A Genome-Wide Association Study Identifies New Loci Involved in Wound-Induced Lateral Root Formation in Arabidopsis thaliana

Root systems can display variable architectures that contribute to nutrient foraging or to increase the tolerance of abiotic stress conditions. Root tip excision promotes the developmental progression of previously specified lateral root (LR) founder cells, which allows to easily measuring the branching capacity of a given root as regards its genotype and/or growth conditions. Here, we describe the natural variation among 120 Arabidopsis thaliana accessions in root system architecture (RSA) after root tip excision. Wound-induced changes in RSA were associated with 19 genomic loci using genome-wide association mapping. Three candidate loci associated with wound-induced LR formation were investigated. Sequence variation in the hypothetical protein encoded by the At4g01090 gene affected wound-induced LR development and its loss-of-function mutants displayed a reduced number of LRs after root tip excision. Changes in a histidine phosphotransfer protein putatively involved in cytokinin signaling were significantly associated with LR number variation after root tip excision. Our results provide a better understanding of some of the genetic components involved in LR capacity variation among accessions

Estudio de los mecanismos fisiológicos y moleculares implicados en la formación y desarrollo de las raíces adventicias en distintas especies vegetales

The adventitious root formation is critical for the plant survival during its vegetative propagation through cuttings, hence the study and understanding of this process is strategic during mass propagation of the new elite varieties obtained by genetic breeding. This Thesis pursues genetic, morphological and hormonal studies of de novo root formation in the Arabidopsis thaliana (L.) Heynh. model, and in two other plant species of agronomic interest in the Mediterranean region, such as the cultivated carnation (Dianthus caryophyllus L.) and the argan tree (Argania spinosa [L.] Skeels). To identify some genetic determinants involved in woundinduced root formation process, we carried out a genomewide association (GWA) study in a collection of 174 Arabidopsis thaliana natural accessions. We found statistically significant associations between woundinduced root system architectural traits and 19 genomic loci. We then studied three candidate loci associated with woundinduced lateral root formation. Lossoffunction mutants in one of these genes, At4g01090, which encodes a protein of unknown function, were affected in woundinduced lateral root development. We identified null alleles in the At5g19710 gene, which encodes a histidine phosphotransfer protein putatively involved in cytokinin signalling, with a reduced number of lateral roots after root tip excision. In addition, we found an epistatic interaction between several nonsynonymous single nucleotide polymorphisms in the coding region of K+ UPTAKE TRANSPORTER 5 (KUP5), which encodes a potassium uptake transporter; and At5g19710, which would point to a functional relationship between both processes and woundinduced root formation. We studied morphological changes occurring in the basal region of stem cuttings during rooting in a collection of 159 F1 lines derived from a cross between two carnation hybrid cultivars, 210102 MFR and 2003 R 8, with contrasting rooting performances. We provided molecular evidence that the badrooting behaviour of 2003 R 8 cuttings was caused by an early inactivation of active auxin, indole3acetic acid, through its conjugation with aspartic acid by GRETCHEN HAGEN 3 proteins. Our analyses further elucidated the relationship between the physiological state of carnation cuttings, estimated by the proportion of salicylic acid and jasmonate, and the formation of adventitious roots. In addition, we developed an effective method for argan micropropagation from in vitro germinated seeds and we considerably increased the adventitious rooting from lignified cuttings obtained from adult plants. When combined with a suitable genetic selection, our procedure will allow the efficient production of elite varieties of argan trees for intensive cultivation.La formación de raíces adventicias supone un paso crítico y de vital importancia para la supervivencia de las plantas que se propagan vegetativamente mediante esquejes, por lo que el estudio y la comprensión de este proceso es clave para optimizar la propagación vegetativa de las nuevas variedades élite obtenidas por métodos convencionales de mejora genética. En esta Tesis se propone el estudio a nivel genético, morfológico y hormonal de la formación de raíces de novo en la crucífera modelo Arabidopsis thaliana (L.) Heynh., y en dos especies de interés agronómico en la región mediterránea: el clavel cultivado (Dianthus caryophyllus L.) y el argán (Argania spinosa [L.] Skeels). Para identificar algunos de los determinantes genéticos implicados en la formación de raíces en respuesta a herida, hemos llevado a cabo un estudio de asociación a genoma completo o GWA (genomewide association study) en una colección de 174 estirpes silvestres de Arabidopsis thaliana. Nuestros resultados indican que existen asociaciones estadísticamente significativas entre los cambios en la arquitectura radicular en respuesta a herida y 19 regiones genómicas concretas. Nos hemos centrado en el análisis genético de 6 polimorfismos de un solo nucleótido o SNPs (single nucleotide polymorphisms) que generan cambios no conservados de residuos aminoacídicos en la región codificante de tres genes. Los mutantes de pérdida de función en At4g01090, que codifica una proteína de función desconocida, muestran un menor número de raíces laterales en respuesta a herida. Hemos identificado alelos nulos en el gen At5g19710, que codifica una proteína con un dominio de histidina para la fosfotransferencia implicado en la señalización de las citoquininas, con un número reducido de raíces laterales en respuesta a herida. Además, hemos encontrado una interacción epistática entre los SNPs de la región codificante de K+ UPTAKE TRANSPORTER 5 (KUP5), cuyo producto participa en la asimilación del potasio, y At5g19710, lo que apuntaría a una relación funcional entre ambos procesos y con la formación de raíces en respuesta a herida. Por otra parte, hemos llevado a cabo un análisis detallado de la formación de raíces adventicias a partir de esquejes de clavel cultivado en una colección de 159 líneas F1 derivadas del cruzamiento entre dos variedades comerciales, 210102 MFR y 2003 R 8, que difieren de forma significativa en su capacidad de enraizamiento adventicio. Hemos proporcionado evidencias moleculares de que la baja capacidad de enraizamiento adventicio de los esquejes en la estirpe 2003 R 8 se debe a una inactivación temprana de la auxina activa, el ácido indolacético, en la base del esqueje mediante su conjugación con ácido aspártico por enzimas de la familia GRETCHEN HAGEN 3. Nuestros análisis han permitido dilucidar, además, la relación existente entre el estado fisiológico de los esquejes de clavel, estimada por la proporción de ácidos salicílico y jasmónico, y la formación de raíces adventicias. Adicionalmente, hemos desarrollado un método eficaz para la micropropagación del argán a partir de semillas germinadas in vitro, además de un protocolo que ha permitido incrementar considerablemente la formación de raíces adventicias a partir de esquejes lignificados obtenidos de plantas adultas. Combinado con una selección genética adecuada, nuestro procedimiento permitirá la producción eficiente de variedades élite de argán para su uso en cultivo intensivo

