RESUMEN
The ultraviolet B (UVB) sensitivity of rice cultivated in Asia and Africa varies greatly, with African rice cultivars (Oryza glaberrima Steud. and O. barthii A. Chev.) being more sensitive to UVB because of their low cyclobutane pyrimidine dimer (CPD) photolyase activity, which is a CPD repair enzyme, relative to Asian rice cultivars (O. sativa L.). Hence, the production of UVB-resistant African rice with augmented CPD photolyase activity is of great importance, although difficulty in transforming the African rice cultivars to this end has been reported. Here, we successfully produced overexpressing transgenic African rice with higher CPD photolyase activity by modifying media conditions for callus induction and regeneration using the parental line (PL), UVB-sensitive African rice TOG12380 (O. glaberrima). The overexpressing transgenic African rice carried a single copy of the CPD photolyase enzyme, with a 4.4-fold higher level of CPD photolyase transcripts and 2.6-fold higher activity than its PL counterpart. When the plants were grown for 21 days in a growth chamber under visible radiation or with supplementary various UVB radiation, the overexpressing transgenic plants have a significantly increased UVB resistance index compared to PL plants. These results strongly suggest that CPD photolyase remains an essential factor for tolerating UVB radiation stress in African rice. As a result, African rice cultivars with overexpressed CPD photolyase may survive better in tropical areas more prone to UVB radiation stress, including Africa. Collectively, our results provide strong evidence that CPD photolyase is a useful biotechnological tool for reducing UVB-induced growth inhibition in African rice crops of O. glaberrima.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Oryza , Reparación del ADN , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Oryza/enzimología , Oryza/genética , Oryza/efectos de la radiación , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/efectos de la radiación , Dímeros de Pirimidina , Rayos UltravioletaRESUMEN
The quest to engineer increasingly complex synthetic gene networks in mammalian and plant cells requires an ever-growing portfolio of orthogonal gene expression systems. To control gene expression, light is of particular interest due to high spatial and temporal resolution, ease of dosage and simplicity of administration, enabling increasingly sophisticated man-machine interfaces. However, the majority of applied optogenetic switches are crowded in the UVB, blue and red/far-red light parts of the optical spectrum, limiting the number of simultaneously applicable stimuli. This problem is even more pertinent in plant cells, in which UV-A/B, blue, and red light-responsive photoreceptors are already expressed endogenously. To alleviate these challenges, we developed a green light responsive gene switch, based on the light-sensitive bacterial transcription factor CarH from Thermus thermophilus and its cognate DNA operator sequence CarO. The switch is characterized by high reversibility, high transgene expression levels, and low leakiness, leading to up to 350-fold induction ratios in mammalian cells. In this chapter, we describe the essential steps to build functional components of the green light-regulated gene switch, followed by detailed protocols to quantify transgene expression over time in mammalian cells. In addition, we expand this protocol with a description of how the optogenetic switch can be implemented in protoplasts of A. thaliana.
Asunto(s)
Arabidopsis/efectos de la radiación , Proteínas Bacterianas/efectos de la radiación , Ingeniería Celular , Genes de Cambio , Luz , Optogenética , Plantas Modificadas Genéticamente/efectos de la radiación , Thermus thermophilus/genética , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas de Cultivo de Célula , Células Cultivadas , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genes Reporteros , Humanos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Thermus thermophilus/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
