Mechanism Enhancing Arabidopsis Resistance to Cadmium: The Role of NRT1.5 and Proton Pump

Aim: Heavy metal pollution is serious in China, and abscisic acid (ABA) is an important stress hormone. How it regulates plant tolerance to cadmium remains unclear, so we aimed to explore the molecular mechanism responsible for enhanced cadmium resistance in Arabidopsis wild-type and mutant plants and Brassica napus seedlings.Methods: Arabidopsis/B. napus were cultured hydroponically for 28/15 days and then treated with 20/10 μM Cd/Cd+ABA (5 μM) for 3/4 days. Chlorophyll degradation rate, SPAD values, proline, MDA, ABA, NO3−, and Cd concentrations were measured in root vacuoles and protoplasts; root to shoot NO3− and Cd concentration ratios were determined and NRT1.5-, NRT1.8-, BnNRT1.5-, and BnNRT1.8-related gene expression was studied.Results: Cytoplasmic ABA levels in root cells of bglu10 and bglu18 Arabidopsis mutants were significantly lower than those in the wild-type, apparently making the latter more resistant to Cd. NO3− long-distance transporter NRT1.5 responded to ABA signaling by downregulating its own expression, while NRT1.8 did not respond. Concomitantly, proton pump activity in wild-type plants was higher than in the bglu10 and bglu18 mutants; thus, more NO3− and Cd accumulated in the vacuoles of wild-type root cells. ABA application inhibited Cd absorption by B. napus. BnNRT1.5 responded to exogenous ABA signal by downregulating its own expression, while the lack of response by BnNRT1.8 resulted in increased amount of NO3− accumulating in the roots to participate in the anti-cadmium reaction.Conclusion:NRT1.5 responds to the ABA signal to inhibit its own expression, whereas unresponsiveness of NRT1.8 causes accumulation of NO3− in the roots; thus, enhancing Cd resistance. In Arabidopsis, because of proton pump action, more NO3− and Cd accumulate in the vacuoles of Arabidopsis root cells, thereby reducing damage by Cd toxicity. However, in B. napus, the addition of exogenous ABA inhibited Cd absorption. Our data provide a sound basis to the theoretical molecular mechanism involved in hormone signaling during response of plants to heavy metal stress

A High-Density Genetic Map Identifies a Novel Major QTL for Boron Efficiency in Oilseed Rape (Brassica napus L.)

Low boron (B) seriously limits the growth of oilseed rape (Brassica napus L.), a high B demand species that is sensitive to low B conditions. Significant genotypic variations in response to B deficiency have been observed among B. napus cultivars. To reveal the genetic basis for B efficiency in B. napus, quantitative trait loci (QTLs) for the plant growth traits, B uptake traits and the B efficiency coefficient (BEC) were analyzed using a doubled haploid (DH) population derived from a cross between a B-efficient parent, Qingyou 10, and a B-inefficient parent, Westar 10. A high-density genetic map was constructed based on single nucleotide polymorphisms (SNPs) assayed using Brassica 60 K Infinium BeadChip Array, simple sequence repeats (SSRs) and amplified fragment length polymorphisms (AFLPs). The linkage map covered a total length of 2139.5 cM, with 19 linkage groups (LGs) and an average distance of 1.6 cM between adjacent markers. Based on hydroponic evaluation of six B efficiency traits measured in three separate repeated trials, a total of 52 QTLs were identified, accounting for 6.14–46.27 % of the phenotypic variation. A major QTL for BEC, qBEC-A3a, was co-located on A3 with other QTLs for plant growth and B uptake traits under low B stress. Using a subset of substitution lines, qBEC-A3a was validated and narrowed down to the interval between CNU384 and BnGMS436. The results of this study provide a novel major locus located on A3 for B efficiency in B. napus that will be suitable for fine mapping and marker-assisted selection breeding for B efficiency in B. napus

Genome-Wide Identification and Characterization of the Aquaporin Gene Family and Transcriptional Responses to Boron Deficiency in Brassica napus