Effects of Auxin (Indole-3-butyric Acid) on Adventitious Root Formation in Peach-Based Prunus Rootstocks

18 Pags.- 5 Figs.- 3 Tabls. © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.Several Prunus species are among the most important cultivated stone fruits in the Mediterranean region, and there is an urgent need to obtain rootstocks with specific adaptations to challenging environmental conditions. The development of adventitious roots (ARs) is an evolutionary mechanism of high relevance for stress tolerance, which has led to the development of environmentally resilient plants. As a first step towards understanding the genetic determinants involved in AR formation in Prunus sp., we evaluated the rooting of hardwood cuttings from five Prunus rootstocks (Adafuel, Adarcias, Cadaman, Garnem, and GF 677) grown in hydroponics. We found that auxin-induced callus and rooting responses were strongly genotype-dependent. To investigate the molecular mechanisms involved in these differential responses, we performed a time-series study of AR formation in two rootstocks with contrasting rooting performance, Garnem and GF 677, by culturing in vitro microcuttings with and without auxin treatment (0.9 mg/L of indole-3-butyric acid [IBA]). Despite showing a similar histological structure, Garnem and GF677 rootstocks displayed dynamic changes in endogenous hormone homeostasis involving metabolites such as indole-3-acetic acid (IAA) conjugated to aspartic acid (IAA-Asp), and these changes could explain the differences observed during rooting.This research received no external funding. M.M. is a research fellow of the Generalitat Valenciana (GRISOLIAP/2019/098).Peer reviewe

Transcriptomic and hormonal analysis of the roots of maize seedlings grown hydroponically at low temperature

