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Genetic variation of BnaA3.NIP5;1 expressing in the lateral root cap contributes to boron deficiency tolerance in Brassica napus

['Mingliang He', 'National Key Laboratory Of Crop Genetic Improvement', 'Huazhong Agricultural University', 'Wuhan', 'Microelement Research Centre', 'Sheliang Wang', 'Cheng Zhang', 'Liu Liu', 'College Of Life Science', 'Technology']

Date: 2021-09

Abstract Boron (B) is essential for vascular plants. Rapeseed (Brassica napus) is the second leading crop source for vegetable oil worldwide, but its production is critically dependent on B supplies. BnaA3.NIP5;1 was identified as a B-efficient candidate gene in B. napus in our previous QTL fine mapping. However, the molecular mechanism through which this gene improves low-B tolerance remains elusive. Here, we report genetic variation in BnaA3.NIP5;1 gene, which encodes a boric acid channel, is a key determinant of low-B tolerance in B. napus. Transgenic lines with increased BnaA3.NIP5;1 expression exhibited improved low-B tolerance in both the seedling and maturity stages. BnaA3.NIP5;1 is preferentially polar-localized in the distal plasma membrane of lateral root cap (LRC) cells and transports B into the root tips to promote root growth under B-deficiency conditions. Further analysis revealed that a CTTTC tandem repeat in the 5’UTR of BnaA3.NIP5;1 altered the expression level of the gene, which is tightly associated with plant growth and seed yield. Field tests with natural populations and near-isogenic lines (NILs) confirmed that the varieties carried BnaA3.NIP5;1Q allele significantly improved seed yield. Taken together, our results provide novel insights into the low-B tolerance of B. napus, and the elite allele of BnaA3.NIP5;1 could serve as a direct target for breeding low-B-tolerant cultivars.

Author summary Boron (B) deficiency severely rapeseed (Brassica napus) yields in most high rainfall areas worldwide, and genetic improvement is an effective strategy for addressing the problem. Here we show that BnaA3.NIP5;1, encoding a boric acid channel, is a key determinant of the low-B tolerance in B. napus. Our results demonstrate that BnaA3.NIP5;1 is preferentially located in the distal side plasma membrane of lateral root cap (LRC) cells and transports B into meristem zone to promote root growth under B limitation, which provide insights into the LRC’s function in mineral nutrition. A CTTTC tandem repeat in the 5’UTR of BnaA3.NIP5;1 altered the expression level of the gene, which is tightly associated with plant growth and seed yield under low-B conditions. The functional gene and elite allele could be useful in rapeseed breeding.

Citation: He M, Wang S, Zhang C, Liu L, Zhang J, Qiu S, et al. (2021) Genetic variation of BnaA3.NIP5;1 expressing in the lateral root cap contributes to boron deficiency tolerance in Brassica napus. PLoS Genet 17(7): e1009661. https://doi.org/10.1371/journal.pgen.1009661 Editor: Zhixi Tian, Chinese Academy of Sciences, CHINA Received: March 18, 2021; Accepted: June 10, 2021; Published: July 1, 2021 Copyright: © 2021 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: This research was funded by the National Key Research and Development Program of China: 2016YFD0100700, National Natural Science Foundation of China (NSFC): 31772380, 31572185 and Fundamental Research Funds for the Central Universities of China: 2662019PY058, 2662019PY013 to F.S.X. and NSFC: 31670267, 31770283 to S.W.X. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Boron (B) is an essential micronutrient for all higher plants. Plant roots take up B in the form of boric acid (H 3 BO 3 ). However, the natural abundance of B is relatively low in the soil [1], and B is very leachable, especially in areas of high rainfall (South-East Asia, Brazil, China). The leaching of B from soil leads to a decrease in the availability of B to plants. B deficiency is a worldwide agricultural production problem that has been reported in the field for at least 132 crops from 80 countries [2]. B acts as a cross-link between the pectin polysaccharide rhamnogalacturonan II (RG-II) via borate-diol ester bonds in vascular plants and is necessary for plant growth [3–6]. B deficiency during early vegetative stages leads to slow growth and low biomass, whereas B deficiency can considerably diminish productivity [7]. Application of B fertilizer can alleviate B-deficiency problems, but borate rock is a non-renewable mineral resource. Thus, genetic improvement of B efficiency of crops is a promising and cost-efficient strategy in B-deficient regions. Under low-B conditions, B can be taken up via two different mechanisms in plants. Channel-mediated facilitated transport via NOD26-LIKE MAJOR INTRINSIC PROTEIN5;1 (NIP5;1) encodes a channel protein belonging to the aquaporin family [8]. Energy-dependent active transport against concentration gradients via AtBOR1, an anion co-exchanger that functions as an efflux transporter for xylem loading of B under B-limiting conditions [9]. Likely, the coordinated functions of both the channel protein and the transporter are essential for the growth of plants under B-limiting conditions [10]. Overexpression of AtBOR1 or increased expression of AtNIP5;1 can improve B-deficiency symptoms in Arabidopsis [11,12], but techniques for improving crop tolerance to low-B conditions have rarely been reported. Rice OsNIP3;1 is a homolog of AtNIP5;1, and the Osnip3;1 mutant shows typical B-deficiency symptoms under B deficiency [13–15]. OsBOR1 was reported to be involved in both B uptake and xylem loading [16]. In maize, TLS1 encodes a protein that is a member of the aquaporin family and is co-orthologous to AtNIP5;1, and RTE encodes a B-efflux transporter that is co-orthologous to AtBOR1. Both tls1 and rte mutants showed vegetative and reproductive defects in low-B soils [17,18]. It was recently reported that BnaC4.BOR1;1c is essential for the inflorescence development of rapeseed under B deficiency [19]. Allotetraploid rapeseed (Brassica napus L., AnAnCnCn, 2n = 38), one of the main oil crop species worldwide, has a high B demand and is highly sensitive to B deficiency [20,21]. Under B deficiency, B. napus exhibit evident growth defects in both vegetative and reproductive organs, including inhibited root growth, curved leaves, multiple branches, necrosis and protruding stigmas, all of which lead to severe losses in seed yield [22]. Therefore, one potential solution is the identification of target genes for low-B tolerance for breeding B-efficient rapeseed. Previous studies have indicated that natural rapeseed varieties significantly vary in their tolerance to low-B conditions [23,24]. Quantitative trait locus (QTL) analysis revealed BnaA3.NIP5;1 to be the candidate gene for qBEC-A3a, a major quantitative trait locus for low-B tolerance from B-efficient (low-B tolerant) Qingyou 10 (QY10) no difference was found in the amino acid sequence of BnaA3.NIP5;1 between QY10 and the B-inefficient (low-B sensitive) Westar 10 (W10), while the expression level of BnaA3.NIP5;1 was significantly higher in QY10 than in W10 [25,26]. However, the molecular mechanism of BnaA3.NIP5;1 in response to B deficiency is still unknown. Here, we found that BnaA3.NIP5;1 is a boric acid channel responsible for B uptake into the root tips due to the specific distal polar localization in lateral root cap (LRC) cells. Further investigation revealed that genetic variation in the 5’UTR of BnaA3.NIP5;1 dictates the distinct transcript abundance, thus leading to different B efficiency-dependent root growth and development and seed yields. Our study provides new insight into the biological role of BnaA3.NIP5;1 for low-B tolerance and a novel perspective on the genetic improvement of B efficiency in B. napus.