DNA damage is one of the most impactful events in living organisms, leading to DNA sequence changes (mutation) and disruption of biological processes. A study has identified a protein called Damage Suppressor Protein (Dsup) in the tardigrade Ramazzotius varieornatus that has shown to reduce the effects of radiation damage in human cell cultures (Hashimoto in Nature Communications 7:12808, 2016). We have generated tobacco plants that express the codon-optimized tardigrade Dsup gene and examined their responses when treated with mutagenic chemicals, ultraviolet (UV) and ionizing radiations. Our studies showed that compared to the control plants, the Dsup-expressing plants grew better in the medium containing mutagenic ethylmethane sulfonate (EMS). RT-qPCR detected distinct expression patterns of endogenous genes involved in DNA damage response and repair in the Dsup plants in response to EMS, bleomycin, UV-C and X-ray radiations. Comet assays revealed that the nuclei from the Dsup plants appeared more protected from UV and X-ray damages than the control plants. Overall, our studies demonstrated that Dsup gene expression enhanced tolerance of plants to genomutagenic stress. We suggest the feasibility of exploring genetic resources from extremotolerant species such as tardigrades to impart plants with tolerance to stressful environments for future climate changes and human space endeavors.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/genética , Metanosulfonato de Etilo/efectos adversos , Nicotiana/crecimiento & desarrollo , Tardigrada/genética , Animales , Bleomicina/efectos adversos , Clonación Molecular , Daño del ADN , Estudios de Factibilidad , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genoma de Planta , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/efectos de la radiación , Nicotiana/efectos de los fármacos , Nicotiana/genética , Nicotiana/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Rayos X/efectos adversosRESUMEN
Flowering time is an important agronomic trait that greatly influences plant architecture and grain yield in cereal crops. The present study identified a light-regulated gene, TaLWD1L-A, from hexaploid wheat that encodes a WD40 domain-containing protein. TaLWD1L-A was localized in the nucleus. Phenotypic analysis demonstrated that TaLWD1L-A overexpression in transgenic wheat led to an obvious early flowering phenotype. Upregulation of the floral activator gene TaFT1 caused the early flowering phenotype in transgenic wheat plants. TaLWD1L-A also affected the expression of circadian clock genes, including TaTOC1, TaLHY, TaPRR59, TaPRR73 and TaPRR95, and indirectly regulated the expression of the TaFT1 in transgenic plants by affecting the expression of vernalization-related genes TaVRN1 and TaVRN2 and photoperiod-related genes TaPpd-1 and TaGI. The early flowering phenotype in TaLWD1L-A-overexpressing transgenic lines led to a relatively shorter phenotype and yield reduction. Our results revealed that TaLWD1L-A affected the expression of circadian clock-related genes and played an important role in wheat flowering regulation by influencing the expression of genes related to vernalization and photoperiod pathways.
Asunto(s)
Luz , Proteínas Circadianas Period/genética , Proteínas de Plantas/genética , Triticum/genética , Secuencia de Aminoácidos , Flores/genética , Flores/crecimiento & desarrollo , Proteínas Circadianas Period/química , Proteínas Circadianas Period/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Alineación de Secuencia , Triticum/metabolismo , Triticum/efectos de la radiaciónRESUMEN
Although Ultraviolet-B (UV-B)-plant interactions have been extensively analysed in the past years, many physiological aspects of the complex plant response mechanisms still need to be elucidated. Depending on the energy dose, this part of the electromagnetic spectrum can induce detrimental or beneficial effects in plant and fruit. In the present work, active thermography is used to analyse in real time the response of plants under different doses of artificial UV-B. In particular, we investigated the temporal variations of the leaf surface temperature (LST) to UV-B exposure by Long Pulse and Lock-in thermography in Epipremnum aureum and in Arabidopsis plants overexpressing or knockout mutants of UVR8, the known UV-B photoreceptor. In both cases, UV-B irradiation triggers a cooling effect, namely a thermal response characterised by a LST lower respect to the initial value. Lock-in thermography demonstrated that the cooling effect is associated with an immediate mobilization and accumulation of water in the leaves. Also, we demonstrated that thermographic responses change according to the different capability of plants to tolerate high UV-B radiation. Our study highlights new physiological and physical aspects of the plants response to UV-B radiation and, more in general, it opens new opportunities for the use of the thermography as smart tool for real-time monitoring of plant environmental interactions.
