RESUMEN
KEY MESSAGE: OsLec-RLK overexpression enhances cell signalling and salt stress tolerance in pigeon pea, enhancing seed yield and harvest index and thus, enabling marginal lands to increase food and nutritional security. Lectin Receptor-like kinases (Lec-RLKs) are highly effective cell signaling molecules that counteract various stresses, including salt stress. We engineered pigeon pea by overexpressing OsLec-RLK gene for enhancing salt tolerance. The OsLec-RLK overexpression lines demonstrated superior performance under salt stress, from vegetative to reproductive phase, compared to wild types (WT). The overexpression lines had significantly higher K+/Na+ ratio than WT exposed to 100 mM NaCl. Under salt stress, transgenic lines showed higher levels of chlorophyll, proline, total soluble sugars, relative water content, and peroxidase and catalase activity than WT plants. Membrane injury index and lipid peroxidation were significantly reduced in transgenic lines. Analysis of phenological and yield attributes confirmed that the OsLec-RLK pigeon pea lines maintain plant vigor, with 10.34-fold increase in seed yield (per plant) and 4-5-fold increase in harvest index of overexpression lines, compared to wild type. Meanwhile, the overexpression of OsLec-RLK up-regulated the expression levels of histone deacetylase1, acyl CoA, ascorbate peroxidase, peroxidase, glutathione reductase and catalase, which were involved in the K+/Na+ homeostasis pathway. This study showed the potential of OsLec-RLK gene for increasing crop productivity and yields under salt stress and enabling the crops to be grown on marginal lands for increasing food and nutritional security.
Asunto(s)
Cajanus , Clorofila , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas , Plantas Modificadas Genéticamente , Tolerancia a la Sal , Semillas , Semillas/genética , Semillas/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Cajanus/genética , Cajanus/fisiología , Cajanus/crecimiento & desarrollo , Tolerancia a la Sal/genética , Clorofila/metabolismo , Oryza/genética , Oryza/fisiología , Oryza/crecimiento & desarrollo , Oryza/enzimología , Estrés Salino/genética , Potasio/metabolismoRESUMEN
BACKGROUND: Sulphotransferase (SOT) enzyme (encoded by a conserved family of SOT genes) is involved in sulphonation of a variety of compounds, through transfer of a sulphuryl moiety from 3'phosphoadenosine- 5'phosphosulphate (PAPS) to a variety of secondary metabolites. The PAPS itself is derived from 3'adenosine-5'phosphosulphate (APS) that is formed after uptake of sulphate ions from the soil. The process provides tolerance against abiotic stresses like drought and heat in plants. Therefore, a knowledge of SOT genes in any crop may help in designing molecular breeding methods for improvement of tolerance for drought and heat. METHODS: Sequences of rice SOT genes and SOT domain (PF00685) of corresponding proteins were both used for identification of SOT genes in wheat and six related species (T. urartu, Ae. tauschii, T. turgidum, Z. mays, B. distachyon and Hordeum vulgare), although detailed analysis was conducted only in wheat. The wheat genes were mapped on individual chromosomes and also subjected to synteny and collinearity analysis. The proteins encoded by these genes were examined for the presence of a complete SOT domain using 'Conserved Domain Database' (CDD) search tool at NCBI. RESULTS: In wheat, 107 TaSOT genes, ranging in length from 969 bp to 7636 bp, were identified and mapped onto individual chromosomes. SSRs (simple sequence repeats), microRNAs, long non-coding RNAs (lncRNAs) and their target sites were also identified in wheat SOT genes. SOT proteins were also studied in detail. An expression assay of TaSOT genes via wheat RNA-seq data suggested engagement of these genes in growth, development and responses to various hormones and biotic/abiotic stresses. CONCLUSIONS: The results of the present study should help in further functional characterization of SOT genes in wheat and other related crops.
