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. 2015 Oct;27(10):2880-93.
doi: 10.1105/tpc.15.00370. Epub 2015 Oct 13.

Arabidopsis CBP1 Is a Novel Regulator of Transcription Initiation in Central Cell-Mediated Pollen Tube Guidance

Hong-Ju Li  1 , Shan-Shan Zhu  1 , Meng-Xia Zhang  2 , Tong Wang  2 , Liang Liang  2 , Yong Xue  2 , Dong-Qiao Shi  1 , Jie Liu  1 , Wei-Cai Yang  3
Affiliations

Arabidopsis CBP1 Is a Novel Regulator of Transcription Initiation in Central Cell-Mediated Pollen Tube Guidance

Hong-Ju Li et al. Plant Cell. 2015 Oct.

Abstract

In flowering plants, sperm cells are delivered to the embryo sac by a pollen tube guided by female signals. Both the gametic and synergid cells contribute to pollen tube attraction. Synergids secrete peptide signals that lure the tube, while the role of the gametic cells is unknown. Previously, we showed that CENTRAL CELL GUIDANCE (CCG) is essential for pollen tube attraction in Arabidopsis thaliana, but the molecular mechanism is unclear. Here, we identified CCG BINDING PROTEIN1 (CBP1) and demonstrated that it interacts with CCG, Mediator subunits, RNA polymerase II (Pol II), and central cell-specific AGAMOUS-like transcription factors. In addition, CCG interacts with TATA-box Binding Protein 1 and Pol II as a TFIIB-like transcription factor. CBP1-knockdown ovules are defective in pollen tube attraction. Expression profiling revealed that cysteine-rich peptide (CRP) transcripts were downregulated in ccg ovules. CCG and CBP1 coregulate a subset of CRPs in the central cell and the synergids, including the attractant LURE1. CBP1 is extensively expressed in multiple vegetative tissues and specifically in the central cell in reproductive growth. We propose that CBP1, via interaction with CCG and the Mediator complex, connects transcription factors and the Pol II machinery to regulate pollen tube attraction.

