Genetic and molecular mechanisms involved in protein compensation in wheat grain: implications for obtaining varieties with low-immunogenic gluten

Prolamins, or gluten proteins, play an important role in the bread-making quality of wheat. These proteins are divided into two main fractions: gliadins and glutenins. In addition to their viscoelastic properties, gluten proteins are responsible for triggering the autoimmune-response in patients suffering from celiac disease (CD), a chronic enteropathy given in genetically predisposed individuals that affects 0.7% of the global population. The only available treatment is to follow a life-long gluten-free diet, which is difficult due to the large number of products that are based on wheat or present it as an additive. Biotechnological tools have been applied to obtain wheat varieties with lowgluten content. RNA of interference (RNAi) has been one of those, providing the silencing of the three groups of gliadins: ω-, α- and γ-gliadins. The synthesis of gluten proteins is influenced by the environment: among the factors that affect the grain composition, temperature and nitrogen (N) stand out, especially during the grain-filling stage. In the second chapter of this thesis, gliadins and glutenins from four RNAi lines with low-gluten content have been studied under heat stress and two nitrogen (N) treatments. Quantification was performed by reverse-phase high-performance liquid chromatography (RP-HPLC). In addition to the effect of heat stress decreasing grain weight in all the lines tested, an increase in the prolamin content was observed in the control lines (without the silencing fragment) due to temperature. Except for ω-gliadins, the rest of the prolamins of the RNAi lines did not present any effect on their content in the environmental conditions tested, while the proteins of the control lines were affected. This indicates great stability of the silencing character in the RNAi lines, which gives them value in the context of increased temperature and heat stress events, and environments with different N availability. In this work, as well as in previous ones, the RNAi lines showed a total grain N content similar to that of the control lines, despite the great decrease in the gliadin content. This phenomenon of compensation or readjustment of the grain protein composition gave rise to the next two chapters. The third chapter presents the results of gene expression by RT-qPCR and RNAseq from two RNAi lines with two different endosperm promoters driving the silencing fragment. In both lines, the compensation was produced by the increase in the nongluten proteins (NGPs) and the high molecular weight (HMW) subunits of glutenins. The increase in the expression of the genes that encode these subunits is already notable in the RNAi lines at 18 days after anthesis (DAA), as well as the transcription factors (TFs) related to the regulation of prolamins synthesis. In addition, the expression of genes encoding NGPs (globulins, serpins, ns-LTPs, and triticins) was also higher in the RNAi lines. Moreover, other changes at the transcriptome level were observed in the RNAi lines, particularly those related to enzyme regulation activity, carbohydrate metabolism, and stress response. Regarding the latter, many of the genes differentially expressed in the RNAi lines involved in this process are regulated by the abscisic acid (ABA), which could suggest the involvement of this phytohormone in the stress response observed at the expression level in the RNAi lines.Las prolaminas, o proteínas del gluten, tienen un papel fundamental en la calidad harino-panadera del trigo. Estas proteínas se dividen en dos grandes fracciones: gliadinas y gluteninas. Además de sus propiedades viscoelásticas, las proteínas del gluten son las responsables de desencadenar la respuesta autoinmune en pacientes con enfermedad celíaca (EC), una enteropatía crónica en individuos genéticamente predispuestos que afecta al 0.7% de la población mundial. El único tratamiento posible es seguir una dieta libre de gluten de por vida, lo que resulta difícil debido a la gran cantidad de productos basados en trigo o que lo presentan como aditivo. La aplicación de herramientas biotecnológicas ha permitido obtener variedades de trigo con bajo contenido en gluten. El ARN de interferencia (ARNi) ha sido una de las utilizadas, teniendo como objetivo las tres fracciones de gliadinas: ω-, α- y γ. La síntesis de estas proteínas está influenciada por el ambiente: entre los factores que afectan a la composición del grano, destacan la temperatura y la disponibilidad de nitrógeno (N), especialmente durante el estadio de llenado del grano. En el segundo capítulo de este trabajo, gliadinas y gluteninas de cuatro líneas ARNi con bajo contenido en gluten han sido cuantificadas bajo estrés térmico y dos tratamientos de N. La cuantificación se realizó mediante cromatografía líquida de alta resolución en fase reversa (RP-HPLC, de sus siglas en inglés). Además del efecto del estrés térmico disminuyendo el peso del grano en todas las líneas ensayadas, se observó un incremento de las prolaminas en las líneas control (sin el fragmento de silenciamiento) debido a la temperatura. A excepción de las ω-gliadinas, el resto de las prolaminas de las líneas ARNi no se vieron afectadas por los cambios ambientales ensayados, mientras que estos sí influyeron en las proteínas de las líneas control. Esto indica una gran estabilidad del carácter de silenciamiento en