Sara Simonini | PubMed Health

PubMed2025

Chromatin Immunoprecipitation for Standard, Rare, or Weakly Binding Proteins.

Various proteins interact with specific genome regions, playing crucial roles in gene regulation. Chromatin Immunoprecipitation (ChIP) is the most commonly used method to study protein-DNA interactions in vivo. By combining ChIP with high-throughput sequencing, ChIP-seq allows for studying the genome-wide localization of proteins. Although several ChIP protocols are available for plant tissues, they are primarily designed for histone modifications and abundant proteins with high DNA-binding affinity, which are considered as the "standard targets." Here we describe a ChIP protocol for plant tissues not only optimized for the standard targets but also adapted for proteins with low abundances or weak DNA-binding ability. Successful execution of the protocol enables reliable generation of DNA templates for quantitative PCR or libraries for next-generation sequencing, which makes it an effective tool for analyzing genomic interactions of a wide range of proteins.

Chromatin Immunoprecipitation↗

PubMed2025

Functional genetics of rice PISTILLATA genes reveals new roles and target genes in flowering time, female fertility, and parthenocarpy.

Floral organ identity is controlled largely by the combinatorial action of MADS domain homeotic transcription factors. Lodicules are specialized plant organs in cereals and grasses that are involved in floret opening and facilitate pollination and fertility in rice (Oryza sativa L.). To understand the mechanisms underlying the specification of the rice lodicule, we investigated the developmental functions of the rice PISTILLATA (PI) paralogs, OsMADS2, and OsMADS4. Null osmads2 mutants reiterated OsMADS2 nonredundant lodicule specification roles and revealed new roles in flowering time and floral organ number and fate. Doubly perturbed osmads2 osmads4kd florets had severe abnormalities, were female infertile, yet could initiate parthenocarpy. Ubiquitous OsMADS4 overexpression rescued osmads2 abnormalities. We also utilized genome-wide binding analyses and transcriptome profiling to identify putative target genes contributing to OsMADS2 functions. In osmads2d8/d8 null mutant, we observed deregulated genes in a plethora of processes including lodicule and stamen development, floral organ number, and cell wall development. Some examples are cell division regulators (Cyclin D6, Cyclin-P4-1-like), an aquaporin (PIP1A), a peptide transporter, a vascular developmental regulator (HOX1), and a cell wall modulator (GH9B16). The deregulation of these genes may be associated with the disrupted cell division, tissue differentiation, and physiology of the malformed lodicules in osmads2 and osmads2 osmads4kd florets. Altogether, we reveal novel roles for the rice PI paralogs in flowering time, panicle exsertion, and embryo sac differentiation, identify gene targets for lodicule development, and provide mechanistic insights on the functional diversification of rice PI paralogs.

Oryza↗