Characterization of the phenylboronic acid-induced defects in primary roots of maize (Zea mays L.) and the association of boron homeostasis with the benzoxazinoid pathway
Characterization of the phenylboronic acid-induced defects in primary roots of maize (Zea mays L.) and the association of boron homeostasis with the benzoxazinoid pathway
8985.pdf (7.5MB)Type of Scholarly Publication
DissertationDate of Exam
20.03.2026Date of Publication
19.05.2026Advisor
Matthes, MichaelaCo-Referee
Schaaf, GabrielMetadata
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Abstract
Boron is an essential micronutrient for plants. In planta, boron predominantly resides in the primary cell wall, crosslinking the pectic polysaccharide rhamnogalacturonan II (RG-II), which is vital for maintaining cell wall integrity. Non-optimal levels of boron negatively affect the development of plants and reduce crop yield. In adaptation to variable soil boron conditions, plants have developed mechanisms to maintain boron homeostasis, of which the best known are boron transporters. In the cereal crop and model plant maize (Zea mays L.), the importance of boron during reproductive development is well studied, and genes encoding boron transporters have been characterized. However, the effects of boron deficiency on the maize root, and regulators of boron homeostasis in maize next to boron transporters, remain largely obscure.
To assess boron deficiency-induced defects in maize roots, one strategy is to induce boron deficiency using a chemical approach. Phenylboronic acid (PBA) was hypothesized to inhibit RG-II dimerization and therefore might mimic boron deficiency. However, the characterization of PBA as a boron deficiency mimic in planta has not been demonstrated.
To identify new regulators of boron homeostasis in maize, one strategy is to assess natural variation of boron concentration in association panels, and to subsequently analyze the phenotypic variation with the genetic variation through genome-wide association analysis (GWAS).
This thesis characterized the boron deficiency-induced defects in maize roots, tested the usability of PBA as a boron deficiency mimic, and identified additional regulators of boron homeostasis in maize leaves using GWAS.
In chapter 2, the primary root defects induced by boron deficiency and by PBA were characterized and compared. Both boron deficiency and PBA induced defects in primary root length, lateral root density, auxin levels and reactive oxygen species levels. Although the PBA-induced defects were similar to the boron deficiency-induced defects, the severity was different. Notably, PBA did not inhibit nor promote RG-II crosslinking in vitro, and PBA did not incorporate into pectin in vivo. Specifically, the PBA-induced lateral root density defects appeared linked to functions of the boric acid moiety. Using the ratios of primary root length and lateral root density between PBA treatment and no-PBA control, putative targets of PBA related to phytohormones, cell wall modification, endocytosis, and root development were detected through GWAS.
In chapter 3, a GWAS of boron levels in maize ear leaves was conducted to identify novel genetic regulators of boron homeostasis. Integrated analysis of the GWAS results and gene expression data highlighted benzoxazinless3 (bx3) as a promising candidate, suggesting an association between leaf boron homeostasis and the benzoxazinoid pathway, a well-characterized defense pathway. The loss-of-function mutation of bx3 in maize and the overexpression of BX1 and BX2 in Arabidopsis, a species that does not endogenously express the benzoxazinoid pathway, both resulted in elevated boron levels in leaves. Furthermore, the product of the BX3 enzyme function, 3-hydroxy-indolin-2-one (HION), was found to form a complex with boric acid in vitro. Our study, therefore, detected a novel connection between boron homeostasis and the benzoxazinoid pathway, likely through bx3 and the direct substrate and product of BX3, furthermore suggesting a potential target for crop engineering for better adaptability to low soil boron levels. Bor ist ein essentieller Mikronährstoff für Pflanzen. In Pflanzen befindet sich Bor überwiegend in der primären Zellwand, wo es das pektische Polysaccharid Rhamnogalacturonan II (RG-II) vernetzt. Diese Vernetzung ist entscheidend für die Aufrechterhaltung der Zellwandintegrität. Nicht-optimale Konzentrationen von Bor beeinträchtigen die Pflanzenentwicklung und verringern den Ernteertrag. Zur Aufrechterhaltung der Borhomöostase haben Pflanzen unterschiedliche Mechanismen entwickelt, von denen die bekanntesten über Bortransporter wirken. Bei der Nutz- und Modellpflanze Mais (Zea mays L.) ist die Bedeutung von Bor während der reproduktiven Entwicklung gut untersucht. Zusätzlich sind auch die Bortransporter in Mais charakterisiert. Dennoch bleiben die Auswirkungen von Bormangel auf die Maiswurzel sowie Regulatoren der Bor-Homöostase neben den Transportern weitgehend unklar.
