Dual-transgenic BiFC vector systems for protein-protein interaction analysis in plants.

Protein-protein interaction (PPI) play a pivotal role in cellular signal transduction. The bimolecular fluorescence complementation (BiFC) assay offers a rapid and intuitive means to ascertain the localization and interactions of target proteins within living cells. BiFC is based on fluorescence complementation by reconstitution of a functional fluorescent protein by co-expression of N- and C-terminal fragments of this protein. When fusion proteins interact, the N- and C-terminal fragments come into close proximity, leading to the reconstitution of the fluorescent protein. In the conventional approach, the N-terminal and C-terminal fragments of the fluorescent protein are typically expressed using two separate vectors, which largely relies on the efficiency of the transformation of the two vectors in the same cells. Furthermore, issues of vector incompatibility can often result in loss of one plasmid. To address these challenges, we have developed novel dual-transgenic BiFC vectors, designed as pDTQs, derived from the previously published pDT1 vector. This set of BiFC vectors offers the following advantages: 1) Both fluorescent fusion proteins are expressed sequentially within a single vector, enhancing expression efficiency; 2) Independent promoters and terminators regulate the expression of the two proteins potentially mitigating vector compatibility issues; 3) A long linker is inserted between the fluorescent protein fragment and the gene of interest, facilitating the recombination of the fused fluorescent protein into an active form; 4) Four distinct types of fluorescent proteins, namely, EYFP, mVenus, mRFP1Q66T and mCherry are available for BiFC analysis. We assessed the efficiency of the pDTQs system by investigating the oligomerization of Arabidopsis CRY2 and CRY2-BIC2 interactions in N. benthamiana. Notably, the pDTQs were found to be applicable in rice, underscoring their potential utility across various plant species.

ATP binding cassette transporters ABCG1 and ABCG16 affect reproductive development via auxin signalling in Arabidopsis.

Angiosperm reproductive development is a complex event that includes floral organ development, male and female gametophyte formation and interaction between the male and female reproductive organs for successful fertilization. Previous studies have revealed the redundant function of ATP binding cassette subfamily G (ABCG) transporters ABCG1 and ABCG16 in pollen development, but whether they are involved in other reproductive processes is unknown. Here we show that ABCG1 and ABCG16 were not only expressed in anthers and stamen filaments but also enriched in pistil tissues, including the stigma, style, transmitting tract and ovule. We further demonstrated that pistil-expressed ABCG1 and ABCG16 promoted rapid pollen tube growth through their effects on auxin distribution and auxin flow in the pistil. Moreover, disrupted auxin homeostasis in stamen filaments was associated with defective filament elongation. Our work reveals the key functions of ABCG1 and ABCG16 in reproductive development and provides clues for identifying ABCG1 and ABCG16 substrates in Arabidopsis.

KLU suppresses megasporocyte cell fate through SWR1-mediated activation of WRKY28 expression in Arabidopsis.

Germ-line specification is essential for sexual reproduction. In the ovules of most flowering plants, only a single hypodermal cell enlarges and differentiates into a megaspore mother cell (MMC), the founder cell of the female germ-line lineage. The molecular mechanisms restricting MMC specification to a single cell remain elusive. We show that the Arabidopsis transcription factor WRKY28 is exclusively expressed in hypodermal somatic cells surrounding the MMC and is required to repress these cells from acquiring MMC-like cell identity. In this process, the SWR1 chromatin remodeling complex mediates the incorporation of the histone variant H2A.Z at the WRKY28 locus. Moreover, the cytochrome P450 gene KLU, expressed in inner integument primordia, non-cell-autonomously promotes WRKY28 expression through H2A.Z deposition at WRKY28. Taken together, our findings show how somatic cells in ovule primordia cooperatively use chromatin remodeling to restrict germ-line cell specification to a single cell.

The THO Complex Non-Cell-Autonomously Represses Female Germline Specification through the TAS3-ARF3 Module.

In most sexually reproducing plants, a single somatic, sub-epidermal cell in an ovule is selected to differentiate into a megaspore mother cell, which is committed to giving rise to the female germline. However, it remains unclear how intercellular signaling among somatic cells results in only one cell in the sub-epidermal layer differentiating into the megaspore mother cell. Here we uncovered a role of the THO complex in restricting the megaspore mother cell fate to a single cell. Mutations in TEX1, HPR1, and THO6, components of the THO/TREX complex, led to the formation of multiple megaspore mother cells, which were able to initiate gametogenesis. We demonstrated that TEX1 repressed the megaspore mother cell fate by promoting the biogenesis of TAS3-derived trans-acting small interfering RNA (ta-siRNA), which represses ARF3 expression. The TEX1 protein was present in epidermal cells, but not in the germline, and, through TAS3-derived ta-siRNA, restricted ARF3 expression to the medio domain of ovule primordia. Expansion of ARF3 expression into lateral epidermal cells in a TAS3 ta-siRNA-insensitive mutant led to the formation of supernumerary megaspore mother cells, suggesting that TEX1- and TAS3-mediated restriction of ARF3 expression limits excessive megaspore mother cell formation non-cell-autonomously. Our findings reveal the role of a small-RNA pathway in the regulation of female germline specification in Arabidopsis.

Genome-Wide Identification and Expression Profiling of ATP-Binding Cassette (ABC) Transporter Gene Family in Pineapple (Ananas comosus (L.) Merr.) Reveal the Role of AcABCG38 in Pollen Development.

Pineapple (Ananas comosus L.) cultivation commonly relies on asexual reproduction which is easily impeded by many factors in agriculture production. Sexual reproduction might be a novel approach to improve the pineapple planting. However, genes controlling pineapple sexual reproduction are still remain elusive. In different organisms a conserved superfamily proteins known as ATP binding cassette (ABC) participate in various biological processes. Whereas, till today the ABC gene family has not been identified in pineapple. Here 100 ABC genes were identified in the pineapple genome and grouped into eight subfamilies (5 ABCAs, 20 ABCBs, 16 ABCCs, 2 ABCDs, one ABCEs, 5 ABCFs, 42 ABCGs and 9 ABCIs). Gene expression profiling revealed the dynamic expression pattern of ABC gene family in various tissues and different developmental stages. AcABCA5, AcABCB6, AcABCC4, AcABCC7, AcABCC9, AcABCG26, AcABCG38 and AcABCG42 exhibited preferential expression in ovule and stamen. Over-expression of AcABCG38 in the Arabidopsis double mutant abcg1-2abcg16-2 partially restored its pollen abortion defects, indicating that AcABCG38 plays important roles in pollen development. Our study on ABC gene family in pineapple provides useful information for developing sexual pineapple plantation which could be utilized to improve pineapple agricultural production.