las líneas ARNi, lo que las pone en valor en un contexto de cambio climático con incremento de temperatura y eventos de estrés térmico, y ambientes con distinta disponibilidad de N. En este trabajo, así como en otros anteriores, las líneas ARNi presentaron un contenido de N total en el grano similar al de las líneas control, pese a la gran disminución en el contenido de gliadinas. Este fenómeno de compensación o reajuste de la composición proteica del grano dio lugar a los dos siguientes capítulos. En el tercer capítulo se exponen los resultados del análisis de la expresión génica mediante RT-qPCR y RNA-seq de dos líneas ARNi con silenciamiento de gliadinas donde el fragmento de silenciamiento estaba bajo el control de dos promotores de endospermo distintos. En ambas líneas, la compensación se produjo por el incremento de las proteínas no pertenecientes al gluten (NGPs, de sus siglas en inglés) y de las subunidades de gluteninas de alto peso molecular (HMW, de sus siglas en inglés). El incremento de la expresión de los genes que codifican estas subunidades ya es notable en las líneas ARNi a los 18 días después de la antesis (DAA, de sus siglas en inglés), al igual que factores de transcripción (FTs) relacionados con la regulación de la síntesis de las prolaminas. Además, la expresión de los genes que codifican para las NGPs (globulinas, serpinas, ns-LTPs y triticinas) también fue mayor en las líneas con silenciamiento de gliadinas. También se observaron otros cambios a nivel de transcriptoma en las líneas ARNi, particularmente los relacionados con la regulación de la actividad enzimática, el metabolismo de los carbohidratos y la respuesta a estrés. En relación con este último, muchos de los genes implicados en este proceso, y diferencialmente expresados en las líneas ARNi, están regulados por el ácido abscísico (ABA), lo que podría sugerir la implicación de esta fitohormona en la respuesta a estrés observada a nivel de expresión en las líneas ARNi. El transcriptoma de otra línea ARNi con fuerte silenciamiento de todas las fracciones de gliadinas fue analizada en el cuarto capítulo. En esta ocasión se escogió la línea E82 para el análisis de expresión diferencial y se tomaron muestras de dos tejidos: el grano y la hoja en un estadio intermedio del llenado del grano. La línea E82 es una de las líneas ARNi con mayor reducción de gluten (ppm) y que, además, ha sido utilizada en ensayos clínicos con pacientes celíacos y sensibles al trigo. En este trabajo, el foco se puso principalmente en los elementos reguladores a nivel transcripcional, obteniéndose así una red de FTs candidatos a regular la síntesis de las proteínas de reserva de la semilla, incluyendo las prolaminas, y el almidón en el grano en las líneas ARNi. Tanto los genes codificantes de los FTs de la red, como los de las prolaminas y otras proteínas de reserva del grano, estuvieron subexpresados en la línea E82. Los resultados obtenidos en la hoja de la línea ARNi mostraron una gran cantidad de genes subexpresados relacionados con la actividad de las proteasas, el metabolismo de los amino-ácidos y de los azúcares, y su transporte. En relación a este último, cabe destacar que los transportadores específicos de prolina estuvieron subexpresados en la línea E82, mientras que los de lisina-histidina estuvieron sobreexpresados. Además del menor contenido en gluten, esta línea difiere de las analizadas en el capítulo anterior en que la compensación proteica en el grano se produce principalmente por las NGPs, concretamente las globulinas ricas en lisina, y no por las HMW. Los genes que codifican para las globulinas no estuvieron subexpresados en la línea ARNi ni presentaron relación alguna con la red de FTs, sugiriendo que son reguladas independientemente del resto de proteínas de reserva en el grano. Tanto la red de FTs descrita como la información relativa a las globulinas y los procesos en la hoja, contribuyen a incrementar el conocimiento relativo a la regulación de las prolaminas y la compensación proteica en las líneas con silenciamiento de las gliadinas. Además del ARNi, se ha utilizado la tecnología CRISPR/Cas para la obtención de líneas con bajo contenido en gluten. Debido a la complejidad de las regiones genómicas que codifican los genes de prolaminas y a que no todo el gluten es igual de inmunogénico, la caracterización de estas regiones es importante para diseñar programas de mejora genética de precisión, tanto tradicionales como basados en las nuevas herramientas de mejora como CRISPR/Cas. Con este objetivo se realizó el quinto capítulo, donde los complejos inmunogénicos de las - y -gliadinas fueron caracterizados mediante secuenciación masiva en un conjunto de líneas de trigo harinero, trigo duro y tritórdeo, incluyendo líneas de trigo con la translocación de centeno 1BL/1RS. Se identificaron y cuantificaron los epítopos y péptidos inmunogénicos relacionados con la EC en los amplicones de las α- y γ-gliadinas, incluyendo también las 40k-γ-secalinas. Los resultados de este capítulo permitieron conocer la complejidad estructural de estos complejos inmunogénicos en trigo, y clasificar las líneas con mayor y menor potencial inmunogénicos, así como cuantificar la abundancia de los epítopos relacionados con le EC, incluyendo el 33-mer, uno de los péptidos más inmunodominantes que se conocen. Estos resultados permitirán seleccionar variedades y su incorporación en programas de mejora genética clásica o su uso directo mediante edición genética con CRISPR/Cas