Asunto(s)
Sequías , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas , Sulfotransferasas , Triticum , Triticum/genética , Triticum/enzimología , Regulación de la Expresión Génica de las Plantas/genética , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estrés Fisiológico/genética , Filogenia , Mapeo Cromosómico/métodos , Calor , Hordeum/genética , Hordeum/enzimología , Cromosomas de las Plantas/genética , Oryza/genética , Oryza/enzimología , Genes de PlantasRESUMEN
MAIN CONCLUSION: This review focuses on HATs and HDACs that modify non-histone proteins, summarizes functional mechanisms of non-histone acetylation as well as the roles of HATs and HDACs in rice and Arabidopsis. The growth and development of plants, as well as their responses to biotic and abiotic stresses, are governed by intricate gene and protein regulatory networks, in which epigenetic modifying enzymes play a crucial role. Histone lysine acetylation levels, modulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), are well-studied in the realm of transcriptional regulation. However, the advent of advanced proteomics has unveiled that non-histone proteins also undergo acetylation, with its underlying mechanisms now being clarified. Indeed, non-histone acetylation influences protein functionality through diverse pathways, such as modulating protein stability, adjusting enzymatic activity, steering subcellular localization, influencing interactions with other post-translational modifications, and managing protein-protein and protein-DNA interactions. This review delves into the recent insights into the functional mechanisms of non-histone acetylation in plants. We also provide a summary of the roles of HATs and HDACs in rice and Arabidopsis, and explore their potential involvement in the regulation of non-histone proteins.
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Arabidopsis , Histona Acetiltransferasas , Histona Desacetilasas , Oryza , Proteínas de Plantas , Procesamiento Proteico-Postraduccional , Histona Desacetilasas/metabolismo , Histona Desacetilasas/genética , Acetilación , Oryza/genética , Oryza/metabolismo , Oryza/enzimología , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimología , Histona Acetiltransferasas/metabolismo , Histona Acetiltransferasas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Histonas/metabolismoRESUMEN
Anthocyanins are natural flavonoids with a high antioxidant power and many associated health benefits, but most rice produce little amounts of these compounds. In this study, 141 MYB transcription factors in 15 chromosomes, including the nucleus-localised ZlMYB1 (Zla03G003370) and ZlMYB2 (Zla15G015220), were discovered in Zizania latifolia. Overexpression of ZlMYB1 or ZlMYB2 in rice seeds induced black pericarps, and flavonoid content, antioxidant capacity, and α-glucosidase and tyrosinase inhibition effects significantly increased compared to those in the control seeds. ZlMYB1 and ZlMYB2 overexpression induced the upregulation of 764 and 279 genes, respectively, and the upregulation of 162 and 157 flavonoids, respectively, linked to a black pericarp phenotype. The expression of flavonoid 3'-hydroxylase and UDP-glycose flavonoid glycosyltransferase, as well as the activities of these enzymes, increased significantly in response to ZlMYB1 or ZlMYB2 overexpression. This study systematically confirmed that the overexpression of ZlMYB1 and ZlMYB2 promotes flavonoid biosynthesis (especially of anthocyanins) in rice.
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Antioxidantes , Flavonoides , Monofenol Monooxigenasa , Oryza , Proteínas de Plantas , Semillas , alfa-Glucosidasas , Semillas/química , Semillas/genética , Semillas/metabolismo , Semillas/enzimología , Oryza/genética , Oryza/química , Oryza/metabolismo , Oryza/enzimología , Flavonoides/metabolismo , Flavonoides/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , alfa-Glucosidasas/genética , alfa-Glucosidasas/metabolismo , alfa-Glucosidasas/química , Antioxidantes/metabolismo , Antioxidantes/química , Monofenol Monooxigenasa/metabolismo , Monofenol Monooxigenasa/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Regulación de la Expresión Génica de las Plantas , Inhibidores de Glicósido Hidrolasas/química , Inhibidores de Glicósido Hidrolasas/farmacología , Inhibidores de Glicósido Hidrolasas/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/enzimologíaRESUMEN
Rubisco activity is highly regulated and frequently limits carbon assimilation in crop plants. In the chloroplast, various metabolites can inhibit or modulate Rubisco activity by binding to its catalytic or allosteric sites, but this regulation is complex and still poorly understood. Using rice Rubisco, we characterised the impact of various chloroplast metabolites which could interact with Rubisco and modulate its activity, including photorespiratory intermediates, carbohydrates, amino acids; as well as specific sugar-phosphates known to inhibit Rubisco activity - CABP (2-carboxy-d-arabinitol 1,5-bisphosphate) and CA1P (2-carboxy-d-arabinitol 1-phosphate) through in vitro enzymatic assays and molecular docking analysis. Most metabolites did not directly affect Rubisco in vitro activity under both saturating and limiting concentrations of Rubisco substrates, CO2 and RuBP (ribulose-1,5-bisphosphate). As expected, Rubisco activity was strongly inhibited in the presence of CABP and CA1P. High physiologically relevant concentrations of the carboxylation product 3-PGA (3-phosphoglyceric acid) decreased Rubisco activity by up to 30%. High concentrations of the photosynthetically derived hexose phosphates fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) slightly reduced Rubisco activity under limiting CO2 and RuBP concentrations. Biochemical measurements of the apparent Vmax and Km for CO2 and RuBP (at atmospheric O2 concentration) and docking interactions analysis suggest that CABP/CA1P and 3-PGA inhibit Rubisco activity by binding tightly and loosely, respectively, to its catalytic sites (i.e. competing with the substrate RuBP). These findings will aid the design and biochemical modelling of new strategies to improve the regulation of Rubisco activity and enhance the efficiency and sustainability of carbon assimilation in rice.
