Boron demanding tissues of Brassica napus express specific sets of functional Nodulin26-like Intrinsic Proteins and BOR1 transporters.

The sophisticated uptake and translocation regulation of the essential element boron (B) in plants is ensured by two transmembrane transporter families: the Nodulin26-like Intrinsic Protein (NIP) and BOR transporter family. Though the agriculturally important crop Brassica napus is highly sensitive to B deficiency and NIPs and BORs have been suggested to be responsible for B efficiency in this species, functional information of these transporter subfamilies is extremely rare. Here, we molecularly characterized the NIP and BOR1 transporter family in the European winter-type cv. Darmor-PBY018. Our transport assays in the heterologous oocyte and yeast expression systems as well as in growth complementation assays in planta demonstrated B transport activity of NIP5, NIP6, NIP7 and BOR1 isoforms. Moreover, we provided functional and quantitative evidence that also members of the NIP2, NIP3 and NIP4 groups facilitate the transport of B. A detailed B- and tissue-dependent B-transporter expression map was generated by qPCR. We showed that NIP5 isoforms are highly up-regulated under B-deficient conditions in roots, but also in shoot tissues. Moreover, we detected transcripts of several B-permeable NIPs from various groups in floral tissues that contribute to the B distribution within the highly B deficiency-sensitive flowers. This article is protected by copyright. All rights reserved

Boron demanding tissues of Brassica napus

The sophisticated uptake and translocation regulation of the essential element boron (B) in plants is ensured by two transmembrane transporter families: the Nodulin26-like Intrinsic Protein (NIP) and BOR transporter family. Though the agriculturally important crop Brassica napus is highly sensitive to B deficiency and NIPs and BORs have been suggested to be responsible for B efficiency in this species, functional information of these transporter subfamilies is extremely rare. Here, we molecularly characterized the NIP and BOR1 transporter family in the European winter-type cv. Darmor-PBY018. Our transport assays in the heterologous oocyte and yeast expression systems as well as in growth complementation assays in planta demonstrated B transport activity of NIP5, NIP6, NIP7 and BOR1 isoforms. Moreover, we provided functional and quantitative evidence that also members of the NIP2, NIP3 and NIP4 groups facilitate the transport of B. A detailed B- and tissue-dependent B-transporter expression map was generated by qPCR. We showed that NIP5 isoforms are highly up-regulated under B-deficient conditions in roots, but also in shoot tissues. Moreover, we detected transcripts of several B-permeable NIPs from various groups in floral tissues that contribute to the B distribution within the highly B deficiency-sensitive flowers. This article is protected by copyright. All rights reserved

Functional evolution of nodulin 26-like intrinsic proteins: from bacterial arsenic detoxification to plant nutrient transport.

Nodulin 26-like intrinsic proteins (NIPs) play essential roles in transporting the nutrients silicon and boron in seed plants, but the evolutionary origin of this transport function and the co-permeability to toxic arsenic remains enigmatic. Horizontal gene transfer of a yet uncharacterised bacterial AqpN-aquaporin group was the starting-point for plant NIP evolution. We combined intense sequence, phylogenetic and genetic context analyses and a mutational approach with various transport assays in oocytes and plants to resolve the transorganismal and functional evolution of bacterial and algal and terrestrial plant NIPs and to reveal their molecular transport specificity features. We discovered that aqpN genes are prevalently located in arsenic resistance operons of various prokaryotic phyla. We provided genetic and functional evidence that these proteins contribute to the arsenic detoxification machinery. We identified NIPs with the ancestral bacterial AqpN selectivity filter composition in algae, liverworts, moss, hornworts and ferns and demonstrated that these archetype plant NIPs and their prokaryotic progenitors are almost impermeable to water and silicon but transport arsenic and boron. With a mutational approach, we demonstrated that during evolution, ancestral NIP selectivity shifted to allow subfunctionalisations. Together, our data provided evidence that evolution converted bacterial arsenic efflux channels into essential seed plant nutrient transporters

Functional Evolution of Nodulin 26-like Intrinsic Proteins: From bacterial arsenic detoxification to plant nutrient transport

