Class I BASIC PENTACYSTEINE factors regulate HOMEOBOX genes involved in meristem size maintenance

Abstract

The BASIC PENTACYSTEINE (BCP) family is a poorly characterized plant transcription factor family of GAGA BINDING PROTEINS. In Arabidopsis, there are seven members (BPC1-7) that are broadly expressed, and they can potentially bind more than 3000 Arabidopsis GAGA-repeat-containing genes. To date, BPCs are known to be direct regulators of the INNER NO OUTER (INO), SEEDSTICK (STK), and LEAFY COTYLEDON 2 (LEC2) genes. Because of the high functional redundancy, neither single knockout nor double bpc mutant combinations cause aberrant phenotypes. The bpc1-2 bpc2 bpc3 triple mutant shows several pleiotropic developmental defects, including enlargement of the inflorescence meristem and flowers with supernumerary floral organs. Here, we demonstrated through expression analysis and chromatin immunoprecipitation assays that this phenotype is probably due to deregulation of the expression of the SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS/KNAT1 (BP) genes, which are both direct targets of BPCs. Moreover, we assigned a role to BPCs in the fine regulation of the cytokinin content in the meristem, as both ISOPENTENYLTRANSFERASE 7 (IPT7) and ARABIDOPSIS RESPONSE REGULATOR 7 (ARR7) genes were shown to be overexpressed in the bpc1-2 bpc2 bpc3 triple mutant.

Keywords: Arabidopsis; BPC; HOMEOBOX; cytokinin; meristem..

Fig. 1.

The bpc1-2 bpc2 bpc3 mutant has enlarged IMs. (A) SEM of a wild-type inflorescence apex. Asterisks indicate developing FMs. (B) SEM of wild-type flower. One sepal has been removed. (C) A bpc1-2 bpc2 bpc3 inflorescence (right) compared a wild-type inflorescence (left). (D) SEM of a bpc1-2 bpc2 bpc3 inflorescence apex showing the IM with several developing floral primordia (arrow). Young floral buds presented fused extra sepals (arrowhead). Asterisks indicate developing FMs. (E) Frequency of divergence angle of siliques in wild-type plants: the majority of the angles fell in the 120–150° class. (F) Frequency of divergence angle of siliques in bpc1-2 bpc2 bpc3 mutant plants: the siliques were randomly distributed along the stem. (G) A bpc1-2 bpc2 bpc3 mutant flower displaying eight stamens (white numbers) and five petals (black numbers). (H) SEM of a bpc1-2 bpc2 bpc3 mutant flower. One sepal has been removed to reveal a petaloid extra stamen developing from the second whorl (arrowhead). (I) SEM of a bpc1-2 bpc2 bpc3 pistil with a third fused carpel (arrowhead). (J) DAPI staining of a longitudinal section of a wild-type inflorescence. The arrowhead indicates the IM. (K) DAPI staining of a longitudinal section of a bpc1-2 bpc2 bpc3 inflorescence. Compare the IM (arrowhead) size with the wild-type one in (J). (L, M) mPS-PI staining of a wild-type (L) and bpc1-2 bpc2 bpc3 (M) inflorescence apex in transversal (upper panel) and longitudinal (lower panel) sections. Note the increased meristem dimension in the bpc1-2 bpc2 bpc3 triple mutant. (N, O) Magnification of mPS-PI staining of the L1 and L2 layers of an inflorescence apex of a wild-type (N) and bpc1-2 bpc2 bpc3 triple mutant (O): the cells of the bpc1-2 bpc2 bpc3 mutant were slightly bigger than those of the wild type. (P) Expression of pCLV3::GUS in the BPC1/bpc1-2 bpc2 bpc3 background (plant does not present the bpc1-2 bpc2 bpc3 phenotype). The arrowhead indicates the IM. (Q) Expression of pCLV3::GUS in the bpc1-2 bpc2 bpc3 background. Note the increase of GUS expression at the IM (arrowhead) compared with that in (L). (R) In situ hybridization with a WUS-specific antisense (as) probe in wild-type IMs (arrowhead) and FMs. (S) In situ hybridization with WUS specific antisense (as) probe in the bpc1-2 bpc2 bpc3 IM (arrowhead) and FMs. Ca, carpel; Pe, petal; Pi, pistil; PS, petaloid stamen; Se, sepal; St, stamen. Bars, 100 µm. (This figure is available in colour at JXB online.)

