Heritable Virus-Induced Genome Editing (VIGE) in Nicotiana attenuata.

The CRISPR/Cas9 system is an extremely powerful tool for targeted mutagenesis in plants. However, plant genome editing relies on the labor-intensive plant regeneration method for generating gene-edited plants. To overcome this bottleneck, several virus-induced genome editing (VIGE) techniques have been developed. The VIGE system aims to induce targeted mutations in germ cells without plant regeneration. However, due to the delivery issues of a large Cas9 protein, scientists focus on developing a virus-mediated delivery system for guide RNA into Cas9-overproducing plants. Here, we describe how to induce heritable targeted mutations in a non-model plant, Nicotiana attenuata, using VIGE system. This method will be applied for manipulating the target genes in any plants that scientists are interested in.

High Ambient Temperature Accelerates Leaf Senescence via PHYTOCHROME-INTERACTING FACTOR 4 and 5 in Arabidopsis.

Leaf senescence is a developmental process by which a plant actively remobilizes nutrients from aged and photosynthetically inefficient leaves to young growing ones by disassembling organelles and degrading macromolecules. Senescence is accelerated by age and environmental stresses such as prolonged darkness. Phytochrome B (phyB) inhibits leaf senescence by inhibiting phytochrome-interacting factor 4 (PIF4) and PIF5 in prolonged darkness. However, it remains unknown whether phyB mediates the temperature signal that regulates leaf senescence. We found the light-activated form of phyB (Pfr) remains active at least four days after a transfer to darkness at 20°C but is inactivated more rapidly at 28°C. This faster inactivation of Pfr further increases PIF4 protein levels at the higher ambient temperature. In addition, PIF4 mRNA levels rise faster after the transfer to darkness at high ambient temperature via a mechanism that depends on ELF3 but not phyB. Increased PIF4 protein then binds to the ORE1 promoter and activates its expression together with ABA and ethylene signaling, accelerating leaf senescence at high ambient temperature. Our results support a role for the phy-PIF signaling module in integrating not only light signaling but also temperature signaling in the regulation of leaf senescence.

Association between pre-and postnatal growth and longitudinal trends in serum uric acid levels and blood pressure in children aged 3 to 7 years.

BACKGROUND: Uric acid has been identified as an important factor in the development of hypertension. If low birth weight (LBW) combined with catch-up growth (CUG) is associated with continuously elevated serum uric acid levels (SUA) level trajectories, LBW children who experience CUG may have an increased risk of hypertension later in life. Therefore, this cohort study analyzed longitudinal trends in SUA levels and changes in blood pressure in relation to pre- and postnatal growth over an extended follow-up period. METHODS: This prospective cohort study of 364 children from the Ewha Birth and Growth Cohort assessed the effects of pre- and postnatal growth status on SUA at 3, 5, and 7 years of age using a linear mixed model and the change in blood pressure over the 7-year follow-up period using a generalized linear model (analysis of covariance). CUG was defined as a change in weight (between birth and age 3) with a z-score > 0.67 for LBW subjects. The multivariate model considered sex, gestational age, and uric acid, height, and weight at 3 years of age. RESULTS: Children with LBW and CUG had higher SUA for the first 7 years of life compared to the normal birth weight group. This trend was particularly evident when comparing LBW children at term to children with normal birth weight. Within the group with LBW at term, children with greater CUG had higher SUA than children with normal birth weight, and this difference increased with age. Changes in the systolic blood pressure between 3 and 7 years of age were higher by 7.9 mmHg in children who experienced LBW and CUG compared with those who had a normal birth weight after adjusting for sex, gestational age, and height, weight, and uric acid at 3 years of age (p-value = 0.08). CONCLUSIONS: The uric acid levels and changes in systolic blood pressure were consistently higher among LBW children who experienced CUG compared with NBW children for the first 7 years of life. LBW children who experienced greater weight gain from birth to age 3 had even higher uric acid levels compared with NBW children.

PHYTOCHROME INTERACTING FACTOR8 Inhibits Phytochrome A-Mediated Far-Red Light Responses in Arabidopsis.