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Cloroplastos , Simulación del Acoplamiento Molecular , Oryza , Ribulosa-Bifosfato Carboxilasa , Ribulosa-Bifosfato Carboxilasa/metabolismo , Ribulosa-Bifosfato Carboxilasa/química , Cloroplastos/metabolismo , Cloroplastos/enzimología , Oryza/metabolismo , Oryza/enzimología , Fotosíntesis , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Dióxido de Carbono/metabolismo , Ribulosafosfatos/metabolismo , Fructosafosfatos/metabolismoRESUMEN
Lactate dehydrogenase (Ldh, EC 1.1.1.27), an oxidoreductase enzyme catalyses the interconversion of pyruvate to L-lactate and vice-versa with concomitant oxidation and reduction of NADH and NAD+. The enzyme functions as a ROS sensor and mitigates stress response by maintaining NAD+/NADH homeostasis. In this study, we delineated the role of the Ldh enzyme in imparting cadmium stress tolerance in rice. Previously, we identified a putatively active Ldh in rice (OsLdh7) through insilico modelling. Biochemical characterization of the OsLdh7 enzyme revealed it to be optimally active at pH 6.6 in the forward direction and pH 9 in the reverse direction. Overexpression of OsLdh7 in rice cv. IR64, increased tolerance of the transgenic lines to cadmium stress compared to the wild type (WT) at both seedling and reproductive stages. The transgenic lines showed increased enzyme activity in the reverse direction under cadmium stress, attributed to elevated cytosolic pH resulting from increased calcium concentration. This increased NADH content is highly essential for functioning of the ROS scavenging enzymes, RbohD and MPK6. qPCR analysis revealed that the overexpression lines had increased transcript abundance of these genes indicating an effective ROS scavenging mechanism. Additionally, the overexpression lines showed an efficient cadmium sequestration mechanism compared to the WT by increasing the transcript levels of the vacuolar transporters of cadmium as well as total phytochelatin content. Thus, our findings indicated OsLdh7 imparts cadmium stress tolerance in rice through a two-pronged approach by mitigating ROS and sequestering cadmium ions, highlighting its potential for crop improvement programs.
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Cadmio , NAD , Oryza , Proteínas de Plantas , Estrés Fisiológico , Oryza/genética , Oryza/metabolismo , Oryza/enzimología , Oryza/efectos de los fármacos , Cadmio/metabolismo , Cadmio/toxicidad , NAD/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Estrés Fisiológico/genética , Plantas Modificadas Genéticamente , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa/genéticaRESUMEN
Geranylgeranyl diphosphate synthase (GGPPS) is the crucial bottleneck in carotenoid biosynthesis. However, low activity limits the broad application of GGPPS. In this study, OsGGPPS1 in rice was engineered based on ancestral sequence reconstruction (ASR) and semirational design to improve the catalytic performances of existing GGPPS. The better mutant of A22R/A26P with improved enzyme activity was generated based on ASR. Additionally, the improved enzyme activity of mutants as V162A/M218S/F227Y was designed using a semirational design. The combinatorial assembly of the d-OsGGPPS1 mutant (A22R/A26P/V162A/M218S/F227Y) exhibited higher conversion of IPP and each cosubstrate of DMAPP for 9.8-fold in GPP production, GPP for 6.4-fold in FPP production, and FPP for 1.4-fold in GGPP production relative to wild-type OsGGPPS1 at 25 °C, which showed higher conversion than wild-type OsGGPPS1 at temperatures as high as 50 °C. The successful design of OsGGPPS1 was representative of protein engineering, which will shed new light on GGPPS engineering and active plant pigment resource utilization.
