HANABA TARANU regulates the shoot apical meristem and leaf development in cucumber (Cucumis sativus L.)

Abstract

The shoot apical meristem (SAM) is essential for continuous organogenesis in higher plants, while the leaf is the primary source organ and the leaf shape directly affects the efficiency of photosynthesis. HANABA TARANU (HAN) encodes a GATA3-type transcription factor that functions in floral organ development, SAM organization, and embryo development in Arabidopsis, but is involved in suppressing bract outgrowth and promoting branching in grass species. Here the function of the HAN homologue CsHAN1 was characterized in cucumber, an important vegetable with great agricultural and economic value. CsHAN1 is predominantly expressed at the junction of the SAM and the stem, and can partially rescue the han-2 floral organ phenotype in Arabidopsis. Overexpression and RNAi of CsHAN1 transgenic cucumber resulted in retarded growth early after embryogenesis and produced highly lobed leaves. Further, it was found that CsHAN1 may regulate SAM development through regulating the WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) pathways, and mediate leaf development through a complicated gene regulatory network in cucumber.

Keywords: CsHAN; CsSTM; CsWUS; cucumber; leaf development; shoot apical meristem..

Fig. 1.

Gene structure and phylogenetic analyses of CsHAN. (A) Structural analysis of HAN genes in cucumber and Arabidopsis. Grey boxes represent the 3′- or 5′-untranslated regions, black boxes indicate the exon, and black lines represent the introns. Cs, Cucumis sativus; At, Arabidopsis thaliana. (B) Protein alignment of HANs from Arabidopsis, rice (Oryza sativa), maize (Zea mays), and cucumber. The single and double underlines indicate the conserved GATA zinc finger domain and HAN motif, respectively. The asterisks indicate the conserved cysteine residues present in type IV zinc finger domains (C-X2-C-X17-20-C-X2-C). (C) Phylogenetic analysis of CsHAN genes (boxed) and HAN-like genes. MEGA 5.0 software was used to construct the Neighbor–Joining tree. Homologues of CsHAN genes from 11 dicotyledon species (double underlines) and four monocotyledon species (single underline) were used for the analyses and formed distinct clades (dicotyledon group and monocotyledon group). Vv, Vitis vinifera; Cr, Capsella rubella; Pt, Populus trichocarpa; Rc, Ricinus communis; Fv, Fragaria vesca subsp. vesca; Gm, Glycine max; Pp, Prunus persica; Mt, Medicago truncatula; Sl, Solanum lycopersicum; Bf, Botryotinia fuckeliana; Aet, Aegilops tauschii; Cl, Citrullus lanatus; Hv, Hordeum vulgare, Zm, Zea mays; Os, Oryza sativa; At, Arabidopsis thaliana. (This figure is available in colour at JXB online.)

Fig. 2.

Expression analysis of CsHAN1 in cucumber. (A) Quantitative RT–PCR (qRT–PCR) analysis of CsHAN1 in different tissues of cucumber. YL, young leaves; FB, female buds; MB, male buds; FF, female flowers; MF, male flowers; F-4, young fruits 4 d before anthesis; F, fruit at anthesis; F+3, fruits 3 d after anthesis. The Ubiquitin extension protein (UBI-ep) gene was used as an internal reference to normalize the expression data. (B–K) In situ hybridization with the CsHAN1 antisense probe (B–J) and sense probe (K). (B) In the ucumber shoot apex, CsHAN1 is expressed in the junction region of the inflorescence meristem (IM) and stem (arrow), the junction regions of the floral meristem (FM) and stem (asterisk), and the axil of leaf primordia (arrowhead). (C–E) Floral buds at stage 2 (C), 3 (D), and 4 (E). Asterisks show the expression domain of CsHAN1 at the junction of the meristem and stem, and arrows indicate the expression of CsHAN1 at the boundary between the petal and stamen, and the boundary between the stamen and initiating carpel primordia. (F–G’) Male flowers at stage 9 (F) and stage 11 (G); (G’) is a high magnification view of the anther in (G). The signal of CsHAN1 was detected in the developing anther, tapetum cell layer, and the uninuclear pollen. (H) Female flower in stage 8. CsHAN1 is expressed in the ovary (arrow). (I, J) Cross-sections of the female ovary in stage 9 (I) and stage 10 (J) showing the expression domain of CsHAN1 in the ovules and the base of the embryo sac. (K) No signal was found on hybridization with the sense CsHAN1 probe. S, sepal; P, petal; St, stamen; C, carpel; Ov, ovule; In, integument; Es, embryo sac. Bar=100 μm. (This figure is available in colour at JXB online.)