Fig. 2.

Expression of the BPC1–EAR chimeric protein causes strong developmental defects. (A) A 35S::BPC1–EAR plant with wild-type phenotype. (B) A 35S::BPC1–EAR plant with a severe phenotype. Leaves are small and curled (arrowhead). (C) Leaf of a 35S::BPC1–EAR plants with both abaxial and adaxial sides covered by trichomes. (D) Inflorescence of a 35S::BPC1–EAR plant with a severe phenotype. Flowers displayed petaloid sepals (arrowhead). (E) A pALC::BPC1–EAR plant after 6 d of ethanol induction in which the IM arrested prematurely (arrowhead). (F) A flower of a pALC::BPC1–EAR plant after 4 d of ethanol induction with petaloid sepals (arrowhead), no petals, and short and aberrant stamens. Pi, pistil; Se, sepal; St, stamen; Ps, petaloid sepals; Up, upper side; Lo, lower side. (This figure is available in colour at JXB online.)

Fig. 3.

STM and BP are direct targets of class I BPCs. (A) qRT-PCR of the wild type and bpc1-2 bpc2 bpc3 triple mutant to determine STM and BP expression levels. (B–E) In situ hybridization using an STM-specific antisense probe of transversal sections of (B, D) and wild-type (C, E) bpc1-2 bpc2 bpc3 inflorescences. The signal was more intense and expanded in the meristems of the mutant (arrowhead in B). In the wild-type flower, the signal was localized in the replum and at the base of the flower, whereas in the triple mutant it was also in the petal and ovules (arrowheads in E). (F, G) GUS staining of BPC1/bpc1-2 bpc2 bpc3 (F) and bpc1-2 bpc2 bpc3 (G) inflorescences of plants containing the pBP::GUS construct. In the homozygous triple mutant, the signal was expanded and more persistent in both the pedicels and stems (arrowhead in G). (H) ChIP analysis revealing that BPCs of class I directly bind the STM and BP promoter; the bpc1-2 bpc2 bpc3 was used as a negative control. Each bar shows the average of three independent ChIP experiments (±standard deviation). Pe, petals; Se, sepals; St, stamen; Re, replum; Ov, ovules; Sm, stem; Pd, pedicel. Bars, 50 µm. (This figure is available in colour at JXB online.)

Fig. 4.

HOMEOBOX and cytokinin pathway genes are regulated by class I BPCs. (A) ChIP analysis revealing that class I BPCs directly bind the GA-rich sites in the promoter of different HOMEOBOX transcription factors; the bpc1-2 bpc2 bpc3 triple mutant was used as a negative control. Each bar shows the average of three independent ChIP experiments (±standard deviation). (B) Expression analyses of IPT7 in wild-type and bpc1-2 bpc2 bpc3 young inflorescences. (C, D) In situ hybridization with ARR7-specific antisense probe using wild-type (C) and bpc1-2 bpc2 bpc3 (D) inflorescences. A stronger and more diffuse signal was detectable in the mutant IM (arrowhead), FM, pedicels, and stem (arrow). (E) ChIP analysis revealing that class I BPCs directly bind the GA-rich site in the ARR7 promoter; the bpc1-2 bpc2 bpc3 triple mutant was used as a negative control. Each bar shows the average of three independent ChIP experiments (±standard deviation). Pd, pedicel; Sm, stem. Bars, 50 µm. (This figure is available in colour at JXB online.)