Prolonged cold stress has a strong effect on plant growth and development, especially in subtropical crops such as maize. Soil temperature limits primary root elongation, mainly during early seedling establishment. However, little is known about how moderate temperature fluctuations affect root growth at the molecular and physiological levels. We have studied root tips of young maize seedlings grown hydroponically at 30 ºC and after a short period (up to 24 h) of moderate cooling (20 ºC). We found that both cell division and cell elongation in the root apical meristem are affected by temperature. Time-course analyses of hormonal and transcriptomic profiles were achieved after temperature reduction from 30 ºC to 20 ºC. Our results highlighted a complex regulation of endogenous pathways leading to adaptive root responses to moderate cooling conditionsWork in JS lab was supported by the Junta de Extremadura and Fondo Europeo de Desarrollo Regional (FEDER) "Una manera de hacer Europa" (grants no. GR21167, GR21196, IB18035 and CCESAGROS). Work in JMPP lab was supported by the Conselleria d’Educació, Cultura i Sport of the Generalitat Valenciana and Fondo Europeo de Desarrollo Regional (FEDER) "Una manera de hacer Europa" (grants no. IDIFEDER 2018/016 and PROMETEO/2019/117)Peer reviewe

Enhanced Conjugation of Auxin by GH3 Enzymes Leads to Poor Adventitious Rooting in Carnation Stem Cuttings

Commercial carnation (Dianthus caryophyllus) cultivars are vegetatively propagated from axillary stem cuttings through adventitious rooting; a process which is affected by complex interactions between nutrient and hormone levels and is strongly genotype-dependent. To deepen our understanding of the regulatory events controlling this process, we performed a comparative study of adventitious root (AR) formation in two carnation cultivars with contrasting rooting performance, “2101–02 MFR” and “2003 R 8”, as well as in the reference cultivar “Master”. We provided molecular evidence that localized auxin response in the stem cutting base was required for efficient adventitious rooting in this species, which was dynamically established by polar auxin transport from the leaves. In turn, the bad-rooting behavior of the “2003 R 8” cultivar was correlated with enhanced synthesis of indole-3-acetic acid conjugated to aspartic acid by GH3 proteins in the stem cutting base. Treatment of stem cuttings with a competitive inhibitor of GH3 enzyme activity significantly improved rooting of “2003 R 8”. Our results allowed us to propose a working model where endogenous auxin homeostasis regulated by GH3 proteins accounts for the cultivar dependency of AR formation in carnation stem cuttings

Integration of phenotype and hormone data during adventitious rooting in carnation (Dianthus caryophyllus L.) stem cuttings

The rooting of stem cuttings is a highly efficient procedure for the vegetative propagation of ornamental plants. In cultivated carnations, an increased auxin level in the stem cutting base produced by active auxin transport from the leaves triggers adventitious root (AR) formation from the cambium. To provide additional insight into the physiological and genetic basis of this complex trait, we studied AR formation in a collection of 159 F1 lines derived from a cross between two hybrid cultivars (2003 R 8 and 2101-02 MFR) showing contrasting rooting performances. In three different experiments, time-series for several stem and root architectural traits were quantified in detail in a subset of these double-cross hybrid lines displaying extreme rooting phenotypes and their parental genotypes. Our results indicate that the water content and area of the AR system directly contributed to the shoot water content and shoot growth. Moreover, morphometric data and rooting quality parameters were found to be associated with some stress-related metabolites such as 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene precursor, and the conjugated auxin indol-3-acetic acid-aspartic acid (IAA-Asp).This research was funded by the Ministerio de Economía, Industria y Competitividad (MINECO) of Spain (grants no. AGL2012-33610 and BIO2015-64255-R), by the Center for the Development of Industrial Technology (CARNOMICS Eurostars-EUREKA Project E! 6384), and by the European Regional Development Fund (ERDF) of the European Commission.http://creativecommons.org/licenses/by/4.0/Peer reviewe

Image_2_Enhanced Conjugation of Auxin by GH3 Enzymes Leads to Poor Adventitious Rooting in Carnation Stem Cuttings.TIF

<p>Commercial carnation (Dianthus caryophyllus) cultivars are vegetatively propagated from axillary stem cuttings through adventitious rooting; a process which is affected by complex interactions between nutrient and hormone levels and is strongly genotype-dependent. To deepen our understanding of the regulatory events controlling this process, we performed a comparative study of adventitious root (AR) formation in two carnation cultivars with contrasting rooting performance, “2101–02 MFR” and “2003 R 8”, as well as in the reference cultivar “Master”. We provided molecular evidence that localized auxin response in the stem cutting base was required for efficient adventitious rooting in this species, which was dynamically established by polar auxin transport from the leaves. In turn, the bad-rooting behavior of the “2003 R 8” cultivar was correlated with enhanced synthesis of indole-3-acetic acid conjugated to aspartic acid by GH3 proteins in the stem cutting base. Treatment of stem cuttings with a competitive inhibitor of GH3 enzyme activity significantly improved rooting of “2003 R 8”. Our results allowed us to propose a working model where endogenous auxin homeostasis regulated by GH3 proteins accounts for the cultivar dependency of AR formation in carnation stem cuttings.</p