Pommerrenig B, Diehn TA, Bernhardt N, et al. Functional Evolution of Nodulin 26-like Intrinsic Proteins: From bacterial arsenic detoxification to plant nutrient transport. New Phytologist. 2020;225(3):1383-1396.Nodulin 26-like-intrinsic-proteins (NIPs) play essential roles in transporting the nutrients, silicon and boron, in seed plants, but the evolutionary origin of this transport function and the co-permeability to toxic arsenic remains enigmatic. Horizontal gene transfer of a yet uncharacterized bacterial AqpN-aquaporin group was the starting-point for plant NIP evolution. We combined intense sequence-, phylogenetic and genetic context analyses and a mutational approach with various transport assays in oocytes and plants to resolve the trans-organismal and functional evolution of bacterial and algal and terrestrial plant NIPs and to reveal their molecular transport specificity features. We discovered that aqpN genes are prevalently located in arsenic-resistance-operons of various prokaryotic phyla. We provided genetic and functional evidence that these proteins contribute to the arsenic detoxification machinery. We identified NIPs with the ancestral bacterial AqpN selectivity filter composition in algae, liverworts, moss, hornworts and ferns and demonstrated that these archetype plant NIPs and their prokaryotic progenitors are almost impermeable to water and silicon but transport arsenic and boron. With a mutational approach, we demonstrated that during evolution, ancestral NIP selectivity shifted to allow subfunctionalizations. Together, our data provided evidence that evolution converted bacterial arsenic efflux channels into essential seed plant nutrient transporters. © 2019 The Authors New Phytologist © 2019 New Phytologist Trust

Data_Sheet_9_Identification of Rapeseed (Brassica napus) Cultivars With a High Tolerance to Boron-Deficient Conditions.docx

<p>Boron (B) is an essential micronutrient for seed plants. Information on B-efficiency mechanisms and B-efficient crop and model plant genotypes is very scarce. Studies evaluating the basis and consequences of B-deficiency and B-efficiency are limited by the facts that B occurs as a trace contaminant essentially everywhere, its bioavailability is difficult to control and soil-based B-deficiency growth systems allowing a high-throughput screening of plant populations have hitherto been lacking. The crop plant Brassica napus shows a very high sensitivity toward B-deficient conditions. To reduce B-deficiency-caused yield losses in a sustainable manner, the identification of B-efficient B. napus genotypes is indispensable. We developed a soil substrate-based cultivation system which is suitable to study plant growth in automated high-throughput phenotyping facilities under defined and repeatable soil B conditions. In a comprehensive screening, using this system with soil B concentrations below 0.1 mg B (kg soil)^-1, we identified three highly B-deficiency tolerant B. napus cultivars (CR2267, CR2280, and CR2285) among a genetically diverse collection comprising 590 accessions from all over the world. The B-efficiency classification of cultivars was based on a detailed assessment of various physical and high-throughput imaging-based shoot and root growth parameters in soil substrate or in in vitro conditions, respectively. We identified cultivar-specific patterns of B-deficiency-responsive growth dynamics. Elemental analysis revealed striking differences only in B contents between contrasting genotypes when grown under B-deficient but not under standard conditions. Results indicate that B-deficiency tolerant cultivars can grow with a very limited amount of B which is clearly below previously described critical B-tissue concentration values. These results suggest a higher B utilization efficiency of CR2267, CR2280, and CR2285 which would represent a unique trait among so far identified B-efficient B. napus cultivars which are characterized by a higher B-uptake capacity. Testing various other nutrient deficiency treatments, we demonstrated that the tolerance is specific for B-deficient conditions and is not conferred by a general growth vigor at the seedling stage. The identified B-deficiency tolerant cultivars will serve as genetic and physiological “tools” to further understand the mechanisms regulating the B nutritional status in rapeseed and to develop B-efficient elite genotypes.</p

Data_Sheet_8_Identification of Rapeseed (Brassica napus) Cultivars With a High Tolerance to Boron-Deficient Conditions.docx

<p>Boron (B) is an essential micronutrient for seed plants. Information on B-efficiency mechanisms and B-efficient crop and model plant genotypes is very scarce. Studies evaluating the basis and consequences of B-deficiency and B-efficiency are limited by the facts that B occurs as a trace contaminant essentially everywhere, its bioavailability is difficult to control and soil-based B-deficiency growth systems allowing a high-throughput screening of plant populations have hitherto been lacking. The crop plant Brassica napus shows a very high sensitivity toward B-deficient conditions. To reduce B-deficiency-caused yield losses in a sustainable manner, the identification of B-efficient B. napus genotypes is indispensable. We developed a soil substrate-based cultivation system which is suitable to study plant growth in automated high-throughput phenotyping facilities under defined and repeatable soil B conditions. In a comprehensive screening, using this system with soil B concentrations below 0.1 mg B (kg soil)^-1, we identified three highly B-deficiency tolerant B. napus cultivars (CR2267, CR2280, and CR2285) among a genetically diverse collection comprising 590 accessions from all over the world. The B-efficiency classification of cultivars was based on a detailed assessment of various physical and high-throughput imaging-based shoot and root growth parameters in soil substrate or in in vitro conditions, respectively. We identified cultivar-specific patterns of B-deficiency-responsive growth dynamics. Elemental analysis revealed striking differences only in B contents between contrasting genotypes when grown under B-deficient but not under standard conditions. Results indicate that B-deficiency tolerant cultivars can grow with a very limited amount of B which is clearly below previously described critical B-tissue concentration values. These results suggest a higher B utilization efficiency of CR2267, CR2280, and CR2285 which would represent a unique trait among so far identified B-efficient B. napus cultivars which are characterized by a higher B-uptake capacity. Testing various other nutrient deficiency treatments, we demonstrated that the tolerance is specific for B-deficient conditions and is not conferred by a general growth vigor at the seedling stage. The identified B-deficiency tolerant cultivars will serve as genetic and physiological “tools” to further understand the mechanisms regulating the B nutritional status in rapeseed and to develop B-efficient elite genotypes.</p