PHYTOCHROME INTERACTING FACTORs (PIFs) are a group of basic helix-loop-helix (bHLH) transcription factors that repress plant light responses. PIF8 is one of the less-characterized Arabidopsis (Arabidopsis thaliana) PIFs, whose putative orthologs are conserved in other plant species. PIF8 possesses a bHLH motif and an active phytochrome B motif but not an active phytochrome A motif. Consistent with this motif composition, PIF8 binds to G-box elements and interacts with the Pfr form of phyB but only very weakly, if at all, with that of phyA. PIF8 differs, however, from other PIFs in its protein accumulation pattern and functional roles in different light conditions. First, PIF8 inhibits phyA-induced seed germination, suppression of hypocotyl elongation, and randomization of hypocotyl growth orientation in far-red light, but it does not inhibit phyB-induced red light responses. Second, PIF8 protein accumulates more in far-red light than in darkness or red light. This is distinct from the pattern observed with PIF3, which accumulates more in darkness. This PIF8 accumulation pattern requires degradation of PIF8 by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in darkness, inhibition of COP1 by phyA in far-red light, and promotion of PIF8 degradation by phyB in red light. Together, our results indicate that PIF8 is a genuine PIF that represses phyA-mediated light responses.

Phytochrome B Requires PIF Degradation and Sequestration to Induce Light Responses across a Wide Range of Light Conditions.

Phytochrome B (phyB) inhibits the function of phytochrome-interacting factors (PIFs) by inducing their degradation and sequestration, but the relative physiological importance of these two phyB activities is unclear. In an analysis of published Arabidopsis thaliana phyB mutations, we identified a point mutation in the N-terminal half of phyB (phyBG111D) that abolishes its PIF sequestration activity without affecting its PIF degradation activity. We also identified a point mutation in the phyB C-terminal domain, which, when combined with a deletion of the C-terminal end (phyB990G767R), does the opposite; it blocks PIF degradation without affecting PIF sequestration. The resulting phyB proteins, phyB990G767R and phyBG111D, are equally capable of inducing light responses under continuous red light. However, phyBG111D, which exhibits only the PIF degradation activity, induces stronger light responses than phyB990G767R under white light with prolonged dark periods (i.e., diurnal cycles). In contrast, phyB990G767R, which exhibits only the PIF sequestration activity, induces stronger light responses in flickering light (a condition that mimics sunflecks). Together, our results indicate that both of these separable phyB activities are required for light responses in varying light conditions.

Epidermal Phytochrome B Inhibits Hypocotyl Negative Gravitropism Non-Cell-Autonomously.

Seedling hypocotyls display negative gravitropism in the dark but agravitropism in the light. The Arabidopsis thaliana pif quadruple mutant (pifQ), which lacks four PHYTOCHROME-INTERACTING FACTORS (PIFs), is agravitropic in the dark. Endodermis-specific expression of PIF1 rescues gravitropism in pifQ mutant seedlings. Since phytochromes induce light responses by inhibiting PIFs and the COP1-SPA ubiquitin E3 ligase complex in the nucleus, we asked whether phyB can cell autonomously inhibit hypocotyl negative gravitropism in the endodermis. We found that while epidermis-specific expression of PHYB rescues hypocotyl negative gravitropism and all other phyB mutant phenotypes, endodermis-specific expression of PHYB does not. Epidermal phyB induces the phosphorylation and degradation of endodermal PIFs in response to red light. This induces a global gene expression pattern similar to that induced by red light treatment of seedlings expressing PHYB under the control of its own endogenous promoter. Our results imply that epidermal phyB generates an unidentified mobile signal that travels to the endodermis where it promotes PIF degradation and inhibits hypocotyl negative gravitropism.

The Arabidopsis RING Domain Protein BOI Inhibits Flowering via CO-dependent and CO-independent Mechanisms.