Asunto(s)
Arabidopsis/metabolismo , Rayos Ultravioleta , Arabidopsis/crecimiento & desarrollo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Araceae/crecimiento & desarrollo , Araceae/metabolismo , Araceae/efectos de la radiación , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Temperatura , TermografíaRESUMEN
Photosynthetic efficiency is a major target for improvement of crop yield potential under agricultural field conditions. Inefficiencies can occur in many steps of the photosynthetic process, from chloroplast biogenesis to functioning of the light harvesting and carbon fixation reactions. Nuclear-encoded GOLDEN2-LIKE (GLK) transcription factors regulate some of the earliest steps by activating target genes encoding chloroplast-localized and photosynthesis-related proteins. Here we show that constitutive expression of maize GLK genes in rice leads to enhanced levels of chlorophylls and pigment-protein antenna complexes, and that these increases lead to improved light harvesting efficiency via photosystem II in field-grown plants. Increased levels of xanthophylls further buffer the negative effects of photoinhibition under high or fluctuating light conditions by facilitating greater dissipation of excess absorbed energy as heat. Significantly, the enhanced photosynthetic capacity of field-grown transgenic plants resulted in increased carbohydrate levels and a 30-40% increase in both vegetative biomass and grain yield.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Productos Agrícolas/metabolismo , Oryza/metabolismo , Fotosíntesis , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Factores de Transcripción/metabolismo , Biomasa , Metabolismo de los Hidratos de Carbono/genética , Metabolismo de los Hidratos de Carbono/efectos de la radiación , Clorofila/genética , Clorofila/metabolismo , Productos Agrícolas/genética , Productos Agrícolas/crecimiento & desarrollo , Productos Agrícolas/efectos de la radiación , Regulación de la Expresión Génica de las Plantas , Luz , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismo , Oryza/genética , Oryza/crecimiento & desarrollo , Fotosíntesis/genética , Fotosíntesis/efectos de la radiación , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/efectos de la radiación , Estaciones del Año , Factores de Transcripción/genéticaRESUMEN
Although natural variations in rice flavonoids exist, and biochemical characterization of a few flavonoid glycosyltransferases has been reported, few studies focused on natural variations in tricin-lignan-glycosides and their underlying genetic basis. In this study, we carried out metabolic profiling of tricin-lignan-glycosides and identified a major quantitative gene annotated as a UDP-dependent glycosyltransferase OsUGT706C2 by metabolite-based genome-wide association analysis. The putative flavonoid glycosyltransferase OsUGT706C2 was characterized as a flavonoid 7-O-glycosyltransferas in vitro and in vivo. Although the in vitro enzyme activity of OsUGT706C2 was similar to that of OsUGT706D1, the expression pattern and induced expression profile of OsUGT706C2 were very different from those of OsUGT706D1. Besides, OsUGT706C2 was specifically induced by UV-B. Constitutive expression of OsUGT706C2 in rice may modulate phenylpropanoid metabolism at both the transcript and metabolite levels. Furthermore, overexpressing OsUGT706C2 can enhance UV-B tolerance by promoting ROS scavenging in rice. Our findings might make it possible to use the glycosyltransferase OsUGT706C2 for crop improvement with respect to UV-B adaptation and/or flavonoid accumulation, which may contribute to stable yield.
Asunto(s)
Flavonoides/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/efectos de la radiación , Oryza/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Adaptación Fisiológica , Vías Biosintéticas , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genes de Plantas/efectos de la radiación , Estudio de Asociación del Genoma Completo , Glicósidos/metabolismo , Lignanos/metabolismo , Metaboloma/efectos de la radiación , MutaciónRESUMEN
Ion beams are physical mutagens used for plant and microbe breeding that cause mutations via a mechanism distinct from those of chemical mutagens or gamma rays. We utilized whole-exome sequencing of rice DNA in order to understand the properties of ion beam-induced mutations in a genome-wide manner. DNA libraries were constructed from selected carbon-ion-beam-induced rice mutants by capturing with a custom probes covering 66.3â¯M bases of nearly all exons and miRNAs predicted in the genome. A total of 56 mutations, including 24 single nucleotide variations, 23 deletions, and 5 insertions, were detected in five mutant rice lines (two dwarf and three early-heading-date mutants). The mutations were distributed among all 12 chromosomes, and the average mutation frequency in the M1 generation was estimated to be 2.7â¯×â¯10-7 per base. Many single base insertions and deletions were associated with homopolymeric repeats, whereas larger deletions up to seven base pairs were observed at polynucleotide repeats in the DNA sequences of the mutation sites. Of the 56 mutations, six were classified as high-impact mutations that caused a frame shift or loss of exons. A gene that was functionally related to the phenotype of the mutant was disrupted by a high-impact mutation in four of the five lines tested, suggesting that whole-exome sequencing of ion-beam-irradiated mutants could facilitate the detection of candidate genes responsible for the mutant phenotypes.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genoma de Planta , Iones Pesados/efectos adversos , Mutación , Oryza/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Rayos gamma , Tasa de Mutación , Oryza/efectos de la radiación , Fenotipo , Plantas Modificadas Genéticamente/efectos de la radiaciónRESUMEN