Um Bormangel-bedingte Defekte in Maiswurzeln zu untersuchen, besteht eine Strategie darin, Bor-Mangelbedingungen chemisch zu induzieren. Es wird angenommen, dass Boronsäuren, wie Phenylborsäure (PBA), die Dimerisierung von RG-II blockieren und dadurch Bormangel imitieren. Eine Charakterisierung von PBA in Bezug auf dessen Fähigkeit Bormangel in planta zu induzieren wurde jedoch bisher nicht durchgeführt.
Zur Identifizierung neuer Regulatoren der Bor-Homöostase in Mais können beispielsweise genomweite Assoziationsanalysen (GWAS) durchgeführt werden. Dabei wird die natürliche Variation von Merkmalen, z. B. die Borkonzentration, in genetischen Populationen erfasst und anschließend die phänotypische Variation mit genetischen Informationen der Population assoziiert.
Diese Dissertation charakterisierte die durch Bormangel verursachten Defekte in Maiswurzeln, prüfte die Verwendbarkeit von PBA zur Induzierung von Bormangel und identifizierte neue Regulatoren der Bor-Homöostase in Mais.
In Kapitel 2 wurden die durch Bormangel und durch PBA induzierten Primärwurzeldefekte charakterisiert und verglichen. Sowohl Bormangel als auch PBA führten zu Defekten in der Primärwurzel-Länge, der Dichte der Seitenwurzeln, sowie dem Gehalt an Auxin und reaktiver Sauerstoffspezies. Obwohl die durch PBA induzierten Defekte denjenigen des Bormangels ähnelten, unterschieden sie sich in ihrer Ausprägung. Auffällig war, dass PBA in vitro weder die RG-II-Vernetzung hemmte noch förderte und in vivo nicht in Pektin eingebaut wurde. Insbesondere schienen die durch PBA hervorgerufenen Defekte in der Seitenwurzel-Dichte mit Funktionen der Borsäure-Gruppe zusammenzuhängen. Durch die Analyse der Verhältnisse von Primärwurzel-Länge und Seitenwurzel-Dichte zwischen PBA-Behandlung und Kontrollbedingungen konnten mittels GWAS potenzielle PBA-Zielgene identifiziert werden, die im Zusammenhang mit Phytohormonen, Zellwandmodifikation, Endozytose und Wurzelentwicklung standen.
In Kapitel 3 wurde eine GWAS der Borgehalte in Mais-Kolbenblättern durchgeführt, um neue genetische Regulatoren der Bor-Homöostase zu identifizieren. Die integrierte Analyse der GWAS-Ergebnisse mit öffentlich verfügbaren Genexpressionsdaten hob benzoxazinless3 (bx3) als vielversprechendes Kandidatengen hervor und deutete auf eine Verbindung zwischen der Bor-Homöostase in Blättern und dem Benzoxazinoid-Stoffwechselweg hin. Sowohl die Funktionsverlust-Mutation von bx3 in Mais als auch die gleichzeitige Überexpression von BX1 und BX2 in Arabidopsis, einer Art, die den Benzoxazinoid-Stoffwechselweg endogen nicht exprimiert, führten zu erhöhten Borgehalten in Blättern. Darüber hinaus wurde festgestellt, dass das Produkt der BX3-Enzymfunktion, 3-Hydroxy-Indolin-2-on, in vitro einen Komplex mit Borsäure bilden kann. Somit zeigte unsere Studie, dass der Benzoxazinoid-Stoffwechselweg in Mais mit der Bor-Homöostase verknüpft ist, wahrscheinlich über bx3 sowie das direkte Substrat und Produkt von BX3. Somit wurde in dieser Dissertation ein potenzielles Ziel für die Pflanzenzüchtung zur besseren Anpassungsfähigkeit von Mais an geringe Borgehalte im Boden identifiziert.
To assess boron deficiency-induced defects in maize roots, one strategy is to induce boron deficiency using a chemical approach. Phenylboronic acid (PBA) was hypothesized to inhibit RG-II dimerization and therefore might mimic boron deficiency. However, the characterization of PBA as a boron deficiency mimic in planta has not been demonstrated.
