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      Rootstock mediates transcriptional regulation of citrulline metabolism in grafted watermelon Translated title: Mediação do porta-enxerto na regulação transcricional do metabolismo da citrulina na melancia enxertada

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          Abstract

          Abstract Citrulline is a non-essential amino acid, involved in key biological functions in plants and humans. Rootstocks have a major impact on citrulline accumulation in grafted watermelon. Information regarding rootstock induced changes in citrulline metabolism is elusive. To understand the regulatory mechanism, parallel changes in the expression profiles of citrulline metabolic genes and citrulline content of watermelon were monitored during the development of self-rooted watermelon and watermelon grafted onto pumpkin, wild and bottle gourd rootstocks. Results demonstrated that rootstocks regulated the expression profiles in different ways to influence the citrulline content. GAT, NAGPR, ASS3 ASS2 and Asl2 showed the negative correlation with citrulline content in pumpkin grafted watermelon. Pumpkin rootstock promoted the citrulline content by high down-regulation and synergistic effect of ASS2, ASS3, ASL1 and ASl2 genes. In wild grafted watermelon, citrulline was accumulated as a result of down regulation of GAT, NAGS and ASL2 genes, which showed an inverse correlation with citrulline. In gourd grafted watermelon, changes in citrulline content were observed to be linked with lower expressions of GAT, NAGK, ASS2, ASS3, ASL1 and ARG which were negatively correlated with citrulline content. Our study will provide the basis to understand the molecular mechanism of citrulline accumulation in various rootstocks.

          Translated abstract

          Resumo A citrulina é um aminoácido não essencial, envolvida em importantes funções biológicas de plantas e seres humanos. Os porta-enxertos têm um grande impacto no acúmulo de citrulina na melancia enxertada. Informações sobre alterações induzidas por porta-enxertos no metabolismo da citrulina ainda não foram descritas. Para entender o mecanismo regulatório, foram monitoradas mudanças paralelas nos perfis de expressão dos genes metabólicos de citrulina e no teor de citrulina da melancia durante o desenvolvimento da melancia e da melancia enxertada em porta-enxertos de abóbora, silvestre e cabaça. Os resultados demonstraram que o porta-enxerto regulou os perfis de expressão de diferentes maneiras para influenciar no conteúdo de citrulina. GAT, NAGPR, ASS3, ASS2 e ASL2 apresentaram correlação negativa com o teor de citrulina em melancia enxertada de abóbora. O porta-enxerto de abóbora promoveu o conteúdo de citrulina por meio de baixa regulação e efeito sinérgico de duas famílias de genes ASS e ASL. Na melancia enxertada, a acumulação de citrulina resultou na regulação negativa de GAT, NAGS e ASL2, que mostraram uma correlação inversa com a citrulina. Na melancia enxertada, observou-se que as alterações no conteúdo de citrulina foram associadas a menores expressões de GAT, NAGK, ASS2, ASS3, ASL1 e ARG, que foram negativamente correlacionadas com o conteúdo de citrulina. Esses resultados fornecem a base para identificar o mecanismo molecular do acúmulo de citrulina em vários porta-enxertos.

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction

            Background Choosing appropriate primers is probably the single most important factor affecting the polymerase chain reaction (PCR). Specific amplification of the intended target requires that primers do not have matches to other targets in certain orientations and within certain distances that allow undesired amplification. The process of designing specific primers typically involves two stages. First, the primers flanking regions of interest are generated either manually or using software tools; then they are searched against an appropriate nucleotide sequence database using tools such as BLAST to examine the potential targets. However, the latter is not an easy process as one needs to examine many details between primers and targets, such as the number and the positions of matched bases, the primer orientations and distance between forward and reverse primers. The complexity of such analysis usually makes this a time-consuming and very difficult task for users, especially when the primers have a large number of hits. Furthermore, although the BLAST program has been widely used for primer target detection, it is in fact not an ideal tool for this purpose as BLAST is a local alignment algorithm and does not necessarily return complete match information over the entire primer range. Results We present a new software tool called Primer-BLAST to alleviate the difficulty in designing target-specific primers. This tool combines BLAST with a global alignment algorithm to ensure a full primer-target alignment and is sensitive enough to detect targets that have a significant number of mismatches to primers. Primer-BLAST allows users to design new target-specific primers in one step as well as to check the specificity of pre-existing primers. Primer-BLAST also supports placing primers based on exon/intron locations and excluding single nucleotide polymorphism (SNP) sites in primers. Conclusions We describe a robust and fully implemented general purpose primer design tool that designs target-specific PCR primers. Primer-BLAST offers flexible options to adjust the specificity threshold and other primer properties. This tool is publicly available at http://www.ncbi.nlm.nih.gov/tools/primer-blast.
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              Physiological implications of arginine metabolism in plants

              Nitrogen is a limiting resource for plant growth in most terrestrial habitats since large amounts of nitrogen are needed to synthesize nucleic acids and proteins. Among the 21 proteinogenic amino acids, arginine has the highest nitrogen to carbon ratio, which makes it especially suitable as a storage form of organic nitrogen. Synthesis in chloroplasts via ornithine is apparently the only operational pathway to provide arginine in plants, and the rate of arginine synthesis is tightly regulated by various feedback mechanisms in accordance with the overall nutritional status. While several steps of arginine biosynthesis still remain poorly characterized in plants, much wider attention has been paid to inter- and intracellular arginine transport as well as arginine-derived metabolites. A role of arginine as alternative source besides glutamate for proline biosynthesis is still discussed controversially and may be prevented by differential subcellular localization of enzymes. Apparently, arginine is a precursor for nitric oxide (NO), although the molecular mechanism of NO production from arginine remains unclear in higher plants. In contrast, conversion of arginine to polyamines is well documented, and in several plant species also ornithine can serve as a precursor for polyamines. Both NO and polyamines play crucial roles in regulating developmental processes as well as responses to biotic and abiotic stress. It is thus conceivable that arginine catabolism serves on the one hand to mobilize nitrogen storages, while on the other hand it may be used to fine-tune development and defense mechanisms against stress. This review summarizes the recent advances in our knowledge about arginine metabolism, with a special focus on the model plant Arabidopsis thaliana, and pinpoints still unresolved critical questions.
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                Author and article information

                Journal
                bjb
                Brazilian Journal of Biology
                Braz. J. Biol.
                Instituto Internacional de Ecologia (São Carlos, SP, Brazil )
                1519-6984
                1678-4375
                February 2021
                : 81
                : 1
                : 125-136
                Affiliations
                [01] Zhengzhou Henan orgnameChinese Academy of Agricultural Sciences orgdiv1Zhengzhou Fruit Research Institute China
                Article
                S1519-69842021000100125 S1519-6984(21)08100100125
                10.1590/1519-6984.223633
                a37a1079-394c-428f-944b-917638e23d88

                This work is licensed under a Creative Commons Attribution 4.0 International License.

                History
                : 07 May 2019
                : 17 October 2019
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 55, Pages: 12
                Product

                SciELO Brazil

                Categories
                Original Article

                qRT-PCR,grafting,Citrullus lanatus,citrulline,gene expression,citrulina,enxerto,expressão gênica

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