Post-transcriptional control is nowadays considered a main checking point for correct gene regulation during development, and RNA binding proteins actively participate in this process. Arabidopsis thaliana FLOWERING LOCUS WITH KH DOMAINS (FLK) and PEPPER (PEP) genes encode RNA-binding proteins that contain three K-homology (KH)-domain, the typical configuration of Poly(C)-binding ribonucleoproteins (PCBPs). We previously demonstrated that FLK and PEP interact to regulate FLOWERING LOCUS C (FLC), a central repressor of flowering time. Now we show that FLK and PEP also play an important role in the maintenance of the C-function during floral organ identity by post-transcriptionally regulating the MADS-box floral homeotic gene AGAMOUS (AG). Previous studies have indicated that the KH-domain containing protein HEN4, in concert with the CCCH-type RNA binding protein HUA1 and the RPR-type protein HUA2, facilitates maturation of the AG pre-mRNA. In this report we show that FLK and PEP genetically interact with HEN4, HUA1, and HUA2, and that the FLK and PEP proteins physically associate with HUA1 and HEN4. Taken together, these data suggest that HUA1, HEN4, PEP and FLK are components of the same post-transcriptional regulatory module that ensures normal processing of the AG pre-mRNA. Our data better delineates the roles of PEP in plant development and, for the first time, links FLK to a morphogenetic process.
Plant floral transition is a major developmental switch regulated by an integrated network of pathways. Arabidopsis FLOWERING LOCUS K (FLK), a protein with three KH RNA-binding domains, operates in the autonomous flowering-promotive pathway by decreasing the transcript levels of the key flowering repressor FLOWERING LOCUS C (FLC). Here we report that PEPPER (PEP), an FLK paralog previously shown to affect vegetative and pistil development, antagonizes FLK by positively regulating FLC. Lack of PEP function rescues the flk late-flowering phenotype with a concomitant decrease in FLC RNA levels. Loss of HUA2, another FLC activator encoding an RNA-binding protein, further rescues flk, being flk hua2 pep triple mutants virtually wild-type regarding flowering time. Consistently, PEP overexpression determines high levels of FLC transcripts and flowering delay. Genetic and molecular analyses indicate that FLK and PEP act independently of FCA, another important FLC repressor in the autonomous pathway. In addition, we present data suggesting that PEP may affect FLC expression at both transcriptional and post-transcriptional levels. Overall, our results uncover PEP as a new factor for FLC upregulation, underscoring the importance of RNA-binding activities during developmental timing of flowering.
SummaryPochonia chlamydosporia has been intensively studied in nematode control of different crops. We have investigated the interaction between P. chlamydosporia and the model system Arabidopsis thaliana under laboratory conditions in the absence of nematodes.This study demonstrates that P. chlamydosporia colonizes A. thaliana. Root colonization monitored with green fluorescent protein-tagged P. chlamydosporia and quantitative PCR (qPCR) quantitation methods revealed root cell invasion. Fungal inoculation reduced flowering time and stimulated plant growth, as determined by total FW increase, faster development of inflorescences and siliques, and a higher yield in terms of seed production per plant.Precocious flowering was associated with significant expression changes in key floweringtime genes. In addition, we also provided molecular and genetic evidence that point towards jasmonate signaling as an important factor to modulate progression of plant colonization by the fungus.Our results indicate that P. chlamydosporia provides benefits to the plant in addition to its nematophagous activity. This report highlights the potential of P. chlamydosporia to improve yield in economically important crops.
Ovules are fundamental for plant reproduction and crop yield as they are the precursors of seeds. Therefore, ovule specification is a critical developmental program. In Arabidopsis thaliana, ovule identity is redundantly conferred by the homeotic D-class genes SHATTERPROOF1 (SHP1), SHP2 and SEEDSTICK (STK), phylogenetically related to the MADS-domain regulatory gene AGAMOUS (AG), essential in floral organ specification. Previous studies have shown that the HUA-PEP activity, comprised of a suite of RNA-binding protein (RBP) encoding genes, regulates AG pre-mRNA processing and thus flower patterning and organ identity. Here, we report that the HUA-PEP activity additionally governs ovule morphogenesis. Accordingly, in severe hua-pep backgrounds ovules transform into flower organ-like structures. These homeotic transformations are most likely due to the dramatic reduction in SHP1, SHP2 and STK activity. Our molecular and genome-wide profiling strategies revealed the accumulation of prematurely terminated transcripts of D-class genes in hua-pep mutants and reduced amounts of their respective functional messengers, which points to pre-mRNA processing misregulation as the origin of the ovule developmental defects in such backgrounds. RNA processing and transcription are coordinated by the RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD). Our results show that HUA-PEP activity members can interact with the CTD regulator C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 (CPL1), supporting a co-transcriptional mode of action for the HUA-PEP activity. Our findings expand the portfolio of reproductive developmental programs in which HUA-PEP activity participates, and further substantiates the importance of RNA regulatory mechanisms (pre-mRNA co-transcriptional regulation) for correct gene expression during plant morphogenesis.
The adaptive success of flowering plants is largely due to their ability to align floral production with optimal conditions. In Arabidopsis thaliana, MADS-box repressors of the FLC/MAF-clade prevent flowering under non-inductive conditions, although the role of some members is not yet clearly defined. Using a genetic strategy, we identified the KH-domain gene HEN4, previously shown to be involved in MADS-box floral homeotic gene regulation, as a modulator of flowering time. Loss-of-function hen4 mutants are early-flowering, and their response to low growth-temperature (16 °C) and day-length is altered. Interestingly, hen4 plants showed dramatic reduction of FLC and MAF4 transcripts, whereas other flowering repressors of the same clade (FLM, MAF2, MAF3, MAF5) remained unaltered. We also determined that hen4, partly due to loss of FLC, accelerates the vegetative phase-change. This report provides insight into flowering time control and highlights the potential of versatile regulators such as HEN4 to coordinate the juvenile-to-adult transition and floral timing.
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