To identify new regulators of boron homeostasis in maize, one strategy is to assess natural variation of boron concentration in association panels, and to subsequently analyze the phenotypic variation with the genetic variation through genome-wide association analysis (GWAS).
This thesis characterized the boron deficiency-induced defects in maize roots, tested the usability of PBA as a boron deficiency mimic, and identified additional regulators of boron homeostasis in maize leaves using GWAS.
In chapter 2, the primary root defects induced by boron deficiency and by PBA were characterized and compared. Both boron deficiency and PBA induced defects in primary root length, lateral root density, auxin levels and reactive oxygen species levels. Although the PBA-induced defects were similar to the boron deficiency-induced defects, the severity was different. Notably, PBA did not inhibit nor promote RG-II crosslinking in vitro, and PBA did not incorporate into pectin in vivo. Specifically, the PBA-induced lateral root density defects appeared linked to functions of the boric acid moiety. Using the ratios of primary root length and lateral root density between PBA treatment and no-PBA control, putative targets of PBA related to phytohormones, cell wall modification, endocytosis, and root development were detected through GWAS.
In chapter 3, a GWAS of boron levels in maize ear leaves was conducted to identify novel genetic regulators of boron homeostasis. Integrated analysis of the GWAS results and gene expression data highlighted benzoxazinless3 (bx3) as a promising candidate, suggesting an association between leaf boron homeostasis and the benzoxazinoid pathway, a well-characterized defense pathway. The loss-of-function mutation of bx3 in maize and the overexpression of BX1 and BX2 in Arabidopsis, a species that does not endogenously express the benzoxazinoid pathway, both resulted in elevated boron levels in leaves. Furthermore, the product of the BX3 enzyme function, 3-hydroxy-indolin-2-one (HION), was found to form a complex with boric acid in vitro. Our study, therefore, detected a novel connection between boron homeostasis and the benzoxazinoid pathway, likely through bx3 and the direct substrate and product of BX3, furthermore suggesting a potential target for crop engineering for better adaptability to low soil boron levels.
Um Bormangel-bedingte Defekte in Maiswurzeln zu untersuchen, besteht eine Strategie darin, Bor-Mangelbedingungen chemisch zu induzieren. Es wird angenommen, dass Boronsäuren, wie Phenylborsäure (PBA), die Dimerisierung von RG-II blockieren und dadurch Bormangel imitieren. Eine Charakterisierung von PBA in Bezug auf dessen Fähigkeit Bormangel in planta zu induzieren wurde jedoch bisher nicht durchgeführt.
Zur Identifizierung neuer Regulatoren der Bor-Homöostase in Mais können beispielsweise genomweite Assoziationsanalysen (GWAS) durchgeführt werden. Dabei wird die natürliche Variation von Merkmalen, z. B. die Borkonzentration, in genetischen Populationen erfasst und anschließend die phänotypische Variation mit genetischen Informationen der Population assoziiert.
Diese Dissertation charakterisierte die durch Bormangel verursachten Defekte in Maiswurzeln, prüfte die Verwendbarkeit von PBA zur Induzierung von Bormangel und identifizierte neue Regulatoren der Bor-Homöostase in Mais.
In Kapitel 2 wurden die durch Bormangel und durch PBA induzierten Primärwurzeldefekte charakterisiert und verglichen. Sowohl Bormangel als auch PBA führten zu Defekten in der Primärwurzel-Länge, der Dichte der Seitenwurzeln, sowie dem Gehalt an Auxin und reaktiver Sauerstoffspezies. Obwohl die durch PBA induzierten Defekte denjenigen des Bormangels ähnelten, unterschieden sie sich in ihrer Ausprägung. Auffällig war, dass PBA in vitro weder die RG-II-Vernetzung hemmte noch förderte und in vivo nicht in Pektin eingebaut wurde. Insbesondere schienen die durch PBA hervorgerufenen Defekte in der Seitenwurzel-Dichte mit Funktionen der Borsäure-Gruppe zusammenzuhängen. Durch die Analyse der Verhältnisse von Primärwurzel-Länge und Seitenwurzel-Dichte zwischen PBA-Behandlung und Kontrollbedingungen konnten mittels GWAS potenzielle PBA-Zielgene identifiziert werden, die im Zusammenhang mit Phytohormonen, Zellwandmodifikation, Endozytose und Wurzelentwicklung standen.