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Farnesiltransferasa , Oryza , Proteínas de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Farnesiltransferasa/genética , Farnesiltransferasa/metabolismo , Farnesiltransferasa/química , Oryza/genética , Oryza/enzimología , Cinética , Ingeniería de Proteínas , BiocatálisisRESUMEN
KEY MESSAGE: The Osckx2 mutant accumulates cytokinin thereby enhancing panicle branching, grain yield, and drought tolerance, marked by improved survival rate, membrane integrity, and photosynthetic function. Cytokinins (CKs) are multifaceted hormones that regulate growth, development, and stress responses in plants. Cytokinins have been implicated in improved panicle architecture and grain yield; however, they are inactivated by the enzyme cytokinin oxidase (CKX). In this study, we developed a cytokinin oxidase 2 (Osckx2)-deficient mutant using CRISPR/Cas9 gene editing in indica rice and assessed its function under water-deficit and salinity conditions. Loss of OsCKX2 function increased grain number, secondary panicle branching, and overall grain yield through improved cytokinin content in the panicle tissue. Under drought conditions, the Osckx2 mutant conserved more water and demonstrated improved water-saving traits. Through reduced transpiration, Osckx2 mutants showed an improved survival response than the wild type to unset dehydration stress. Further, Osckx2 maintained chloroplast and membrane integrity and showed significantly improved photosynthetic function under drought conditions through enhanced antioxidant protection systems. The OsCKX2 function negatively affects panicle grain number and drought tolerance, with no discernible impact in response to salinity. The finding suggests the utility of the beneficial Osckx2 allele in breeding to develop climate-resilient, high-yielding cultivars for future food security.
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Citocininas , Resistencia a la Sequía , Oryza , Oxidorreductasas , Proteínas de Plantas , Citocininas/metabolismo , Resistencia a la Sequía/genética , Grano Comestible/genética , Grano Comestible/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Mutación/genética , Oryza/genética , Oryza/fisiología , Oryza/crecimiento & desarrollo , Oryza/enzimología , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Fotosíntesis/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Estrés Fisiológico/genéticaRESUMEN
Catalase (CAT) is the main reactive oxygen species (ROS)-scavenging enzyme in plants and insects. However, it remains elusive whether and how insect saliva CAT suppresses ROS-mediated plant defense, thereby promoting initial virus transmission by insect vectors. Here, we investigated how leafhopper Recilia dorsalis catalase (RdCAT) was secreted from insect salivary glands into rice phloem, and how it was perceived by rice chaperone NO CATALASE ACTIVITY1 (OsNCA1) to scavenge excessive H2O2 during insect-to-plant virus transmission. We found that the interaction of OsNCA1 with RdCAT activated its enzymatic activity to decompose H2O2 in rice plants during leafhopper feeding. However, initial insect feeding did not significantly change rice CATs transcripts. Knockout of OsNCA1 in transgenic lines decreased leafhopper feeding-activated CAT activity and caused higher H2O2 accumulation. A devastating rice reovirus activated RdCAT expression and promoted the cosecretion of virions and RdCAT into leafhopper salivary cavities and ultimately into the phloem. Virus-mediated increase of RdCAT secretion suppressed excessive H2O2, thereby promoting host attractiveness to insect vectors and initial virus transmission. Our findings provide insights into how insect saliva CAT is secreted and perceived by plant chaperones to suppress the early H2O2 burst during insect feeding, thereby facilitating viral transmission.