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Figures

Figure 1.
Figure 1.
CBP1 Is a CCG Binding Protein. (A) Protein structure of CCG. aa, amino acids. (B) Protein structure of CBP1.1 and CBP1.2. (C) In vitro binding assay with the indicated recombinant proteins showed direct interaction between CBP1 and CCG. Arrow indicates the target protein. (D) Immunoblotting (IB) of GFP immunoprecipitates from lysates of protoplasts transiently transformed with CBP1-GFP and CCG-Flag.
Figure 2.
Figure 2.
Expression Pattern and Subcellular Localization of CBP1. (A) to (C) The genomic fusion of CBP1-GUS under the native promoter showed expression in the seedling, leaf, and inflorescence. (D) to (G) Confocal laser scanning microscopy images of the ovules from ProCBP1:CBP1-3×GFP plants. (D) and (E) No GFP fluorescence was detected in the immature ovules. (F) and (G) The fusion protein CBP1-3×GFP is localized in the nucleus and cytoplasm of the central cell. cc, central cell, s, synergids. (H) to (K) Confocal laser scanning microscopy images showing the nuclear and cytoplasmic localization of CBP1 in the root of Pro35S:GFP-CBP1.1 transgenic plants (H). (I) Fluorescence of DNA by 4′,6-diamidino-2-phenylindole staining of the nucleus. (J) Bright-field image. (K) Merged image. Bars = 20 µm.
Figure 3.
Figure 3.
Interaction of CCG and CBP1 with Components of the Transcription Preinitiation Complex. (A) CCG interacted with TBP1 in a yeast two-hybrid assay. (B) CCG interacted with TBP1 in a GST pull-down assay. (C) CCG interacted with TFIIF in a GST pull-down assay. (D) and (E) CCG and CBP1.1 interacted with the CTD of RNA POLYMERASE II LARGE SUBUNIT NRPB1 (NRPB1_CTD) in a GST pull-down assay. (F) CBP1.1 showed no direct interaction with TFII-B, -E, and -H and TBP1 in a yeast two-hybrid assay. Arrows indicate the target proteins.
Figure 4.
Figure 4.
CBP1 Interacts with MEDs, AGLs, and Itself. (A) CBP1.2 interacted with MED7a, MED7b, and MED9 in a yeast two-hybrid assay. (B) and (C) CBP1.1 and CBP1.2 interacted with Mediator subunits in plants. (D) and (E) CBP1.1 interacted with itself in a yeast two-hybrid assay (D) and in a GST pull-down assay (E). (F) CBP1.1 interacted with itself in planta. (G) Interaction of CBP1 with AGL transcription factors expressed in the central cell detected by a yeast two-hybrid assay. +, Positive interaction; –, no interaction. S, self-activation of the cells expressing the BD-fused AGLs. Arrow in (E) indicates the fusion protein.
Figure 5.
Figure 5.
The Knockdown Ds Insertion Mutant cbp1 Showing Pollen Tube Attraction Defect and Polytubey. (A) Schematic diagram of insertion of two reverse Ds elements in the 3′ untranslated region of CBP1. (B) Real-time quantitative RT-PCR showing knockdown of CBP1 transcript in the cbp1 ovules. Each expression level was normalized to that of eIF1α. The data are the means ± sd of three independent experiments. (C) The wild-type ovule attracted the wild-type pollen tube. (D) The cbp1 ovules show two types of attraction defect to the wild-type pollen tubes: type I, one pollen tube failed to enter the micropyle (white arrow), and the second entered the micropyle (red arrow on the top); type II, two pollen tubes entering the micropyle (red arrows at the bottom). (E) The average ratio (means ± sd) of two types of pollen tube attraction defects of cbp1. Asterisks in (C) and (D) denote the micropyle; asterisks in (B) and (E) denote statistically significant differences to the wild type (Student’s t test, **P < 0.01). Bars = 20 µm.
Figure 6.
Figure 6.
Knockdown of CBP1 by Artificial MicroRNA Causes Pollen Tube Attraction Defect. (A) to (E) Wild-type ovule attracted the pollen tube into the micropyle normally (A). About half of the ovules in amiRCBP1 plants were defective in pollen tube attraction ([B] to [E]). Red arrows indicate the normally attracted pollen tubes and white arrows indicate the misguided pollen tubes. Asterisks indicate the micropyle. Bars = 20 µm. (A) to (C) GUS activity staining of wild-type pollen tubes carrying LAT52:GUS grown for 24 h in the amiRCBP1 pistil. (D) and (E) Aniline blue staining of wild-type pollen tubes grown for 24 h in the pistil. (F) The percentage of ovules with pollen tube attraction defect in different transgenic lines (1# to 6#). (G) The relative expression level of CBP1 in the transgenic lines indicated in (F). 1# to 5# showed significantly severe guidance defect in the T1 generation; 6# with normal transcript level showed no obvious difference to the wild type (n = 230 ovules for each line). Data are presented as means ± sd for three replicates (Student’s t test, ***P < 0.001, **P < 0.01, and *P < 0.05).
Figure 7.
Figure 7.
Distribution and Classification of the Genes Downregulated in ccg. (A) Microarray analysis showing genes that are differentially expressed between ccg and wild-type ovules. Black box: genes primarily downregulated in ccg. (B) Gene Ontology classification of genes downregulated more than 3-fold in ccg ovules. Numbers on the diagram indicate the proportion of each Gene Ontology term. (C) Categories of CRPs downregulated more than 3-fold in ccg ovules.
Figure 8.
Figure 8.
Central Cell-Expressed CRPs Are Downregulated in ccg Ovules. (A) to (I) Ovules of the wild-type transgenic plants transformed with free GFP under the native promoter ([A] to [F]) and genomic fusion GFP ([G] to [I]). (A) ProDD22:GFP; (B) ProDD36:GFP; (C) ProDD66:GFP; (D) ProLCR24:GFP; (E) ProLCR59:GFP; (F) ProAt3g04540:GFP; (G) ProDD22:DD22-GFP; (H) ProDD36:DD36-GFP; (I) ProAt3g04540:At3g04540-GFP. Arrows: cell layers of the integument. Bars = 20 µm. (J) The ratio of ovules expressing CRP genes to the total number (n = 100 for each experiment) of ovules in the wild type and ccg/CCG mutant. The plants analyzed are homozygotes for the transgenes.
Figure 9.
Figure 9.
LURE1 and Synergid Cell-Expressed CRPs Are Downregulated in ccg and cbp1. (A) LURE1, CRP810.2, and ECA1 genes are downregulated in ccg/CCG. Real-time qRT-PCR was performed with cDNA synthesized from total RNA extracted from ccg ovules. (B) LURE1, CRP810.2, and ECA1 genes are downregulated in cbp1 ovules. Real-time qRT-PCR was performed with cDNA synthesized from total RNA extracted from pistils from cbp1 homozygotes. Data are presented as means ± sd for three replicates. Student’s t test, **P < 0.01, *P < 0.05.
Figure 10.
Figure 10.
Proposed Model for CCG-CBP1 Function in Mediator-Mediated Transcription Initiation in the Central Cell. CBP1 recruits CCG, Mediator subunits, and Pol II to AGL transcription factors to promote the transcription of target genes.
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