In Kapitel 3 wurde eine GWAS der Borgehalte in Mais-Kolbenblättern durchgeführt, um neue genetische Regulatoren der Bor-Homöostase zu identifizieren. Die integrierte Analyse der GWAS-Ergebnisse mit öffentlich verfügbaren Genexpressionsdaten hob benzoxazinless3 (bx3) als vielversprechendes Kandidatengen hervor und deutete auf eine Verbindung zwischen der Bor-Homöostase in Blättern und dem Benzoxazinoid-Stoffwechselweg hin. Sowohl die Funktionsverlust-Mutation von bx3 in Mais als auch die gleichzeitige Überexpression von BX1 und BX2 in Arabidopsis, einer Art, die den Benzoxazinoid-Stoffwechselweg endogen nicht exprimiert, führten zu erhöhten Borgehalten in Blättern. Darüber hinaus wurde festgestellt, dass das Produkt der BX3-Enzymfunktion, 3-Hydroxy-Indolin-2-on, in vitro einen Komplex mit Borsäure bilden kann. Somit zeigte unsere Studie, dass der Benzoxazinoid-Stoffwechselweg in Mais mit der Bor-Homöostase verknüpft ist, wahrscheinlich über bx3 sowie das direkte Substrat und Produkt von BX3. Somit wurde in dieser Dissertation ein potenzielles Ziel für die Pflanzenzüchtung zur besseren Anpassungsfähigkeit von Mais an geringe Borgehalte im Boden identifiziert.
Classification (DDC)
570 Biowissenschaften, Biologie 630 Landwirtschaft, VeterinärmedizinRelated Publications
https://doi.org/10.1093/jxb/eraf036https://doi.org/10.1093/plphys/kiae611
Chu, Liuyang: Characterization of the phenylboronic acid-induced defects in primary roots of maize (Zea mays L.) and the association of boron homeostasis with the benzoxazinoid pathway. - Bonn, 2026. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-89855
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-89855
@phdthesis{handle:20.500.11811/14162,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-89855,
author = {{Liuyang Chu}},
title = {Characterization of the phenylboronic acid-induced defects in primary roots of maize (Zea mays L.) and the association of boron homeostasis with the benzoxazinoid pathway},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2026,
month = may,
note = {Boron is an essential micronutrient for plants. In planta, boron predominantly resides in the primary cell wall, crosslinking the pectic polysaccharide rhamnogalacturonan II (RG-II), which is vital for maintaining cell wall integrity. Non-optimal levels of boron negatively affect the development of plants and reduce crop yield. In adaptation to variable soil boron conditions, plants have developed mechanisms to maintain boron homeostasis, of which the best known are boron transporters. In the cereal crop and model plant maize (Zea mays L.), the importance of boron during reproductive development is well studied, and genes encoding boron transporters have been characterized. However, the effects of boron deficiency on the maize root, and regulators of boron homeostasis in maize next to boron transporters, remain largely obscure.
To assess boron deficiency-induced defects in maize roots, one strategy is to induce boron deficiency using a chemical approach. Phenylboronic acid (PBA) was hypothesized to inhibit RG-II dimerization and therefore might mimic boron deficiency. However, the characterization of PBA as a boron deficiency mimic in planta has not been demonstrated.
To identify new regulators of boron homeostasis in maize, one strategy is to assess natural variation of boron concentration in association panels, and to subsequently analyze the phenotypic variation with the genetic variation through genome-wide association analysis (GWAS).
This thesis characterized the boron deficiency-induced defects in maize roots, tested the usability of PBA as a boron deficiency mimic, and identified additional regulators of boron homeostasis in maize leaves using GWAS.
In chapter 2, the primary root defects induced by boron deficiency and by PBA were characterized and compared. Both boron deficiency and PBA induced defects in primary root length, lateral root density, auxin levels and reactive oxygen species levels. Although the PBA-induced defects were similar to the boron deficiency-induced defects, the severity was different. Notably, PBA did not inhibit nor promote RG-II crosslinking in vitro, and PBA did not incorporate into pectin in vivo. Specifically, the PBA-induced lateral root density defects appeared linked to functions of the boric acid moiety. Using the ratios of primary root length and lateral root density between PBA treatment and no-PBA control, putative targets of PBA related to phytohormones, cell wall modification, endocytosis, and root development were detected through GWAS.