Prolamin content and grain weight in RNAi silenced wheat lines under different conditions of temperature and nitrogen availability

Temperature and nitrogen (N) availability are two important environmental factors that may produce important changes in grain composition during grain filling of bread wheat. In this study, four wheat lines with the down-regulation of gliadins by means of RNA interference (RNAi) have been characterized to determine the effect of thermal stress and N availability on grain weight and quality; with focus on gliadin and glutenin protein fractions. Grain weight was reduced with heat stress (HS) in all RNAi lines, whereas gliadin content was increased in the wild-types. With respect to gliadin content, RNAi lines responded to HS and N availability differently from their respective wild-types, except for ω-gliadin content, indicating a very clear stability of silencing under different environmental conditions. In a context of increased temperature and HS events, and in environments with different N availability, the RNAi lines with down-regulated gliadins seem well suited for the production of wheat grain with low gliadin content.The Spanish Ministry of Economy and Competitiveness (Project AGL2016-80566-P) and the European Regional Development Fund (FEDER) supported this research

Oral Consumption of Bread from an RNAi Wheat Line with Strongly Silenced Gliadins Elicits No Immunogenic Response in a Pilot Study with Celiac Disease Patients

Celiac disease (CD) is a genetically predisposed, T cell-mediated and autoimmune-like disorder caused by dietary exposure to the storage proteins of wheat and related cereals. A gluten-free diet (GFD) is the only treatment available for CD. The celiac immune response mediated by CD4+ T-cells can be assessed with a short-term oral gluten challenge. This study aimed to determine whether the consumption of bread made using flour from a low-gluten RNAi wheat line (named E82) can activate the immune response in DQ2.5-positive patients with CD after a blind crossover challenge. The experimental protocol included assessing IFN-γ production by peripheral blood mononuclear cells (PBMCs), evaluating gastrointestinal symptoms, and measuring gluten immunogenic peptides (GIP) in stool samples. The response of PBMCs was not significant to gliadin and the 33-mer peptide after E82 bread consumption. In contrast, PBMCs reacted significantly to Standard bread. This lack of immune response is correlated with the fact that, after E82 bread consumption, stool samples from patients with CD showed very low levels of GIP, and the symptoms were comparable to those of the GFD. This pilot study provides evidence that bread from RNAi E82 flour does not elicit an immune response after a short-term oral challenge and could help manage GFD in patients with CD.This research was funded by The Spanish Ministry of Science and Innovation, Agencia Estatal de Investigación (Project PID2019-110847RB-I00), Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Junta de Andalucía (Project P20_01005), and “ERDF A way of making Europe”, by the “European Union”