KEY MESSAGE: The early flowering system HSP::AtFT allowed a fast evaluation of a gene containment system based on the construct PsEND1::barnase-barstar for poplar. Transgenic lines showed disturbed pollen development and sterility. Vertical gene transfer through pollen flow from transgenic or non-native plant species into their crossable natural relatives is a major concern. Gene containment approaches have been proposed to reduce or even avoid gene flow among tree species. However, evaluation of genetic containment strategies for trees is very difficult due to the long-generation times. Early flowering induction would allow faster evaluation of genetic containment in this case. Although no reliable methods were available for the induction of fertile flowers in poplar, recently, a new early flowering approach was developed. In this study, early flowering poplar lines containing the gene construct PsEND1::barnase-barstar were obtained. The PsEND1 promoter was chosen due to its early expression pattern, its versality and efficiency for generation of male-sterile plants fused to the barnase gene. RT-PCRs confirmed barnase gene activity in flowers, and pollen development was disturbed, leading to sterile flowers. The system developed in this study represents a valuable tool for gene containment studies in forest tree species.
Asunto(s)
Proteínas Bacterianas/genética , Flores/crecimiento & desarrollo , Edición Génica/métodos , Infertilidad Vegetal/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Polen/crecimiento & desarrollo , Populus/crecimiento & desarrollo , Ribonucleasas/genética , Proteínas de Arabidopsis/genética , Proteínas Bacterianas/metabolismo , Flores/genética , Flores/metabolismo , Flores/efectos de la radiación , Regulación de la Expresión Génica de las Plantas , Flujo Génico , Vectores Genéticos , Respuesta al Choque Térmico , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Polen/genética , Populus/genética , Populus/metabolismo , Populus/efectos de la radiación , Regiones Promotoras Genéticas , Ribonucleasas/metabolismo , Temperatura , Transformación GenéticaRESUMEN
Faster onset of photoprotection could potentially increase biomass accumulation. Indeed, this has been realized in tobacco VPZ lines by overexpression of three photoprotective proteins in parallel. To explore the range of application of this approach, we generated Arabidopsis VPZ lines. These lines triggered photoprotection more rapidly, but growth rate and biomass accumulation were impaired under fluctuating light. This implies that the strategy might interfere with other mechanisms controlling excitation energy distribution, or with source-sink relationships or plastid signalling.
Asunto(s)
Arabidopsis/efectos de la radiación , Biomasa , Fotosíntesis/efectos de la radiación , Proteínas de Plantas/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Nicotiana/genética , Transcripción GenéticaRESUMEN
Ascorbate oxidase (AO) plays important roles in plant growth and development. Previously, we reported a cotton AO gene that acts as a positive factor in cell growth. Investigations on Gossypium hirsutum AO (GhAO) family genes and their multiple functions are limited. The present study identified eight GhAO family genes and performed bioinformatic analyses. Expression analyses of the tissue specificity and developmental feature of GhAOs displayed their diverse expression patterns. Interestingly, GhAO1A demonstrated the most rapid significant increase in expression after 1 h of light recovery from the dark. Additionally, the transgenic ao1-1/GhAO1A Arabidopsis lines overexpressing GhAO1A in the Arabidopsis ao1-1 late-flowering mutant displayed a recovery to the normal phenotype of wild-type plants. Moreover, compared to the ao1-1 mutant, the ao1-1/GhAO1A transgenic Arabidopsis presented delayed leaf senescence that was induced by the dark, indicating increased sensitivity to hydrogen peroxide (H2O2) under normal conditions that might be caused by a reduction in ascorbic acid (AsA) and ascorbic acid/dehydroascorbate (AsA/DHA) ratio. The results suggested that GhAOs are functionally diverse in plant development and play a critical role in light responsiveness. Our study serves as a foundation for understanding the AO gene family in cotton and elucidating the regulatory mechanism of GhAO1A in delaying dark-induced leaf senescence.