Aquaporins (AQPs) are an abundant protein family and play important roles to facilitate small neutral molecule transport across membranes. Oilseed rape (Brassica napus L.) is an important oil crop in China and elsewhere in the world, and is very sensitive to low boron (B) stress. Several AQP family genes have been reported to be involved in B transport across plasma membranes in plants. In this study, a total of 121 full-length AQPs were identified and characterized in B. napus (AC genome), and could be classified into four sub-families, including 43 PIPs (plasma membrane intrinsic proteins), 35 TIPs (tonoplast intrinsic proteins), 32 NIPs (NOD26-like intrinsic proteins), and 11 SIPs (small basic intrinsic proteins). The gene characteristics of BnaAQPs were similar to those of BraAQPs (A genome) and BolAQPs (C genome) including the composition of each sub-family, gene structure, and substrate selectivity filters. The BnaNIP was the most complex AQP sub-family, reflecting the composition of substrate selectivity filter structures which affect the permeation of solution molecules. In this study, the seedlings of both B-efficient (QY10) and B-inefficient (W10) cultivars were treated with two boron (B) levels: deficient (0.25 μM B) and sufficient (25 μM B). The transcription of AQP genes in root (R), juvenile leaf (JL), and old leaf (OL) tissues of both cultivars was investigated under B deficient and sufficient conditions. Transcription of most BnaPIPs and BnaTIPs was significantly increased compared with other BnaAQPs in all the three tissues, especially in the roots, of both B-efficient and B-inefficient cultivars under both B conditions. With B deprivation, the expression of the majority of the BnaPIPs and BnaTIPs was down-regulated in the roots. However, the BnaNIPs were up-regulated. In addition, the BnaCnn_random.PIP1;4b, BnaPIP2;4s, BnaC04.TIP4;1a, BnaAnn_random.TIP1;1b, and BnaNIP5;1s (except for BnaA07.NIP5;1c and BnaC06.NIP5;1c) exhibited obvious differences at low B between B-efficient and B-inefficient cultivars. These results will help us to understand boron homeostasis in B. napus

Genome-wide identification and characterization of the Aquaporin gene family and transcriptional responses to boron deficiency in Brassica napus

Aquaporins (AQPs) are an abundant protein family and play important roles to facilitate small neutral molecule transport across membranes. Oilseed rape (Brassica napus L.) is an important oil crop in China and elsewhere in the world, and is very sensitive to low boron (B) stress. Several AQP family genes have been reported to be involved in B transport across plasma membranes in plants. In this study, a total of 121 full-length AQPs were identified and characterized in B. napus (AC genome), and could be classified into four sub-families, including 43 PIPs (plasma membrane intrinsic proteins), 35 TIPs (tonoplast intrinsic proteins), 32 NIPs (NOD26-like intrinsic proteins), and 11 SIPs (small basic intrinsic proteins). The gene characteristics of BnaAQPs were similar to those of BraAQPs (A genome) and BolAQPs (C genome) including the composition of each sub-family, gene structure, and substrate selectivity filters. The BnaNIP was the most complex AQP sub-family, reflecting the composition of substrate selectivity filter structures which affect the permeation of solution molecules. In this study, the seedlings of both B-efficient (QY10) and B-inefficient (W10) cultivars were treated with two boron (B) levels: deficient (0.25 μM B) and sufficient (25 μM B). The transcription of AQP genes in root (R), juvenile leaf (JL), and old leaf (OL) tissues of both cultivars was investigated under B deficient and sufficient conditions. Transcription of most BnaPIPs and BnaTIPs was significantly increased compared with other BnaAQPs in all the three tissues, especially in the roots, of both B-efficient and B-inefficient cultivars under both B conditions. With B deprivation, the expression of the majority of the BnaPIPs and BnaTIPs was down-regulated in the roots. However, the BnaNIPs were up-regulated. In addition, the BnaCnn_random.PIP1;4b, BnaPIP2;4s, BnaC04.TIP4;1a, BnaAnn_random.TIP1;1b, and BnaNIP5;1s (except for BnaA07.NIP5;1c and BnaC06.NIP5;1c) exhibited obvious differences at low B between B-efficient and B-inefficient cultivars. These results will help us to understand boron homeostasis in B. napus