Fig. 3.

Ectopic expression of CsHAN1 in han-2 mutant and wild-type Arabidopsis. (A–E) The inflorescences of Col (A), han-2(Col) (B), 35S:CsHAN1/han-2(Col) line 5 (C), 35S:CsHAN1/han-2(Col) line 2 (D), and 35S:CsHAN1/han-2(Col) line 6 (E). The arrows indicate the flowers with 1–2 petals, and the arrowheads show the flowers with 3–4 petals. (F) The siliques of WT, 35S:CsHAN1/han-2(Col) line 5, 35S:CsHAN1/han-2(Col) line 2, 35S:CsHAN1/han-2(Col) line 6, and the han-2 mutant at the same developmental stage (from bottom to top). (G–I) Rosette leaves of Col (G), han-2(Col) (H), and 35S:CsHAN1/han-2(Col) line 2 (I) show the partially rescued leaf shape. (J) qRT–PCR analyses of CsHAN1 in the three transgenic lines in the han-2(Col) background. Arabidopsis ACTIN2 was used as an internal standard to normalize the templates. (K–N) The whole plants (K), rosette leaves (L), fruits (M), and flowers (N) of Col (left) and the CsHAN1 overexpression line (right) in the Col background. Sepals and petals were removed in (N), and the white arrowhead shows the retarded stamen in the overexpression line. Bar=1mm. (This figure is available in colour at JXB online.)

Fig. 4.

Phenotypes of transgenic cucumber. (A) qRT–PCR analyses of CsHAN1 expression in transgenic overexpression and RNAi lines in cucumber. (B) Plant phenotypes of a transgenic CsHAN1 overexpression line (left), the wild type (middle), and a CsHAN1-RNAi line (right) which are 50 days old. Bar=5cm. (This figure is available in colour at JXB online.)

Fig. 5.

CsHAN1 is required for shoot apical meristem development. (A–D) Embryo phenotypes in the normal torpedo stage of the wild type (A), the heart stage in the CsHAN1-RNAi line 49 (B), the retarded torpedo embryo in the CsHAN1-RNAi line 49 (C), and in the CsHAN1-RNAi line 90 (D) at 16 d after fertilization. Bar=50 μm. (E) Phenotypes of wild-type (left), CsHAN1-RNAi line 49 (middle), and CsHAN1-RNAi line 90 (right) seeds at 36h after germination. Bar=1cm. (F) Seed morphology in the WT and CsHAN1-RNAi line 49. Bar=1cm. (G) qRT–PCR analyses of CsWUS, CsSTM, and CsBP in the shoot apexes of the wild type and the CsHAN1-RNAi line. The UBI-ep gene was used as an internal reference to normalize the expression data. (H–M) The expression of CsWUS (H, I), CsSTM (J, K), and CsBP (L, M) in the wild type (H, J, L) and CsHAN1-RNAi line 49 (I, K, M) in the apex of 6-day-old seedlings as detected by in situ hybridization. Bar=50μm. (This figure is available in colour at JXB online.)

Fig. 6.

CsHAN1 regulates leaf shape development. (A–F) Transgenic cucumber plants (A–C) and representative leaves (D–F) of a CsHAN1 overexpression line (A, D), the wild type (B, E), and a CsHAN1-RNAi line (C, F) which are 20 days old. Arrows showed the notches. Bar=1cm. (G) Diagrammatic data show the position of lobed leaves in the WT and CsHAN1 transgenic lines. Each column represents an individual plant, and each rectangle represents a node. (H) qRT–PCR analyses of leaf developmental genes in CsHAN1 overexpression and RNAi lines. The cucumber UBI-ep gene was used as an internal reference to normalize the expression data, and the experiments were repeated in triplicate independent samples. Error bars represent the SE. (This figure is available in colour at JXB online.)