Asunto(s)
Ascorbato Oxidasa/genética , Oscuridad , Gossypium/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Ascorbato Oxidasa/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Gossypium/efectos de los fármacos , Gossypium/efectos de la radiación , Peróxido de Hidrógeno/farmacología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/efectos de la radiación , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/efectos de los fármacos , Plantas Modificadas Genéticamente/efectos de la radiaciónRESUMEN
Phototropin1 (phot1) perceives low- to high-fluence blue light stimuli and mediates both the first and second positive phototropisms. High-fluence blue light is known to induce autophosphorylation of phot1, leading to the second positive phototropism. However, the phosphorylation status of phot1 by low-fluence blue light that induces the first positive phototropism had not been observed. Here, we conducted a phosphoproteomic analysis of maize coleoptiles to investigate the fluence-dependent phosphorylation status of Zmphot1. High-fluence blue light induced phosphorylation of Zmphot1 at several sites. Notably, low-fluence blue light significantly increased the phosphorylation level of Ser291 in Zmphot1. Furthermore, Ser291-phosphorylated and Ser369Ser376-diphosphorylated peptides were found to be more abundant in the low-fluence blue light-irradiated sides than in the shaded sides of coleoptiles. The roles of these phosphorylation events in phototropism were explored by heterologous expression of ZmPHOT1 in the Arabidopsis thaliana phot1phot2 mutant. The first positive phototropism was restored in wild-type ZmPHOT1-expressing plants; however, plants expressing S291A-ZmPHOT1 or S369AS376A-ZmPHOT1 showed significantly reduced complementation rates. All transgenic plants tested in this study exhibited a normal second positive phototropism. These findings provide the first indication that low-fluence blue light induces phosphorylation of Zmphot1 and that this induced phosphorylation is crucial for the first positive phototropism.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Luz , Fototropismo/fisiología , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Fosforilación/genética , Fosforilación/efectos de la radiación , Fototropismo/genética , Fototropismo/efectos de la radiación , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Zea mays/genética , Zea mays/metabolismo , Zea mays/efectos de la radiaciónRESUMEN
Stomatal movements are regulated by many environmental signals, such as light, CO2, temperature, humidity, and drought. Recently, we showed that photoperiodic flowering components have positive effects on light-induced stomatal opening in Arabidopsis thaliana. In this study, we determined that light-induced stomatal opening and increased stomatal conductance were larger in plants grown under long-day (LD) conditions than in those grown under short-day (SD) conditions. Gene expression analyses using purified guard cell protoplasts revealed that FT and SOC1 expression levels were significantly increased under LD conditions. Interestingly, the enhancement of light-induced stomatal opening and increased SOC1 expression in guard cells due to LD conditions persisted for at least 1 week after plants were transferred to SD conditions. We then investigated histone modification using chromatin immunoprecipitation-PCR, and observed increased trimethylation of lysine 4 on histone 3 (H3K4) around SOC1. We also found that LD-dependent enhancement of light-induced stomatal opening and H3K4 trimethylation in SOC1 were suppressed in the ft-2 mutant. These results indicate that photoperiod is an important environmental cue regulating stomatal opening, and that LD conditions enhance light-induced stomatal opening and epigenetic modification (H3K4 trimethylation) around SOC1, a positive regulator of stomatal opening, in an FT-dependent manner. Thus, this study provides novel insights into stomatal responses to photoperiod.