Data_Sheet_6_Identification of Rapeseed (Brassica napus) Cultivars With a High Tolerance to Boron-Deficient Conditions.docx

<p>Boron (B) is an essential micronutrient for seed plants. Information on B-efficiency mechanisms and B-efficient crop and model plant genotypes is very scarce. Studies evaluating the basis and consequences of B-deficiency and B-efficiency are limited by the facts that B occurs as a trace contaminant essentially everywhere, its bioavailability is difficult to control and soil-based B-deficiency growth systems allowing a high-throughput screening of plant populations have hitherto been lacking. The crop plant Brassica napus shows a very high sensitivity toward B-deficient conditions. To reduce B-deficiency-caused yield losses in a sustainable manner, the identification of B-efficient B. napus genotypes is indispensable. We developed a soil substrate-based cultivation system which is suitable to study plant growth in automated high-throughput phenotyping facilities under defined and repeatable soil B conditions. In a comprehensive screening, using this system with soil B concentrations below 0.1 mg B (kg soil)^-1, we identified three highly B-deficiency tolerant B. napus cultivars (CR2267, CR2280, and CR2285) among a genetically diverse collection comprising 590 accessions from all over the world. The B-efficiency classification of cultivars was based on a detailed assessment of various physical and high-throughput imaging-based shoot and root growth parameters in soil substrate or in in vitro conditions, respectively. We identified cultivar-specific patterns of B-deficiency-responsive growth dynamics. Elemental analysis revealed striking differences only in B contents between contrasting genotypes when grown under B-deficient but not under standard conditions. Results indicate that B-deficiency tolerant cultivars can grow with a very limited amount of B which is clearly below previously described critical B-tissue concentration values. These results suggest a higher B utilization efficiency of CR2267, CR2280, and CR2285 which would represent a unique trait among so far identified B-efficient B. napus cultivars which are characterized by a higher B-uptake capacity. Testing various other nutrient deficiency treatments, we demonstrated that the tolerance is specific for B-deficient conditions and is not conferred by a general growth vigor at the seedling stage. The identified B-deficiency tolerant cultivars will serve as genetic and physiological “tools” to further understand the mechanisms regulating the B nutritional status in rapeseed and to develop B-efficient elite genotypes.</p

Data_Sheet_2_Identification of Rapeseed (Brassica napus) Cultivars With a High Tolerance to Boron-Deficient Conditions.docx

<p>Boron (B) is an essential micronutrient for seed plants. Information on B-efficiency mechanisms and B-efficient crop and model plant genotypes is very scarce. Studies evaluating the basis and consequences of B-deficiency and B-efficiency are limited by the facts that B occurs as a trace contaminant essentially everywhere, its bioavailability is difficult to control and soil-based B-deficiency growth systems allowing a high-throughput screening of plant populations have hitherto been lacking. The crop plant Brassica napus shows a very high sensitivity toward B-deficient conditions. To reduce B-deficiency-caused yield losses in a sustainable manner, the identification of B-efficient B. napus genotypes is indispensable. We developed a soil substrate-based cultivation system which is suitable to study plant growth in automated high-throughput phenotyping facilities under defined and repeatable soil B conditions. In a comprehensive screening, using this system with soil B concentrations below 0.1 mg B (kg soil)^-1, we identified three highly B-deficiency tolerant B. napus cultivars (CR2267, CR2280, and CR2285) among a genetically diverse collection comprising 590 accessions from all over the world. The B-efficiency classification of cultivars was based on a detailed assessment of various physical and high-throughput imaging-based shoot and root growth parameters in soil substrate or in in vitro conditions, respectively. We identified cultivar-specific patterns of B-deficiency-responsive growth dynamics. Elemental analysis revealed striking differences only in B contents between contrasting genotypes when grown under B-deficient but not under standard conditions. Results indicate that B-deficiency tolerant cultivars can grow with a very limited amount of B which is clearly below previously described critical B-tissue concentration values. These results suggest a higher B utilization efficiency of CR2267, CR2280, and CR2285 which would represent a unique trait among so far identified B-efficient B. napus cultivars which are characterized by a higher B-uptake capacity. Testing various other nutrient deficiency treatments, we demonstrated that the tolerance is specific for B-deficient conditions and is not conferred by a general growth vigor at the seedling stage. The identified B-deficiency tolerant cultivars will serve as genetic and physiological “tools” to further understand the mechanisms regulating the B nutritional status in rapeseed and to develop B-efficient elite genotypes.</p