Oral consumption of bread from an RNAi wheat line with strongly silenced gliadins elicits no immunogenic response in a pilot study with celiac disease patients

Celiac disease (CD) is a genetically predisposed, T cell-mediated and autoimmune-like disorder caused by dietary exposure to the storage proteins of wheat and related cereals. A gluten-free diet (GFD) is the only treatment available for CD. The celiac immune response mediated by CD4+ T-cells can be assessed with a short-term oral gluten challenge. This study aimed to determine whether the consumption of bread made using flour from a low-gluten RNAi wheat line (named E82) can activate the immune response in DQ2.5-positive patients with CD after a blind crossover challenge. The experimental protocol included assessing IFN-γ production by peripheral blood mononuclear cells (PBMCs), evaluating gastrointestinal symptoms, and measuring gluten immu-nogenic peptides (GIP) in stool samples. The response of PBMCs was not significant to gliadin and the 33-mer peptide after E82 bread consumption. In contrast, PBMCs reacted significantly to Standard bread. This lack of immune response is correlated with the fact that, after E82 bread consump-tion, stool samples from patients with CD showed very low levels of GIP, and the symptoms were comparable to those of the GFD. This pilot study provides evidence that bread from RNAi E82 flour does not elicit an immune response after a short-term oral challenge and could help manage GFD in patients with CD.Ministerio de Ciencia e Innovación PID2019-110847RB-I00Junta de Andalucía P20_0100

Metabolic plasticity is an essential requirement of acquired tyrosine kinase inhibitor resistance in Chronic myeloid leukemia

Tyrosine kinase inhibitors (TKIs) are currently the standard chemotherapeutic agents for the treatment of chronic myeloid leukemia (CML). However, due to TKI resistance acquisition in CML patients, identification of new vulnerabilities is urgently required for a sustained response to therapy. In this study, we have investigated metabolic reprogramming induced by TKIs independent of BCR-ABL1 alterations. Proteomics and metabolomics profiling of imatinib-resistant CML cells (ImaR) was performed. KU812 ImaR cells enhanced pentose phosphate pathway, glycogen synthesis, serine-glycine-one-carbon metabolism, proline synthesis and mitochondrial respiration compared with their respective syngeneic parental counterparts. Moreover, the fact that only 36% of the main carbon sources were utilized for mitochondrial respiration pointed to glycerol-phosphate shuttle as mainly contributors to mitochondrial respiration. In conclusion, CML cells that acquire TKIs resistance present a severe metabolic reprogramming associated with an increase in metabolic plasticity needed to overcome TKI-induced cell death. Moreover, this study unveils that KU812 Parental and ImaR cells viability can be targeted with metabolic inhibitors paving the way to propose novel and promising therapeutic opportunities to overcome TKI resistance in CML

UGR Empática: programa de intervención para el desarrollo de la empatía en el alumnado de Ciencias de la Salud y Ciencias de la Educación

