Seed germination is a key developmental transition that initiates the plant life cycle. The timing of germination is determined by coordinated action of two phytohormones, gibberellin (GA) and abscisic acid (ABA). In particular, ABA plays a key role in integrating environmental information and inhibiting the germination process. Utilization of embryonic lipid reserves contributes to seed germination by acting as an energy source, and ABA suppresses lipid degradation to modulate the germination process. Here, we report that the ABA-responsive R2R3-type MYB transcription factor MYB96, which is highly expressed in embryo, regulates seed germination by controlling the expression of ABA-INSENSITIVE 4 (ABI4). In the presence of ABA, germination was accelerated in MYB96-deficient myb96-1 seeds, whereas the process was significantly delayed in MYB96-overexpressing activation-tagging myb96-ox seeds. Consistently, myb96-1 seeds degraded a larger extent of lipid reserves even in the presence of ABA, while reduced lipid mobilization was observed in myb96-ox seeds. MYB96 directly regulates ABI4, which acts as a repressor of lipid breakdown, to define its spatial and temporal expression. Genetic analysis further demonstrated that ABI4 is epistatic to MYB96 in the control of seed germination. Taken together, the MYB96-ABI4 module regulates lipid mobilization specifically in the embryo to ensure proper seed germination under suboptimal conditions.

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.

Floral transition is influenced by environmental factors such as light and temperature. Plants are capable of integrating photoperiod and ambient temperature signaling into their developmental program. Despite extensive investigations on individual genetic pathways, little is known about the molecular components that integrate both pathways. Here, we demonstrate that the RING finger–containing E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) acts as an integrator of photoperiod and ambient temperature signaling. In addition to the role in photoperiodic destabilization of CONSTANS (CO), COP1 also regulates temperature sensitivity by controlling the degradation of GIGANTEA (GI). COP1-impaired mutants showed reduced sensitivity to low ambient temperature. Notably, COP1 is more stabilized at low temperature and accelerates GI turnover in a 26S proteasome-dependent manner. The direct association of GI with the promoter of FLOWERING LOCUS T (FT) depends on ambient temperature, and thus COP1-triggered GI turnover delays flowering at low temperatures via a CO-independent pathway. Taken together, our findings indicate that environmental conditions regulate the stability of COP1, and conditional specificity of its target selection stimulates proper developmental responses and ensures reproductive success.

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.