Asunto(s)
Arabidopsis/genética , Arabidopsis/efectos de la radiación , Código de Histonas/efectos de la radiación , Fotoperiodo , Estomas de Plantas/efectos de la radiación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Flores/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Histonas/metabolismo , Proteínas de Dominio MADS/genética , Metilación , Estomas de Plantas/fisiología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/efectos de la radiación , Protoplastos/metabolismo , Factores de Transcripción/metabolismoRESUMEN
In light signaling pathways, protein complexes play essential roles in light perception and signal transduction. The phytochrome family of photoreceptors perceives red/far-red region of the light spectrum and then translocates into the nucleus to form protein complexes. Many phytochrome interacting proteins have been identified based on yeast two-hybrid screening and other protein-protein interaction methods. However, it is essential to demonstrate that these proteins interact with phytochromes in vivo to be functionally relevant. In this chapter, a protocol for demonstrating light dependent in vivo interactions between phytochromes and phytochrome interacting proteins is described. This protocol can be adapted for any putative phytochrome interacting protein for validation of their interaction in vivo.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Luz , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Inmunoprecipitación , Fototransducción , Fitocromo/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Unión ProteicaRESUMEN
Artemisinin, the frontline drug against malaria, is a sesquiterpenoid extracted from Artemisia annua. Light has been proposed to play an important role in the activation of artemisinin biosynthesis. Here, we report the basic leucine zipper transcription factor (TF) AaHY5 as a key regulator of light-induced biosynthesis of artemisinin. We show that AaHY5 transcription overlaps with that of artemisinin biosynthesis genes in response to light and in A. annua tissues. Analysis of AaHY5 overexpression and RNAi-suppression lines suggests that AaHY5 is a positive regulator of the expression of artemisinin biosynthesis genes and accumulation of artemisinin. We show that AaHY5 complements the hy5 mutant in Arabidopsis thaliana. Our data further suggest that AaHY5 interacts with AaCOP1, the ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC1 in A. annua. In yeast one-hybrid and transient expression assays, we demonstrate that AaHY5 acts via the TF GLANDULAR TRICHOME-SPECIFIC WRKY 1 (AaGSW1) in artemisinin regulation. In summary, we present a novel regulator of artemisinin gene expression and propose a model in which AaHY5 indirectly controls artemisinin production in response to changing light conditions.
Asunto(s)
Artemisia annua/metabolismo , Artemisininas/metabolismo , Luz , Artemisia annua/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Factores de TranscripciónRESUMEN
MAIN CONCLUSION: The physiological importance of MpUVR8 in UV-B resistance and translocation in a UV-B-dependent manner from the cytosol into the nucleus is characterized in Marchantia polymorpha. UV RESISTANCE LOCUS 8 (UVR8) is an ultraviolet-B (UV-B) light receptor functioning for UV-B sensing and tolerance in Arabidopsis thaliana and other species. It is unclear whether UVR8 physiologically functions in UV-B-induced defense responses in Marchantia polymorpha, which belongs to the earliest diverging group of embryophyte lineages. Here, we demonstrate that UVR8 has a physiological function in UV-B tolerance and that there is a UVR8-dependent pathway involved. In addition, a UVR8-independent pathway is revealed. We examine the tissue-specific expression pattern of M. polymorpha UVR8 (MpUVR8), showing that it is highly expressed in the apical notch in thalli and gametangiophores, as well as in antheridial and archegonial heads. Furthermore, Mpuvr8KO plant transformants, in which the MpUVR8 locus was disrupted, were produced and analyzed to understand the physiological and molecular function of MpUVR8. Analysis using these plants indicates the important roles of MpUVR8 and MpUVR8-regulated genes, and of MpUVR8-independent pathways in UV-B tolerance. Subcellular localization of Citrine-fused MpUVR8 in M. polymorpha cells was also investigated. It was found to translocate from the cytosol into the nucleus in response to UV-B irradiation. Our findings indicate strong conservation of the physiological function of UVR8 and the molecular mechanisms for UVR8-dependent signal transduction through regulation of gene expression in embryophytes.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Marchantia/metabolismo , Marchantia/efectos de la radiación , Proteínas de Plantas/metabolismo , Rayos Ultravioleta , Proteínas Cromosómicas no Histona/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Marchantia/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Transducción de Señal/efectos de la radiaciónRESUMEN
In many plants, anthocyanin biosynthesis is affected by environmental conditions. Ultraviolet-B (UV-B) radiation promotes anthocyanin accumulation and fruit coloration in apple skin, whereas high temperature suppresses these processes. In this study, we characterized a B-box transcription factor, MdCOL4, from 'Fuji' apple, and identified its role in anthocyanin biosynthesis by overexpressing its encoding gene in apple red callus. The expression of MdCOL4 was reduced by UV-B, but promoted by high temperature. We explored the regulatory relationship between heat shock transcription factors (HSFs) and MdCOL4, and found that MdHSF3b and MdHSF4a directly bound to the heat shock element cis-element of the MdCOL4 promoter. MdCOL4 interacted with MdHY5 to synergistically inhibit the expression of MdMYB1, and MdCOL4 directly bound to the promoters of MdANS and MdUFGT, which encode genes in the anthocyanin biosynthetic pathway, to suppress their expression. Our findings shed light on the molecular mechanism by which MdCOL4 suppresses anthocyanin accumulation in apple skin under UV-B and high temperature.