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Catalasa , Hemípteros , Peróxido de Hidrógeno , Insectos Vectores , Oryza , Saliva , Animales , Peróxido de Hidrógeno/metabolismo , Hemípteros/virología , Hemípteros/fisiología , Saliva/virología , Saliva/enzimología , Catalasa/metabolismo , Catalasa/genética , Insectos Vectores/virología , Oryza/virología , Oryza/genética , Oryza/enzimología , Reoviridae/fisiología , Enfermedades de las Plantas/virología , Floema/virologíaRESUMEN
Alpha-amylase (AMY) plays a significant role in regulating the growth, development, and postharvest quality formation in plants. Nevertheless, little is known about the genome-wide features, expression patterns, subcellular localization, and functional regulation of AMY genes (MaAMYs) in the common starchy banana (Musa acuminata). Twelve MaAMY proteins from the banana genome database were clustered into two groups and contained a conserved catalytic domain. These MaAMYs formed collinear pairs with the AMYs of maize and rice. Three tandem gene pairs were found within the MaAMYs and are indicative of putative gene duplication events. Cis-acting elements of the MaAMY promoters were found to be involved in phytohormone, development, and stress responses. Furthermore, MaAMY02, 08, 09, and 11 were actively expressed during fruit development and ripening. Specifically, MaAMY11 showed the highest expression level at the middle and later stages of banana ripening. Subcellular localization showed that MaAMY02 and 11 were predominately found in the chloroplast, whereas MaAMY08 and 09 were primarily localized in the cytoplasm. Notably, transient attenuation of MaAMY11 expression resulted in an obvious increase in the starch content of banana fruit, while a significant decrease in starch content was confirmed through the transient overexpression of MaAMY11. Together, these results reveal new insights into the structure, evolution, and expression patterns of the MaAMY family, affirming the functional role of MaAMY11 in the starch degradation of banana fruit.
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Regulación de la Expresión Génica de las Plantas , Musa , Filogenia , Proteínas de Plantas , alfa-Amilasas , Musa/genética , Musa/enzimología , Musa/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , alfa-Amilasas/genética , alfa-Amilasas/metabolismo , Frutas/genética , Frutas/crecimiento & desarrollo , Frutas/metabolismo , Regiones Promotoras Genéticas , Almidón/metabolismo , Oryza/genética , Oryza/enzimología , Oryza/crecimiento & desarrolloRESUMEN
Amino acids are necessary nutrients for the growth of Oryza sativa (rice), which can be mediated by amino acid transporter; however, our understanding of these transporters is still limited. This study found that the expression levels of amino acid permease gene OsAAP12 differed between indica and japonica rice. Altered expression of OsAAP12 negatively regulated tillering and yield in transgenic rice lines. Subcellular localization revealed that OsAAP12 was primarily localized to the plasma membrane. Moreover, it was indicated that OsAAP12 transported polar neutral amino acids asparagine (Asn), threonine (Thr), and serine (Ser) through experiments involving yeast heterologous complementation, fluorescence amino acid uptake, and amino acid content determination. Additionally, exogenous application of amino acids Asn, Thr, and Ser suppressed axillary buds outgrowth in OsAAP12 overexpression lines compared with wild-type ZH11. Conversely, the opposite trend was observed in CRISPR mutant lines. RNA-seq analysis showed that the expression patterns of genes involved in the nitrogen and cytokinin pathways were generally altered in OsAAP12 modified lines. Hormone assays indicated that OsAAP12 mutant lines accumulated cytokinins in the basal part of rice, whereas overexpression lines had the opposite effect. In summary, CRISPR mutant of OsAAP12 boosted rice tillering and grain yield by coordinating the content of amino acids and cytokinins, which has potential application value in high-yield rice breeding.
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Sistemas de Transporte de Aminoácidos , Aminoácidos , Citocininas , Nitrógeno , Oryza , Proteínas de Plantas , Oryza/genética , Oryza/metabolismo , Oryza/crecimiento & desarrollo , Oryza/enzimología , Citocininas/metabolismo , Aminoácidos/metabolismo , Nitrógeno/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Grano Comestible/genética , Grano Comestible/crecimiento & desarrollo , Grano Comestible/metabolismo , Regulación de la Expresión Génica de las Plantas , Plantas Modificadas Genéticamente/genéticaRESUMEN
CrRLK1L subfamily members are involved in diverse growth- and development-related processes in Arabidopsis. However, the functions of their counterparts in rice are unknown. Here, OsANX expression was detected in developing inflorescences, mature pollen grains, and growing pollen tubes, and it was localized to the plasma membrane in pollen grains and tobacco epidermal cells. Homozygous osanx progeny could not be segregated from the CRISPR/Cas9-edited mutants osanx-c1+/- and osanx-c2+/-, and such progeny were segregated only occasionally from osanx-c3+/-. Further, all three alleles showed osanx male but not female gamete transmission defects, in line with premature pollen tube rupture in osanx-c3. Additionally, osanx-c3 exhibited precocious flowering, excessively branched inflorescences, and an extremely low seed setting rate of 1.4â¯%, while osanx-c2+/- and osanx-c3+/- had no obvious defects in inflorescence development or the seed setting rate compared to wild-type Nipponbare (Nip). Consistent with this, the complemented line pPS1:OsANX-GFP/osanx-c2 (PSC), in which the lack of OsANX expression was inflorescence-specific, showed slightly earlier flowering and overly-branched panicles. Multiple inflorescence meristem transition-related and inflorescence architecture-related genes were expressed at higher levels in osanx-c3 than in Nip; thus, they may partially account for the aforementioned mutant phenotypes. Our findings broaden our understanding of the biological functions of OsANX in rice.