BOTRYTIS SUSCEPTIBLE1 INTERACTOR (BOI) and its three homologs (BOIs) are RING domain-containing proteins that repress flowering. Here, we investigated how BOIs repress flowering. Genetic analysis of the boiQ quadruple mutant indicates that BOIs repress flowering mainly through FLOWERING LOCUS T (FT). BOIs repress the expression of FT by CONSTANS (CO)-dependent and -independent mechanisms: in the CO-dependent mechanism, BOIs bind to CO, inhibit the targeting of CO to the FT locus, and thus repress the expression of FT; in the CO-independent mechanism, BOIs target the FT locus via a mechanism that requires DELLAs but not CO. This dual repression of FT makes BOIs strong repressors of flowering in both CO-dependent and CO-independent pathways in Arabidopsis thaliana. Our finding that BOIs inhibit CO targeting further suggests that, in addition to modulating CO mRNA expression and CO protein stability, flowering regulation can also modulate the targeting of CO to FT.

DELLA proteins and their interacting RING Finger proteins repress gibberellin responses by binding to the promoters of a subset of gibberellin-responsive genes in Arabidopsis.

DELLA proteins, consisting of GA INSENSITIVE, REPRESSOR OF GA1-3, RGA-LIKE1 (RGL1), RGL2, and RGL3, are central repressors of gibberellin (GA) responses, but their molecular functions are not fully understood. We isolated four DELLA-interacting RING domain proteins, previously designated as BOTRYTIS SUSCEPTIBLE1 INTERACTOR (BOI), BOI-RELATED GENE1 (BRG1), BRG2, and BRG3 (collectively referred to as BOIs). Single mutants of each BOI gene failed to significantly alter GA responses, but the boi quadruple mutant (boiQ) showed a higher seed germination frequency in the presence of paclobutrazol, precocious juvenile-to-adult phase transition, and early flowering, all of which are consistent with enhanced GA signaling. By contrast, BOI overexpression lines displayed phenotypes consistent with reduced GA signaling. Analysis of a gai-1 boiQ pentuple mutant further indicated that the GAI protein requires BOIs to inhibit a subset of GA responses. At the molecular level, BOIs did not significantly alter the stability of a DELLA protein. Instead, BOI and DELLA proteins are targeted to the promoters of a subset of GA-responsive genes and repress their expression. Taken together, our results indicate that the DELLA and BOI proteins inhibit GA responses by interacting with each other, binding to the same promoters of GA-responsive genes, and repressing these genes.

Phytochrome B inhibits binding of phytochrome-interacting factors to their target promoters.

Phytochromes are red and far-red light receptors in plants that mediate critical responses to light throughout the lifecycle. They achieve this in part by targeting negatively acting bHLH transcription factors called phytochrome-interacting factors (PIFs) for degradation within the nucleus. However, it is not known whether protein degradation is the primary mechanism by which phytochromes inhibit these repressors of photomorphogenesis. Here, we use chromatin immunoprecipitation to show that phyB inhibits the regulatory activity of PIF1 and PIF3 by releasing them from their DNA targets. The N-terminal fragment of phyB (NG-GUS-NLS; NGB) also inhibits binding of PIF3 to its target promoters. However, unlike full-length phyB, NGB does not promote PIF3 degradation, establishing the activity of NGB reflects its ability to inhibit PIF binding to DNA. We further show that Pfr forms of both full-length phyB and NGB inhibit DNA binding of PIF1 and PIF3 in vitro. Taken together, our results indicate that phyB inhibition of PIF function involves two separate processes: sequestration and protein degradation.

The common NF-κB essential modulator (NEMO) gene rearrangement in Korean patients with incontinentia pigmenti.

Incontinentia pigmenti (IP) is a rare X-linked dominant disorder characterized by highly variable abnormalities of the skin, eyes and central nervous system. A mutation of the nuclear factor-κB essential modulator (NEMO) located at Xq28 is believed to play a role in pathogenesis and the mutation occurs mostly in female patients due to fatal consequence of the mutation in males in utero. This study was designed to identify the common NEMO rearrangement in four Korean patients with IP. Deletion of exons 4 to 10 in the NEMO, the most common mutation in IP patients, was detected in all of the patients by the use of long-range PCR analysis. This method enabled us to discriminate between NEMO and pseudogene rearrangements. Furthermore, all of the patients showed skewed XCI patterns, indicating pathogenicity of IP was due to cells carrying the mutant X chromosome. This is the first report of genetically confirmed cases of IP in Korea.

The association between fetal and postnatal growth status and serum levels of uric acid in children at 3 years of age.