In chapter 3, a GWAS of boron levels in maize ear leaves was conducted to identify novel genetic regulators of boron homeostasis. Integrated analysis of the GWAS results and gene expression data highlighted benzoxazinless3 (bx3) as a promising candidate, suggesting an association between leaf boron homeostasis and the benzoxazinoid pathway, a well-characterized defense pathway. The loss-of-function mutation of bx3 in maize and the overexpression of BX1 and BX2 in Arabidopsis, a species that does not endogenously express the benzoxazinoid pathway, both resulted in elevated boron levels in leaves. Furthermore, the product of the BX3 enzyme function, 3-hydroxy-indolin-2-one (HION), was found to form a complex with boric acid in vitro. Our study, therefore, detected a novel connection between boron homeostasis and the benzoxazinoid pathway, likely through bx3 and the direct substrate and product of BX3, furthermore suggesting a potential target for crop engineering for better adaptability to low soil boron levels.},
url = {https://hdl.handle.net/20.500.11811/14162}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-89855,
author = {{Liuyang Chu}},
title = {Characterization of the phenylboronic acid-induced defects in primary roots of maize (Zea mays L.) and the association of boron homeostasis with the benzoxazinoid pathway},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2026,
month = may,
note = {Boron is an essential micronutrient for plants. In planta, boron predominantly resides in the primary cell wall, crosslinking the pectic polysaccharide rhamnogalacturonan II (RG-II), which is vital for maintaining cell wall integrity. Non-optimal levels of boron negatively affect the development of plants and reduce crop yield. In adaptation to variable soil boron conditions, plants have developed mechanisms to maintain boron homeostasis, of which the best known are boron transporters. In the cereal crop and model plant maize (Zea mays L.), the importance of boron during reproductive development is well studied, and genes encoding boron transporters have been characterized. However, the effects of boron deficiency on the maize root, and regulators of boron homeostasis in maize next to boron transporters, remain largely obscure.
To assess boron deficiency-induced defects in maize roots, one strategy is to induce boron deficiency using a chemical approach. Phenylboronic acid (PBA) was hypothesized to inhibit RG-II dimerization and therefore might mimic boron deficiency. However, the characterization of PBA as a boron deficiency mimic in planta has not been demonstrated.
To identify new regulators of boron homeostasis in maize, one strategy is to assess natural variation of boron concentration in association panels, and to subsequently analyze the phenotypic variation with the genetic variation through genome-wide association analysis (GWAS).
This thesis characterized the boron deficiency-induced defects in maize roots, tested the usability of PBA as a boron deficiency mimic, and identified additional regulators of boron homeostasis in maize leaves using GWAS.
In chapter 2, the primary root defects induced by boron deficiency and by PBA were characterized and compared. Both boron deficiency and PBA induced defects in primary root length, lateral root density, auxin levels and reactive oxygen species levels. Although the PBA-induced defects were similar to the boron deficiency-induced defects, the severity was different. Notably, PBA did not inhibit nor promote RG-II crosslinking in vitro, and PBA did not incorporate into pectin in vivo. Specifically, the PBA-induced lateral root density defects appeared linked to functions of the boric acid moiety. Using the ratios of primary root length and lateral root density between PBA treatment and no-PBA control, putative targets of PBA related to phytohormones, cell wall modification, endocytosis, and root development were detected through GWAS.
In chapter 3, a GWAS of boron levels in maize ear leaves was conducted to identify novel genetic regulators of boron homeostasis. Integrated analysis of the GWAS results and gene expression data highlighted benzoxazinless3 (bx3) as a promising candidate, suggesting an association between leaf boron homeostasis and the benzoxazinoid pathway, a well-characterized defense pathway. The loss-of-function mutation of bx3 in maize and the overexpression of BX1 and BX2 in Arabidopsis, a species that does not endogenously express the benzoxazinoid pathway, both resulted in elevated boron levels in leaves. Furthermore, the product of the BX3 enzyme function, 3-hydroxy-indolin-2-one (HION), was found to form a complex with boric acid in vitro. Our study, therefore, detected a novel connection between boron homeostasis and the benzoxazinoid pathway, likely through bx3 and the direct substrate and product of BX3, furthermore suggesting a potential target for crop engineering for better adaptability to low soil boron levels.},
url = {https://hdl.handle.net/20.500.11811/14162}
}
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