Data_Sheet_5_Identification of Rapeseed (Brassica napus) Cultivars With a High Tolerance to Boron-Deficient Conditions.docx

<p>Boron (B) is an essential micronutrient for seed plants. Information on B-efficiency mechanisms and B-efficient crop and model plant genotypes is very scarce. Studies evaluating the basis and consequences of B-deficiency and B-efficiency are limited by the facts that B occurs as a trace contaminant essentially everywhere, its bioavailability is difficult to control and soil-based B-deficiency growth systems allowing a high-throughput screening of plant populations have hitherto been lacking. The crop plant Brassica napus shows a very high sensitivity toward B-deficient conditions. To reduce B-deficiency-caused yield losses in a sustainable manner, the identification of B-efficient B. napus genotypes is indispensable. We developed a soil substrate-based cultivation system which is suitable to study plant growth in automated high-throughput phenotyping facilities under defined and repeatable soil B conditions. In a comprehensive screening, using this system with soil B concentrations below 0.1 mg B (kg soil)^-1, we identified three highly B-deficiency tolerant B. napus cultivars (CR2267, CR2280, and CR2285) among a genetically diverse collection comprising 590 accessions from all over the world. The B-efficiency classification of cultivars was based on a detailed assessment of various physical and high-throughput imaging-based shoot and root growth parameters in soil substrate or in in vitro conditions, respectively. We identified cultivar-specific patterns of B-deficiency-responsive growth dynamics. Elemental analysis revealed striking differences only in B contents between contrasting genotypes when grown under B-deficient but not under standard conditions. Results indicate that B-deficiency tolerant cultivars can grow with a very limited amount of B which is clearly below previously described critical B-tissue concentration values. These results suggest a higher B utilization efficiency of CR2267, CR2280, and CR2285 which would represent a unique trait among so far identified B-efficient B. napus cultivars which are characterized by a higher B-uptake capacity. Testing various other nutrient deficiency treatments, we demonstrated that the tolerance is specific for B-deficient conditions and is not conferred by a general growth vigor at the seedling stage. The identified B-deficiency tolerant cultivars will serve as genetic and physiological “tools” to further understand the mechanisms regulating the B nutritional status in rapeseed and to develop B-efficient elite genotypes.</p

Data_Sheet_10_Identification of Rapeseed (Brassica napus) Cultivars With a High Tolerance to Boron-Deficient Conditions.docx

<p>Boron (B) is an essential micronutrient for seed plants. Information on B-efficiency mechanisms and B-efficient crop and model plant genotypes is very scarce. Studies evaluating the basis and consequences of B-deficiency and B-efficiency are limited by the facts that B occurs as a trace contaminant essentially everywhere, its bioavailability is difficult to control and soil-based B-deficiency growth systems allowing a high-throughput screening of plant populations have hitherto been lacking. The crop plant Brassica napus shows a very high sensitivity toward B-deficient conditions. To reduce B-deficiency-caused yield losses in a sustainable manner, the identification of B-efficient B. napus genotypes is indispensable. We developed a soil substrate-based cultivation system which is suitable to study plant growth in automated high-throughput phenotyping facilities under defined and repeatable soil B conditions. In a comprehensive screening, using this system with soil B concentrations below 0.1 mg B (kg soil)^-1, we identified three highly B-deficiency tolerant B. napus cultivars (CR2267, CR2280, and CR2285) among a genetically diverse collection comprising 590 accessions from all over the world. The B-efficiency classification of cultivars was based on a detailed assessment of various physical and high-throughput imaging-based shoot and root growth parameters in soil substrate or in in vitro conditions, respectively. We identified cultivar-specific patterns of B-deficiency-responsive growth dynamics. Elemental analysis revealed striking differences only in B contents between contrasting genotypes when grown under B-deficient but not under standard conditions. Results indicate that B-deficiency tolerant cultivars can grow with a very limited amount of B which is clearly below previously described critical B-tissue concentration values. These results suggest a higher B utilization efficiency of CR2267, CR2280, and CR2285 which would represent a unique trait among so far identified B-efficient B. napus cultivars which are characterized by a higher B-uptake capacity. Testing various other nutrient deficiency treatments, we demonstrated that the tolerance is specific for B-deficient conditions and is not conferred by a general growth vigor at the seedling stage. The identified B-deficiency tolerant cultivars will serve as genetic and physiological “tools” to further understand the mechanisms regulating the B nutritional status in rapeseed and to develop B-efficient elite genotypes.</p