The circadian clock control of CONSTANS (CO) transcription and the light regulation of CO stability coordinately regulate photoperiodic flowering by triggering rhythmic expression of the floral integrator FLOWERING LOCUS T (FT). The diurnal pattern of CO accumulation is modulated sequentially by distinct E3 ubiquitin ligases, such as HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) in the morning, FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) in late afternoon, and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) at night. In particular, CO is stabilized by FKF1 in late afternoon only under long days. Here, we show that CO abundance is not simply regulated by the E3 enzymes in a passive manner but also self-regulated actively through dynamic interactions between two CO isoforms. CO alternative splicing produces two protein variants, the full-size COa and the C-terminally truncated COb. Notably, COb, which is resistant to the E3 enzymes, induces the interactions of COa with CO-destabilizing HOS1 and COP1 but inhibits the association of COa with CO-stabilizing FKF1. These observations demonstrate that CO plays an active role in sustaining its diurnal accumulation dynamics in Arabidopsis photoperiodic flowering.

Targeted gene silencing is an essential component of plant biotechnology. RNA interference is often employed for targeted gene silencing in plants. However, it suffers from off-target effects and unstable gene suppression in many cases. In recent years, engineered nuclease-based tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have been developed to induce site-specific genome modifications. However, these approaches require much time and labor for extensive screening of mutants. We have recently reported that the activities of dimeric transcription factors are competently suppressed by genome-encoded small interfering peptides (siPEPs) that competitively form nonfunctional heterodimers in plants. In addition, some splice variants of transcription factors also act in a similar manner to negatively regulate the activities of specific transcription factors. We designated the siPEP-mediated suppression of transcription factors peptide interference (PEPi). Based on our previous observations, we also developed an artificial siPEP (a-siPEP) approach and evaluated its application for the targeted inactivation of transcription factors in the dicot model, Arabidopsis, and monocot model, Brachypodium. We designed a series of potential a-siPEPs of two representative transcription factors SUPPRESSOR OF OVEREXPRESSOR OF CONSTANS 1 (SOC1) and AGAMOUS (AG) that function in flowering induction and floral organogenesis, respectively. Transgenic plants overproducing a-siPEPs displayed phenotypes comparable to those of gene-deficient mutants. Collectively, our data demonstrate that the siPEP tool is an efficient protein knockout system for inactivating specific transcription factors, and other multimeric enzymes and membrane transporters as well, in plants. We will discuss about the global application of the siPEP toll to other plant species and potential advantages over other gene manipulation tools.

Plant breeding is a multidisciplinary science of changing the genetic makeup of plants in order to generate desired traits or characteristics, and thus it can be accomplished through many different techniques ranging from simply selecting plants with desirable traits for propagation to more complex molecular techniques. Both conventional and genetically modified (GM) plant breeding alter or modify the genes of a plant so that a better variety is developed. Breeding using GM tools is achieved for the same reasons as conventional breeding. One prominent distinction is that instead of randomly mixing genes in conventional breeding, which occurs as a result of a sexual cross, a specific gene is directly transferred or selectively inactivated in the new plant variety through GM plant breeding. Site-specific mutagenesis and selection of gene knockout mutants are readily carried out in model plant species, such as Arabidopsis. However, targeted mutation of a specific gene is technically impractical, if not impossible, in most cases. As an alternative approach, RNA interference (RNAi), which is mediated by small interfering RNA (siRNA) and microRNA (miRNA), is routinely employed for targeted silencing of genes in academic and biotechnological studies. Recently, engineered nuclease-based genome editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have been developed to induce site-specific genome modifications in both animals and plants. ZFNs are chimeric DNA restriction enzymes that consist of the nuclease domain of the Fok1 restriction enzyme, which triggers double strand breaks in genomic DNAs, and a custom-designed ZF DNA-binding domain, which guides the ZFNs to specific sequences within genomic DNAs. The double-strand breaks are rejoined by cellular DNA repair machinery, resulting in targeted mutagenesis or targeted gene replacement. In this work, we employed the ZFN tool to specifically inactivate two flowering genes, such as FCA and GI that also mediate high temperature responses and clock output signaling, respectively, in a bioenergy grass crop, Brachypodium distachyon. We designed extensive sets of ZF recognition sequences that recognize target sequences within the FCA and GI genes. The potential ZFN cassettes were transformed into Brachypodium ecotype Bd21-3. The transformants will be screened to identify those carrying targeted gene mutations. We will also discuss the extension of the ZFN tool to other plant species, including crops.