BACKGROUND: Recent studies suggest that uric acid plays a causal role in the development of hypertension in relation to birth weight. The aim of the study was to elucidate the effect of prenatal and/or postnatal growth status on serum uric acid levels in children at 3 years of age. METHODS: A total of 178 children from the birth cohort at Ewha Womans University Hospital were followed. Anthropometric characteristics, blood pressure, and serum uric acid were measured at 3 years of age. RESULTS: The level of serum uric acid was significantly higher in hypertensive children (3.8 mg/dl) than in nonhypertensive children (3.4 mg/dl). Preterm birth and lower placental weight were the preterm factors significantly associated with increased serum uric acid in early childhood. In addition, current weight, body mass index, and change-in-weight s.d. score from birth to current were also associated with serum uric acid level. Both the first- and fourth-quartile groups for weight and weight gain presented higher levels of serum uric acid. We found that the highest uric acid level belonged to the group of preterm or low birth weight (LBW) and higher change in weight s.d. score. Children who were born at full term or with a normal birth weight and lower change in weight s.d. score had the lowest serum uric acid levels. CONCLUSION: Preterm birth and a subsequent high rate of postnatal weight gain are risk factors for increased serum uric acid levels in early childhood.

Transplanted xenogenic bone marrow stem cells survive and generate new bone formation in the posterolateral lumbar spine of non-immunosuppressed rabbits.

Bone marrow stem cells (BMSCs) are pluripotent cells that have been used to facilitate bone repair because of their capability of differentiating into osteoblasts. However, it is well known that the number of BMSCs with osteogenic potential decreases in patients with old age, osteoporosis, and metabolic diseases. In such conditions, xenogenic BMSCs may provide an alternative to autologous BMSCs. In the current study, we investigated the potential of transplanted xenogenic BMSCs to survive and generate new bone formation in the posterolateral lumbar spine of non-immunosuppressed rabbits. The BMSCs were obtained from bilateral femurs of four male rats, cultured and expanded in medium with osteoinduction supplement. The BMSCs (1,000,000 cells) of male rats loaded onto 5 cc compression resistant matrix (CRM; Medtronic Sofamor Danek, USA) were implanted bilaterally onto the L4-5 intertransverse processes of 16 female rabbits (xenogenic BMSCs + CRM group). The 16 female rabbits that received 5 cc CRM alone were used as controls (CRM alone group). To exclude the possibility of migration of BMSCs from the transverse processes of the recipient rabbits, we did not decorticate the transverse processes. No rabbits received any immunosuppressive medications during the experiment. Four rabbits each in both of the experimental and control groups were killed at 1, 2, 4, and 6 months postimplantation, and the lumbar spine underwent radiological and histological analyses for evaluation of new bone formation. The polymerase chain reaction (PCR) for Sry gene (Y-chromosome-specific marker) was used to evaluate the survival of transplanted xenogenic BMSCs. The expression of Sry gene was clearly identified in the lumbar spines of all the 16 rabbits in the xenogenic BMSCs + CRM group at 1-6 months postimplantation. Serial plain radiographs showed gradual resorption of CRM; however, it was difficult to clearly identify the presence of new bone formation due to the radiopacity of the remaining CRM. Histologically, mature lamellar and woven bone with osteoblasts and osteocytes were identified in all eight rabbits in the xenogenic BMSCs + CRM group at 4 and 6 months postimplantation, but in none of the eight rabbits at 1 and 2 months postimplantation. None of CRM alone group showed new bone formation at 1-6 months postimplantation. Mild-to-moderate infiltration of inflammatory cells was identified around the CRM carriers in both the groups. No post-operative wound infection was found in either group. Our results indicate that xenogenic BMSCs loaded onto CRM survive and generate new bone formation when placed into the posterolateral lumbar spine of rabbits without immunosuppression. To determine if a solid fusion can be achieved with such techniques, further studies are needed to investigate the appropriate dose of xenogenic BMSCs, amounts of CRM, and the requisite incubation time.

PIF3 regulates anthocyanin biosynthesis in an HY5-dependent manner with both factors directly binding anthocyanin biosynthetic gene promoters in Arabidopsis.