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Inflorescencia , Oryza , Proteínas de Plantas , Tubo Polínico , Oryza/genética , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Oryza/enzimología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Inflorescencia/genética , Inflorescencia/crecimiento & desarrollo , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/genética , Flores/crecimiento & desarrollo , Flores/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
ent-Kaurene is a biosynthetic intermediate diterpene of phytohormone gibberellins, and is biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP). The successive cyclization is catalyzed by two distinct diterpene synthases, ent-CDP synthase (ent-CPS) and ent-kaurene synthase (KS). Homologs of these diterpene synthase genes have been reported to be involved in the biosynthesis of specialized-metabolic diterpenoids for defense in several plant species, including rice (Oryza sativa). These diterpene synthases consist of three domains, αßγ domains. Active sites of ent-CPS exist at the interface of ß and γ domain, while those of KS are located within the α domain. We herein carried out domain-deletion experiments using several KSs and KS like enzymes (KSLs) to obtain insights into the roles of domains other than active-site domains. As previously reported in taxadiene synthase, deletion of γ or ßγ domains drastically decreased activities of specialized-metabolic OsKSL5, OsKSL8, OsKSL7 and OsKSL10 in O. sativa. However, unexpectedly, only α domains of several gibberellin-biosynthetic KSs, including OsKS1 in O. sativa, AtKS in Arabidopsis thaliana, TaKS in wheat (Triticum aestivum) and BdKS1 in Brachypodium distachyon, retained their original functions. Additionally, the specialized-metabolic OsKSL4, which is closely related to OsKS1, also functioned without its ßγ domains. Domain-swapping experiments showed that replacing ßγ domains in OsKSL7 with those from other KS/KSLs retained the OsKSL7 activity. Moreover, deletion of ßγ domains of bifunctional PpCPS/KS in moss (Physcomitrella patens) drastically impaired its KS-related activity. Thus, we demonstrate that monofunctional gibberellin-biosynthetic KSs are the unique diterpene synthases that retain their functions without ßγ domains.
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Transferasas Alquil y Aril , Giberelinas , Oryza , Proteínas de Plantas , Giberelinas/metabolismo , Transferasas Alquil y Aril/metabolismo , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/química , Oryza/enzimología , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Dominio Catalítico , Diterpenos de Tipo Kaurano/metabolismo , Diterpenos de Tipo Kaurano/química , Arabidopsis/genética , Arabidopsis/enzimología , Arabidopsis/metabolismo , Diterpenos/metabolismo , Diterpenos/química , Dominios Proteicos , CatálisisRESUMEN
Mitochondria and plastids, originated as ancestral endosymbiotic bacteria, contain their own DNA sequences. These organelle DNAs (orgDNAs) are, despite the limited genetic information they contain, an indispensable part of the genetic systems but exist as multiple copies, making up a substantial amount of total cellular DNA. Given this abundance, orgDNA is known to undergo tissue-specific degradation in plants. Previous studies have shown that the exonuclease DPD1, conserved among seed plants, degrades orgDNAs during pollen maturation and leaf senescence in Arabidopsis. However, tissue-specific orgDNA degradation was shown to differ among species. To extend our knowledge, we characterized DPD1 in rice in this study. We created a genome-edited (GE) mutant in which OsDPD1 and OsDPD1-like were inactivated. Characterization of this GE plant demonstrated that DPD1 was involved in pollen orgDNA degradation, whereas it had no significant effect on orgDNA degradation during leaf senescence. Comparison of transcriptomes from wild-type and GE plants with different phosphate supply levels indicated that orgDNA had little impact on the phosphate starvation response, but instead had a global impact in plant growth. In fact, the GE plant showed lower fitness with reduced grain filling rate and grain weight in natural light conditions. Taken together, the presented data reinforce the important physiological roles of orgDNA degradation mediated by DPD1.