Asunto(s)
Frutas/metabolismo , Malus/metabolismo , Antocianinas/metabolismo , Frutas/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Calor , Malus/efectos de la radiación , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/efectos de la radiación , Temperatura , Factores de Transcripción/metabolismo , Rayos UltravioletaRESUMEN
The liverwort species, Marchantia polymorpha, shows environment-dependent morphological plasticity throughout its life cycle. Thalli, representing the predominant body form throughout most of this bryophyte's life cycle, grow with repeated dichotomous branching at the apex and develop horizontally under sufficient light intensity. Spores, after germination, produce a mass of cells, called sporelings, which then grow into thalli. Both thalli and sporelings, if grown under weak light conditions, form narrow shapes, and their apices grow toward the light source. These phototropic responses are specific to blue light and dependent on the blue-light receptor phototropin. This chapter provides several basic procedures, along with some tips, for designing and performing experiments with M. polymorpha to observe their phototropic responses, as well as methods for observing the localization of the phototropin "Mpphot" with a fluorescent protein tag.
Asunto(s)
Luz , Marchantia/efectos de la radiación , Plantas Modificadas Genéticamente/efectos de la radiación , Transformación Genética/efectos de la radiación , Marchantia/genética , Plantas Modificadas Genéticamente/genética , Transformación Genética/genéticaRESUMEN
Light makes carbon fixation possible, allowing plant and animal life on Earth. We have previously shown that light regulates alternative splicing in plants. Light initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing of a subset of Arabidopsis thaliana transcripts. Here, we show that light promotes RNA polymerase II (Pol II) elongation in the affected genes, whereas in darkness, elongation is lower. These changes in transcription are consistent with elongation causing the observed changes in alternative splicing, as revealed by different drug treatments and genetic evidence. The light control of splicing and elongation is abolished in an Arabidopsis mutant defective in the transcription factor IIS (TFIIS). We report that the chloroplast control of nuclear alternative splicing in plants responds to the kinetic coupling mechanism found in mammalian cells, providing unique evidence that coupling is important for a whole organism to respond to environmental cues.
Asunto(s)
Empalme Alternativo/efectos de la radiación , Arabidopsis/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luz , Plantas Modificadas Genéticamente/efectos de la radiación , ARN de Planta/efectos de la radiación , Elongación de la Transcripción Genética/efectos de la radiación , Acetilación , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Oscuridad , Histonas/genética , Histonas/metabolismo , Cinética , Mutación , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN de Planta/biosíntesis , ARN de Planta/genética , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
Silicon (Si) enhances the resistance of rice to biotic and abiotic stress. In rice, the accumulation of Si is controlled by the low silicon rice 1 (Lsi1) gene; overexpression of Lsi1 (Lsi1-OX) increases Si uptake and accumulation, while the reverse is observed in Lsi1-RNA interference (Lsi1-RNAi) transgenic rice. When the two transgenic rice lines and wild-type (WT) rice were exposed to ultraviolet (UV)-B radiation, the Lsi1-OX or Lsi1-RNAi rice showed differential microRNA (miRNA) expression, compared to WT rice. These miRNAs were predicted to target genes involved in light signal transduction and cell detoxification. The greatest capacities of ascorbate peroxidase, superoxide dismutase, peroxidase, and phenylalanine ammonia lyase (PAL) and highest contents of phenolics, flavonoids, and proline were found in Lsi1-OX rice, followed by WT rice and Lsi1-RNAi transgenic rice. A further comparison of the transcript levels of individual PAL genes revealed that the expression of PAL2-2 (Os02g0626400) was positively regulated by Lsi1. Our results demonstrate that Lsi1 overexpression or interference causes changes in both miRNA expression and antioxidant capacity in rice, and therefore modulates rice tolerance to UV-B radiation. Furthermore, we demonstrated that PAL2-2 was positively regulated by Lsi1 during this process.