La capacidad empática es fundamental para garantizar el éxito profesional en las carreras de Ciencias de la Educación y Ciencias de la Salud. En tanto que se trata de una capacidad que puede entrenarse, este proyecto pretende desarrollar la empatía en el estudiantado de estas ramas del conocimiento. Para ello se diseñó una acción de innovación docente consistente en una serie de materiales didácticos en formato multimedia en los que se abordan diferentes aspectos de la empatía y que han sido empleados por los docentes de diferentes materias durante sus clases. Los materiales se han agrupado en 8 sesiones, cada una de ellas conteniendo dos actividades breves. Las actividades han sido presentaciones en videos en torno a 5 minutos y casos prácticos de elaboración propia de los componentes del equipo del PID. En cada sesión se ha incluido un ejercicio de aprendizaje por autoevaluación. La duración ha sido de 8 semanas, a razón de una sesión compuesta por las 2 actividades cada semana. Para determinar la eficacia de la acción innovadora se ha evaluado la capacidad de empatía en el estudiantado antes de iniciar el proyecto y tras la finalización del mismo, a través del Índice de Reactividad Interpersonal (IRI), una escala que mide la empatía de forma multidimensional. El IRI ofrece una medida compuesta de la empatía, incluyendo su dimensión cognitiva y afectiva. La empatía cognitiva se mide mediante el grado en el que la persona comprende el punto de vista de otra persona. El IRI tiene 2 escalas para medir empatía cognitiva: a) la Escala de Toma de Perspectiva, que evalúa cómo la persona intenta adoptar las perspectivas de otras personas y ver las cosas desde su punto de vista; y b) la Escala de fantasía, que mide la tendencia a identificarse con los personajes de las películas, novelas, obras teatrales y otras situaciones ficticias. La de mayor interés para este proyecto es la primera. En cuanto a la empatía afectiva o emocional, el IRI intenta captar la respuesta emocional de la persona cuando observa el estado afectivo de otras personas, y lo hace con otras 2 escalas: c) Escala de Preocupación Empática consta de ítems sobre los sentimientos de simpatía, compasión y preocupación por los demás; y d) la Escala de Angustia Personal, que valora si la persona experimenta ansiedad y malestar cuando observa que otros están atravesando por experiencias negativas. En nuestro estudio, el interés estaba en que aumentase la puntuación en la escala de preocupación empática, pero no en la de angustia personal. Los objetivos específicos fueron la mejora en las dimensiones de la capacidad empática más relevantes, considerando que la toma de perspectiva y la preocupación empática son las más relacionadas con el desempeño profesional del estudiantado al que iba dirigido el proyecto. En general, los resultados indican que el conjunto del alumnado que ha participado en el PID ha mejorado su capacidad empática. Por tanto, podemos considerar que los materiales creados son válidos para tal fin y pueden ser de aplicación práctica a la docencia.Empathic skills are fundamental to ensure professional success in the fields of Education and Health Sciences. As it is an ability that can be trained, this project aims to develop empathy in students of these branches of knowledge. To this end, a teaching innovation action was designed consisting of a series of teaching materials in multimedia format in which different aspects of empathy are addressed and which have been used by teachers of different subjects during their classes. The materials have been grouped into 8 sessions, each containing two short activities. The activities consisted of 5-minute video presentations and case studies developed by the IDP team. Each session included a self-assessment learning exercise. The duration was 8 weeks, with one session consisting of the 2 activities each week. To determine the effectiveness of the innovative action, the students' capacity for empathy was assessed before the start of the project and after its completion, using the Interpersonal Reactivity Index (IRI), a scale that measures empathy in a multidimensional way. The IRI provides a composite measure of empathy, including both cognitive and affective dimensions. Cognitive empathy is measured by the degree to which a person understands another person's point of view. The IRI has 2 scales to measure cognitive empathy: a) the Perspective Taking Scale, which assesses how the person tries to adopt other people's perspectives and see things from their point of view; and b) the Fantasy Scale, which measures the tendency to identify with characters in films, novels, plays and other fictional situations. Of most interest for this project is the first one. As for affective or emotional empathy, the IRI attempts to capture the person's emotional response when observing the affective state of other people, and does so with 2 other scales: c) the Empathic Concern Scale consists of items on feelings of sympathy, compassion and concern for others; and d) the Personal Distress Scale, which assesses whether the person experiences anxiety and discomfort when observing that others are going through negative experiences. In our study, the focus was on increasing scores on the empathic concern scale, but not on the personal distress scale. The specific objectives were to improve the most relevant dimensions of empathic ability, considering that perspective-taking and empathic concern are the most related to the professional performance of the target students. In general, the results indicate that the students who participated in the IDP as a whole have improved their empathic ability. Therefore, we can consider that the materials created are valid for this purpose and can be of practical application in teaching

COVID-19 outbreaks in a transmission control scenario: challenges posed by social and leisure activities, and for workers in vulnerable conditions, Spain, early summer 2020