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Oryza , Oryza/genética , Oryza/metabolismo , Oryza/enzimología , Oryza/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Exonucleasas/metabolismo , Exonucleasas/genética , Edición Génica , Regulación de la Expresión Génica de las Plantas , ADN de Plantas/genética , ADN de Plantas/metabolismo , Polen/genética , Polen/metabolismo , Polen/crecimiento & desarrollo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Genoma de Planta , MutaciónRESUMEN
The effect of inhibiting retrogradation and changes in chain length distribution by AG and BE, which are texture-modifying enzymes, has been clarified. To ascertain in which part of the rice grain retrogradation occurs and which enzymes is most effective, the degree of retrogradation in each part of the rice grain was measured from the surface to the core of the same rice grain using a synchrotron radiation X-ray beam with a beam size of 100 µm. Retrogradation was effectively suppressed at all measurement sites by enzyme addition, although the effect of enzymes was greater at the surface. Rice grain sections were stained with iodine and eosin. A starch layer that does not easily form a complex with iodine was observed inside the protein layer at the surface of cooked rice. A starch layer with a long molecular chain that forms complexes with iodine was observed inside the rice grain.
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Enzima Ramificadora de 1,4-alfa-Glucano , Culinaria , Oryza , alfa-Glucosidasas , Oryza/química , Oryza/enzimología , alfa-Glucosidasas/química , alfa-Glucosidasas/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucano/química , Almidón/química , Almidón/metabolismoRESUMEN
The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops1,2. Often considered to be genome 'parasites', transposable elements (TEs) evolved to insert their DNA seamlessly into genomes3-5. Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type6-9. Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria10-12, we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis. We then translated this system into soybean-a major global crop in need of targeted insertion technology. We have engineered a TE 'parasite' into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.
Asunto(s)
Arabidopsis , Elementos Transponibles de ADN , Ingeniería Genética , Genoma de Planta , Mutagénesis Insercional , Plantas Modificadas Genéticamente , Transposasas , Arabidopsis/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Elementos Transponibles de ADN/genética , Elementos de Facilitación Genéticos/genética , Edición Génica/métodos , Ingeniería Genética/métodos , Genoma de Planta/genética , Mutagénesis Insercional/genética , Sistemas de Lectura Abierta/genética , Oryza/enzimología , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Transposasas/metabolismo , Transposasas/genéticaRESUMEN
Grain size is a crucial agronomic trait that determines grain weight and final yield. Although several genes have been reported to regulate grain size in rice (Oryza sativa), the function of Wall-Associated Kinase family genes affecting grain size is still largely unknown. In this study, we identified GRAIN WEIGHT AND NUMBER 1 (GWN1) using map-based cloning. GWN1 encodes the OsWAK74 protein kinase, which is conserved in plants. GWN1 negatively regulates grain length and weight by regulating cell proliferation in spikelet hulls. We also found that GWN1 negatively influenced grain number by influencing secondary branch numbers and finally increased plant grain yield. The GWN1 gene was highly expressed in inflorescences and its encoded protein is located at the cell membrane and cell wall. Moreover, we identified three haplotypes of GWN1 in the germplasm. GWN1hap1 showing longer grain, has not been widely utilized in modern rice varieties. In summary, GWN1 played a very important role in regulating grain length, weight and number, thereby exhibiting application potential in molecular breeding for longer grain and higher yield.
Asunto(s)
Grano Comestible , Oryza , Proteínas de Plantas , Semillas , Oryza/genética , Oryza/crecimiento & desarrollo , Oryza/enzimología , Grano Comestible/genética , Grano Comestible/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Semillas/crecimiento & desarrollo , Semillas/genética , Fenotipo , Regulación de la Expresión Génica de las Plantas , Clonación Molecular , Mapeo Cromosómico , Haplotipos , Pared Celular/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Genes de PlantasRESUMEN
Cultivated rice (Oryza sativa) produces a variety of diterpenoid-type phytoalexins. Diterpene synthase genes that are responsible for the biosynthesis of momilactones, phytocassanes, and oryzalexins have been identified in O. sativa cv. Nipponbare. OsKSL10 (Os12t0491800 in RAP and LOC_Os12g30824 in MSU) was previously identified as an enzyme catalyzing the conversion of ent-copalyl diphosphate to ent-sandaracopimaradiene for the production of oryzalexins A to F. Our previous study on Oryza rufipogon, a wild progenitor of Asian cultivated rice, showed that both OrKSL10 and OrKSL10ind from O. rufipogon accessions W1943 and W0106, respectively, closely related to the japonica and indica subspecies, converted ent-copalyl diphosphate to ent-miltiradiene. Thus, the functional conversion of ent-miltiradiene synthase into ent-sandaracopimaradiene synthase is implied to have occurred through natural amino acid mutations, the details of which have not been elucidated. In this study, we show that introduction of A654G substitution into OrKSL10 significantly alters its function into more closely resembling that of OsKSL10. Moreover, double substitution V546I/A654G almost completely converts the function of OrKSL10 into that of OsKSL10. On the other hand, the reversed substitution I546V/G654A was insufficient to convert the function of OsKSL10 into OrKSL10, indicating the introduction of additional substitution S522I is required for the functionality of OsKSL10. Lastly, point mutations at the 654A residue in OrKSL10 suggest that hydrophobic side chains at this position have a negative influence on the production of ent-sandaracopimaradiene.
