-2 flower, top view. (C) han-1 clv3-2 double mutant inflorescence. Arrowhead points to an early-arising flower with long peduncle, and arrow points to late-arising bract-like or filamentous organs. (D) han-1 clv3-2 double mutant flower. (E) han-2 clv1-1 double mutant inflorescence. (F) han-2 clv1-1 double mutant flower. (G) han-2 clv2-1 double mutant inflorescence. (H) han-1 clv1-1 double mutant inflorescence. (I) wus-1 single mutant axillary shoot. (J) han-1 wus-1 double mutant axillary shoot. Bars = 1 mm.

Figure 5.

HAN Regulates WUS-Expressing Cells. (A) and (B) WUS expression in wild-type inflorescence SAM is below the outermost three (shown) or two (data not shown) layers. (B) WUS expression in the wild-type floral meristem (FM) is below the outermost two layers. (C) WUS expression in the clv3-2 inflorescence SAM concentrates in the cells below the L2 layer and at lower level in the L2 layer. (D) WUS expression in the clv3-2 floral meristem expands into the L2 layer. (E) and (G) WUS expression in most of the han-1 inflorescence SAMs is diffuse. (F) and (H) WUS expression in the han-1 floral meristems is shifted to include the L2 and L1 layers. (I) WUS expression in the han-1 clv3-2 double mutant inflorescence SAM is concentrated in the L2 layer. (J) WUS expression in the han-1 clv3-2 double mutant floral meristem extends in some cases into the L1 layer. (K) Overview of a clv3-2 inflorescence SAM. (L) Partial view of a han-1 clv3-2 double mutant inflorescence SAM. Bars = 50 μm.

Figure 6.

han Mutant Embryo Defects and Perturbed WUS Expression in han Embryos. (A) Globular stage wild-type embryo. (B) Globular stage han-1 mutant embryo. (C) Late heart stage wild-type embryo. C, cotyledon. (D) Late heart stage han-1 embryo with stunted cotyledons. (E) Walking stick stage wild-type embryo. (F) Walking stick stage han-1 embryo. (G) Mature wild-type embryo. (H) Mature han-1 mutant embryo with three cotyledons. (I) WUS expression in the transition stage wild-type embryo is concentrated in two cells in the subepidermal L2 layer. (J) WUS expression in the transition stage han-1 embryo is located in more than two cells within and beneath the L2 layer. (K) WUS expression in the heart stage wild-type embryo is shifted to two central cells in the corpus. (L) WUS expression in the heart stage han-1 embryo in the L2 layer. (M) WUS expression in the mature stage wild-type embryo is centered below the outermost two layers. (N) WUS expression in the mature han-1 embryo. (O) and (P) WUS expression in the L2 layer of mature han-1 embryos. (P) is an enlarged view of (N). Bars = 50 μm in (A) to (H) and 10 μm in (I) to (P).

Figure 7.

CLV3 Expression in han Mutants. (A) and (B) CLV3 expression in the wild-type inflorescence SAM and floral meristem (FM) is concentrated in the central outermost three layers, at about three to four cells width. (C) and (D) CLV3 expression appears fairly normal in some han inflorescence SAMs and floral meristems. (E) CLV3 expression is slightly diffuse in some han mutants. (F) CLV3 expression is expanded in some han floral meristems. (G) and (H) CLV3 expression is markedly diffuse in some han mutants. Arrows point to the regions of diffuse expression. Bars = 50 μm.

Figure 8.

HAN Is Ectopically Expressed in clv3-2 Mutants. (A) HAN expression in a clv3-2 inflorescence SAM (arrowheads). (B) HAN expression in the undifferentiated center dome cells in a late stage clv3-2 flower (large arrow). Small arrow points to HAN expression in the integuments of ovule. (C) WUS expression in a clv3-2 inflorescence SAM (arrowhead). (D) WUS expression in the undifferentiated cells in a late stage clv3-2 flower (large arrow). Small arrows point to WUS expression in the nucellus. (E) HAN expression in a heart stage clv3-2 embryo. (F) and (G) HAN expression in the SAM and the root of a clv3-2 embryo in late torpedo stage, respectively. Bars = 50 μm.

Figure 9.

Phenotypes of Ectopic HAN Expression. (A) Lobed cauline leaf from a 35S:HAN plant. Inset shows a wild-type cauline leaf. (B) Wild-type inflorescence. (C) 35S:HAN inflorescence. (D) Wild-type (left) and 35S:HAN flowers. (E) Wild-type (right) and 35S:HAN siliques. (F) to (H) 35S:HAN-GR transformants. (F) DEX-treated inflorescence has early opening flowers and a short stem. (G) Seedlings mock-treated from at 11 d of age, shown at 29 d old. (H) Seedlings treated with DEX beginning at 11 d of age, shown at 29 d old. Arrow points to lobed leaf margins. (I) Homozygous 35S:HAN-GR seedling germinated on a DEX plate at 9 d. (J) SAM of 10-d-old wild-type seedling. M, SAM. (K) and (L) SAMs of 10-d-old 35S:HAN-GR seedlings germinated on DEX plates. Arrows point to the SAMs. (M) WUS expression in a 10-d-old 35S:HAN-GR seedling germinated on non-DEX plates. (N) Absence of WUS expression in a 10-d-old 35S:HAN-GR seedling germinated on DEX plates. (O) Non-DEX-induced control stomata with two guard cells. (P) and (Q) Guard cells in 10-d-old and 6-d-old DEX-treated 35S:HAN-GR seedlings, respectively. Arrows point to stomata with five or seven guard cells. (R) Mock-treated 35S:HAN-GR root tip. Arrow points to the root cap. (S) DEX-induced 35S:HAN-GR root tip. Bars in (A) to (I) = 1 mm; bars in (J) to (S) = 50 μm (except for [Q], in which the bar = 10 μm).