Image_4_Enhanced Conjugation of Auxin by GH3 Enzymes Leads to Poor Adventitious Rooting in Carnation Stem Cuttings.TIF

<p>Commercial carnation (Dianthus caryophyllus) cultivars are vegetatively propagated from axillary stem cuttings through adventitious rooting; a process which is affected by complex interactions between nutrient and hormone levels and is strongly genotype-dependent. To deepen our understanding of the regulatory events controlling this process, we performed a comparative study of adventitious root (AR) formation in two carnation cultivars with contrasting rooting performance, “2101–02 MFR” and “2003 R 8”, as well as in the reference cultivar “Master”. We provided molecular evidence that localized auxin response in the stem cutting base was required for efficient adventitious rooting in this species, which was dynamically established by polar auxin transport from the leaves. In turn, the bad-rooting behavior of the “2003 R 8” cultivar was correlated with enhanced synthesis of indole-3-acetic acid conjugated to aspartic acid by GH3 proteins in the stem cutting base. Treatment of stem cuttings with a competitive inhibitor of GH3 enzyme activity significantly improved rooting of “2003 R 8”. Our results allowed us to propose a working model where endogenous auxin homeostasis regulated by GH3 proteins accounts for the cultivar dependency of AR formation in carnation stem cuttings.</p

Image_5_Enhanced Conjugation of Auxin by GH3 Enzymes Leads to Poor Adventitious Rooting in Carnation Stem Cuttings.TIF

<p>Commercial carnation (Dianthus caryophyllus) cultivars are vegetatively propagated from axillary stem cuttings through adventitious rooting; a process which is affected by complex interactions between nutrient and hormone levels and is strongly genotype-dependent. To deepen our understanding of the regulatory events controlling this process, we performed a comparative study of adventitious root (AR) formation in two carnation cultivars with contrasting rooting performance, “2101–02 MFR” and “2003 R 8”, as well as in the reference cultivar “Master”. We provided molecular evidence that localized auxin response in the stem cutting base was required for efficient adventitious rooting in this species, which was dynamically established by polar auxin transport from the leaves. In turn, the bad-rooting behavior of the “2003 R 8” cultivar was correlated with enhanced synthesis of indole-3-acetic acid conjugated to aspartic acid by GH3 proteins in the stem cutting base. Treatment of stem cuttings with a competitive inhibitor of GH3 enzyme activity significantly improved rooting of “2003 R 8”. Our results allowed us to propose a working model where endogenous auxin homeostasis regulated by GH3 proteins accounts for the cultivar dependency of AR formation in carnation stem cuttings.</p

Table_1_Enhanced Conjugation of Auxin by GH3 Enzymes Leads to Poor Adventitious Rooting in Carnation Stem Cuttings.DOCX

<p>Commercial carnation (Dianthus caryophyllus) cultivars are vegetatively propagated from axillary stem cuttings through adventitious rooting; a process which is affected by complex interactions between nutrient and hormone levels and is strongly genotype-dependent. To deepen our understanding of the regulatory events controlling this process, we performed a comparative study of adventitious root (AR) formation in two carnation cultivars with contrasting rooting performance, “2101–02 MFR” and “2003 R 8”, as well as in the reference cultivar “Master”. We provided molecular evidence that localized auxin response in the stem cutting base was required for efficient adventitious rooting in this species, which was dynamically established by polar auxin transport from the leaves. In turn, the bad-rooting behavior of the “2003 R 8” cultivar was correlated with enhanced synthesis of indole-3-acetic acid conjugated to aspartic acid by GH3 proteins in the stem cutting base. Treatment of stem cuttings with a competitive inhibitor of GH3 enzyme activity significantly improved rooting of “2003 R 8”. Our results allowed us to propose a working model where endogenous auxin homeostasis regulated by GH3 proteins accounts for the cultivar dependency of AR formation in carnation stem cuttings.</p