Phytochromes are red/far-red light receptors that regulate various light responses by initiating the transcriptional cascades responsible for changing the expression patterns of 10-30% of the entire plant transcriptome. Several transcription factors that are thought to participate in this process have been identified, but the functional relationships among them have not yet been fully elucidated. Here we investigated the functional relationship between two such transcription factors, PIF3 and HY5, and their effects on anthocyanin biosynthesis. Our results revealed that PIF3 and HY5 do not regulate each other at either the transcriptional or the protein levels in continuous light conditions, suggesting that they are not directly linked within phytochrome-mediated signaling. We found that both PIF3 and HY5 positively regulate anthocyanin biosynthesis by activating the transcription of the same anthocyanin biosynthetic genes, but the positive effects of PIF3 required functional HY5. Chromatin immunoprecipitation analyses indicated that both PIF3 and HY5 regulate anthocyanin biosynthetic gene expression by directly binding to different regions of the gene promoters in vivo. Additional experiments revealed that PIF3 bound the promoters regardless of light and HY5. Collectively, these data show that PIF3 and HY5 regulate anthocyanin biosynthesis by simultaneously binding anthocyanin biosynthetic gene promoters at separate sequence elements.

Paraoxonase gene polymorphism and vitamin levels during pregnancy: Relationship with maternal oxidative stress and neonatal birthweights.

The aim of this study was to determine the association between maternal oxidative stress and adverse birth outcome, and serum vitamin levels and paraoxonase (PON) polymorphism during pregnancy. We investigated 276 pregnant women who visited a hospital for prenatal care during gestational weeks 24 and 28. We measured serum Vitamins C and E levels and urinary levels of 8-hydroxy-2-deoxyguanosine (8-OH-dG). We determined the presence of a maternal PON polymorphism (Q-to-R substitution at a nucleotide located on 7q21.3) using a polymerase chain reaction-restriction fragment length polymorphism assay. High levels (relative to the median) of Vitamins C and E were associated with a reduced concentration of urinary 8-OH-dG, but increased birthweight and gestational age in subjects with the Q/Q and Q/R types of PON polymorphism. Even with the high-risk PON polymorphism (R/R), the results show that birthweight increased with high levels of Vitamins C and E. Our data demonstrate that oxidative stress and neonatal birthweight are affected by maternal levels of Vitamins C and E in subjects with a PON polymorphism.

PIL5, a phytochrome-interacting basic helix-loop-helix protein, is a key negative regulator of seed germination in Arabidopsis thaliana.

The first decision made by an angiosperm seed, whether to germinate or not, is based on integration of various environmental signals such as water and light. The phytochromes (Phys) act as red and far-red light (Pfr) photoreceptors to mediate light signaling through yet uncharacterized pathways. We report here that the PIF3-like 5 (PIL5) protein, a basic helix-loop-helix transcription factor, is a key negative regulator of phytochrome-mediated seed germination. PIL5 preferentially interacts with the Pfr forms of Phytochrome A (PhyA) and Phytochrome B (PhyB). Analyses of a pil5 mutant in conjunction with phyA and phyB mutants, a pif3 pil5 double mutant, and PIL5 overexpression lines indicate that PIL5 is a negative factor in Phy-mediated promotion of seed germination, inhibition of hypocotyl negative gravitropism, and inhibition of hypocotyl elongation. Our data identify PIL5 as the first Phy-interacting protein that regulates seed germination.

Degradation of phytochrome interacting factor 3 in phytochrome-mediated light signaling.

Plant photoreceptors regulate various developmental processes. Among the photoreceptors, phytochromes, red and far-red light receptors, regulate light responses through many signaling components, including phytochrome-interacting proteins. The functional relationships among phytochromes and their interacting proteins, however, have not been clearly established. Here, we sought to identify a functional relationship between phytochromes and phytochrome interacting factor 3 (PIF3). We demonstrate that PIF3 is polyubiquitinated rapidly and subsequently degraded in PHYA and PHYB-mediated light signaling. We also show that the degradation of PIF3 is mediated by the 26S proteasome. Our data indicate that light-stimulated phytochromes cause the degradation of their interacting protein, PIF3, by the 26S proteasome.