Genome-Wide Identification of Brassicaceae Hormone-Related Transcription Factors and Their Roles in Stress Adaptation and Plant Height Regulation in Allotetraploid Rapeseed

Phytohormone-related transcription factors (TFs) are involved in regulating stress responses and plant growth. However, systematic analysis of these TFs in Brassicaceae is limited, and their functions in stress adaptation and plant height (PH) regulation remain unclear. In this study, 2115 hormone-related TFs were identified in nine Brassicaceae species. Specific domains were found in several Brassicaceae hormone-related TFs, which may be associated with diverse functions. Syntenic analysis indicated that expansion of these genes was mainly caused by segmental duplication, with whole-genome duplication occurring in some species. Differential expression analysis and gene co-expression network analysis identified seven phytohormone-related TFs (BnaWRKY7, 21, 32, 38, 52, BnaGL3-4, and BnaAREB2-5) as possible key genes for cadmium (Cd) toxicity, salinity stress, and potassium (K) and nitrogen (N) deficiencies. Furthermore, BnaWRKY42 and BnaARR21 may play essential roles in plant height. Weighted gene co-expression network analysis (WGCNA) identified 15 phytohormone-related TFs and their potential target genes regulating stress adaptation and plant height. Among the above genes, BnaWRKY56 and BnaWRKY60 responded to four different stresses simultaneously, and BnaWRKY42 was identified in two dwarf rapeseeds. In summary, several candidate genes for stress resistance (BnaWRKY56 and BnaWRKY60) and plant height (BnaWRKY42) were identified. These findings should help elucidate the biological roles of Brassicaceae hormone-related TFs, and the identified candidate genes should provide a genetic resource for the potential development of stress-tolerant and dwarf oilseed plants

Increased nitrogen use efficiency via amino acid remobilization from source to sink organs in Brassica napus

Nitrogen (N) is an essential plant growth nutrient whose coordinated distribution from source to sink organs is crucial for seed development and overall crop yield. We compared high and low N use efficiency (NUE) Brassica napus (rapeseed) genotypes. Metabonomics and transcriptomics revealed that leaf senescence induced by N deficiency promoted amino acid allocation from older to younger leaves in the high-NUE genotype at the vegetative growth stage. Efficient source to sink remobilization of amino acids elevated the numbers of branches and pods per plant under a N-deficiency treatment during the reproductive stage. A 15N tracer experiment confirmed that more amino acids were partitioned into seeds from the silique wall during the pod stage in the high-NUE genotype, owing mainly to variation in genes involved in organic N transport and metabolism. We suggest that the greater amino acid source-to-sink allocation efficiency during various growth stages in the high-NUE genotype resulted in higher yield and NUE under N deficiency. These findings support the hypothesis that strong amino acid remobilization in rapeseed leads to high yield, NUE, and harvest index

Summary of the total number of markers, map length and average distance between adjacent loci of the <i>Brassica napus</i> high-density linkage map based on the QW DH population derived from a cross between the B-efficient parent Qingyou 10 and the B-inefficient parent Westar 10.

<p>Summary of the total number of markers, map length and average distance between adjacent loci of the <i>Brassica napus</i> high-density linkage map based on the QW DH population derived from a cross between the B-efficient parent Qingyou 10 and the B-inefficient parent Westar 10.</p

Frequency distributions of plant growth and B uptake traits at high B (left) and low B (right) in the QW DH population derived from a cross between the B-efficient parent Qingyou 10 and the B-inefficient parent Westar 10.

<p>The black arrows indicate Qingyou 10, and the gray arrows indicate Westar 10.</p

Co-location of QTLs for the B efficiency coefficient (BEC), plant growth and B uptake traits in the QW DH population.

<p>Co-location of QTLs for the B efficiency coefficient (BEC), plant growth and B uptake traits in the QW DH population.</p