Severe acute respiratory syndrome coronavirus 2 community-wide transmission declined in Spain by early May 2020, being replaced by outbreaks and sporadic cases. From mid-June to 2 August, excluding single household outbreaks, 673 outbreaks were notified nationally, 551 active (>6,200 cases) at the time. More than half of these outbreaks and cases coincided with: (i) social (family/friends’ gatherings or leisure venues) and (ii) occupational (mainly involving workers in vulnerable conditions) settings. Control measures were accordingly applied

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

RNAi silencing of wheat gliadins alters the network of transcription factors that regulate the synthesis of seed storage proteins toward maintaining grain protein levels

Gluten proteins are responsible for the unique viscoelastic properties of wheat dough, but they also trigger the immune response in celiac disease patients. RNA interference (RNAi) wheat lines with strongly silenced gliadins were obtained to reduce the immunogenic response of wheat. The E82 line presents the highest reduction of gluten, but other grain proteins increased, maintaining a total nitrogen content comparable to that of the wild type. To better understand the regulatory mechanisms in response to gliadin silencing, we carried out a transcriptomic analysis of grain and leaf tissues of the E82 line during grain filling. A network of candidate transcription factors (TFs) that regulates the synthesis of the seed storage proteins (SSPs), α-amylase/trypsin inhibitors, lipid transfer proteins, serpins, and starch in the grain was obtained. Moreover, there were a high number of differentially expressed genes in the leaf of E82, where processes such as nutrient availability and transport were enriched. The source-sink communication between leaf and grain showed that many down-regulated genes were related to protease activity, amino acid and sugar metabolism, and their transport. In the leaf, specific proline transporters and lysine-histidine transporters were down- and up-regulated, respectively. Overall, the silencing of gliadins in the RNAi line is compensated mainly with lysine-rich globulins, which are not related to the proposed candidate network of TFs, suggesting that these proteins are regulated independently of the other SSPs. Results reported here can explain the protein compensation mechanisms and contribute to decipher the complex TF network operating during grain filling.This research was supported by grant PID2019-110847RB-I00 funded by MCIN/AEI/10.13039/501100011033, and grant P20_01005 funded by Consejería de Transformación Económica, Industria, Conocimiento y Universidades (Junta de Andalucía).Peer reviewe

Table_8_RNAi silencing of wheat gliadins alters the network of transcription factors that regulate the synthesis of seed storage proteins toward maintaining grain protein levels.xlsx

Supplementary Table 8. Top 3 Gene Ontology (GO) terms enriched for each candidate transcription factor (TF) summarized from previously published GO enrichment analysis for GENIE3 network genes in Ramírez-González et al., 2018. The black boxes indicate that this is an enriched GO for this TF gene.Gluten proteins are responsible for the unique viscoelastic properties of wheat dough, but they also trigger the immune response in celiac disease patients. RNA interference (RNAi) wheat lines with strongly silenced gliadins were obtained to reduce the immunogenic response of wheat. The E82 line presents the highest reduction of gluten, but other grain proteins increased, maintaining a total nitrogen content comparable to that of the wild type. To better understand the regulatory mechanisms in response to gliadin silencing, we carried out a transcriptomic analysis of grain and leaf tissues of the E82 line during grain filling. A network of candidate transcription factors (TFs) that regulates the synthesis of the seed storage proteins (SSPs), α-amylase/trypsin inhibitors, lipid transfer proteins, serpins, and starch in the grain was obtained. Moreover, there were a high number of differentially expressed genes in the leaf of E82, where processes such as nutrient availability and transport were enriched. The source-sink communication between leaf and grain showed that many down-regulated genes were related to protease activity, amino acid and sugar metabolism, and their transport. In the leaf, specific proline transporters and lysine-histidine transporters were down- and up-regulated, respectively. Overall, the silencing of gliadins in the RNAi line is compensated mainly with lysine-rich globulins, which are not related to the proposed candidate network of TFs, suggesting that these proteins are regulated independently of the other SSPs. Results reported here can explain the protein compensation mechanisms and contribute to decipher the complex TF network operating during grain filling.Peer reviewe