Asunto(s)
Transferasas Alquil y Aril , Diterpenos , Oryza , Fitoalexinas , Proteínas de Plantas , Sesquiterpenos , Oryza/genética , Oryza/metabolismo , Oryza/enzimología , Sesquiterpenos/metabolismo , Sesquiterpenos/química , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Transferasas Alquil y Aril/química , Diterpenos/metabolismo , Diterpenos/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Indoles/metabolismo , Indoles/química , Secuencia de AminoácidosRESUMEN
MAIN CONCLUSION: The rice SnRK2 members SAPK4, SAPK5, SAPK7 and SAPK10 are positive regulators involved in the regulation of rice flowering, while other single mutants exhibited no effect on rice flowering. The rice SnRK2 family, comprising 10 members known as SAPK (SnRK2-Associated Protein Kinase), is pivotal in the abscisic acid (ABA) pathway and crucial for various biological processes, such as drought resistance and salt tolerance. Additionally, these members have been implicated in the regulation of rice heading date, a key trait influencing planting area and yield. In this study, we utilized gene editing technology to create mutants in the Songjing 2 (SJ2) background, enabling a comprehensive analyze the role of each SAPK member in rice flowering. We found that SAPK1, SAPK2, and SAPK3 may not directly participate in the regulatory network of rice heading date, while SAPK4, SAPK5, and SAPK7 play positive roles in rice flowering regulation. Notably, polygene deletion resulted in an additive effect on delaying flowering. Our findings corroborate the previous studies indicating the positive regulatory role of SAPK10 in rice flowering, as evidenced by delayed flowering observed in sapk9/10 double mutants. Moving forward, our future research will focus on analyzing the molecular mechanisms underlying SAPKs involvement in rice flowering regulation, aiming to enhance our understanding of the rice heading date relationship network and lay a theoretical foundation for breeding efforts to alter rice ripening dates.
Asunto(s)
Flores , Regulación de la Expresión Génica de las Plantas , Oryza , Proteínas de Plantas , Oryza/genética , Oryza/crecimiento & desarrollo , Oryza/fisiología , Oryza/enzimología , Flores/genética , Flores/crecimiento & desarrollo , Flores/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mutación , Edición Génica , Estrés Fisiológico/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Ácido Abscísico/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Flooding deprives plants of oxygen and thereby causes severe stress by interfering with energy production, leading to growth retardation. Enzymes and metabolites may help protect plants from waterlogging and hypoxic environmental conditions. Acetolactate synthase (ALS) is a key enzyme in the biosynthesis of branched-chain amino acids (BCAAs), providing the building blocks for proteins and various secondary metabolites. Additionally, under energy-poor conditions, free BCAAs can be used as an alternative energy source by mitochondria through a catabolic enzyme chain reaction. In this study, we characterized ALS-INTERACTING PROTEIN 1 (OsAIP1), which encodes the regulatory subunit of ALS in rice (Oryza sativa). This gene was expressed in all parts of the rice plant, and its expression level was significantly higher in submerged and low-oxygen environments. Rice transformants overexpressing OsAIP1 showed a higher survival rate under hypoxic stress than did non-transgenic control plants under the same conditions. The OsAIP1-overexpressing plants accumulated increased levels of BCAAs, demonstrating that OsAIP1 is an important factor in the hypoxia resistance mechanism. These results suggest that ALS proteins are part of a defense mechanism that improves the tolerance of plants to low-oxygen environments.