Discussion Crop production depends on nutrient uptake, and the reduction in crop yields caused by nutrient deficiency is an important agronomical problem worldwide. A previous study indicated that genetic variations in low-B tolerance exist among different rapeseed varieties [23,24], providing opportunities to improve low-B tolerance in B-inefficient rapeseed by introducing the relevant gene(s) from B-efficient varieties. In this study, we identified the B efficiency-related gene BnaA3.NIP5;1, which is expressed in LRC cells, facilitates B uptake into root tips (Figs 2H and 3E). A CTTTC copy deletion within the 5’UTR increased BnaA3.NIP5;1 expression levels subsequently promoted root growth and increased seed yields under B limitation (Fig 7A, 7C and 7F). Notably, the BnaA3.NIP5;1Q allele effectively improves seed yields under low-B conditions (Fig 6F), indicating that BnaA3.NIP5;1 could serve as a novel target in rapeseed breeding for improved low-B tolerance. Normal plant growth requires an adequate B supply. Under B deficiency, the first symptoms of B deficiency occur in the growing tips of plants; these symptoms include root growth inhibition and shoot apical necrosis [21,30–32]. Thus, sufficient amounts of B must be available for developing tissue. A mathematical model was developed that predicted that the QC region has a high B concentration [33]. However, the mechanisms by which B is taken up or transported in root and shoot apices are still unclear. In the present study, our results showed that BnaA3.NIP5;1 is expressed specifically and polarly localized in the distal plasma membrane of LRC cells to promote B uptake into root tips (Fig 2H), which is necessary for root growth. Under low-B conditions, QY10 with high BnaA3.NIP5;1 expression levels in the LRC cells accumulated more B in the meristem region, which led to better root growth and development than those of W10 under B-deficiency conditions (Fig 3E and 3G). Furthermore, the results of the transgenic plants nip5;1–1 expressing BnaA3.NIP5;1 indicated that B taken up by BnaA3.NIP5;1 was used only for root growth and was not translocated to the shoots (Fig 4A). However, transgenic nip5;1–1 still had a growth defect, which indicates NIP5;1 expressing in the differential region is also important for plant normal growth under B limitation. It was reported that AtBOR2 also had a higher expression level in the LRC cells and with a proximal polar localization [10]. This suggests that BnaA3.NIP5;1 may cooperate with other B transporters to maintain high B concentration in the root meristem region under B-limitation. We conclude that B taken up by LRC cells is used only for local root growth and that high B concentrations are maintained in the meristem region, which is important for root development and growth under B deficiency. With the well-developed root, other boron related transporters uptake more B into roots and jointly promote shoot growth with BnaA3.NIP5;1. Growing roots vary both anatomically and physiologically along their longitudinal axes [34]. Nutrient uptake also varies across the different developmental zones. Generally, the rate of ion uptake per unit root length decreases with increasing distance from the root apex) 34]. It was recently reported that LRC cells are important for Pi uptake, but they have not been shown to influence root growth [35]. The results from our study clearly show that B uptake into root tips is essential for root growth under low-B conditions (Fig 3E and 3G). Previous studies have also demonstrated the presence of transporters of nitrate (NRT1.1) [36], potassium (ATKT3) [37] and iron (IRT1) [38] ions in the root tips. However, the role of these proteins in nutrition is remained to be elucidated. In Arabidopsis, AtNIP5;1 has been shown to be localized in the distal plasma membrane of both LRC cells and epidermal cells of roots [8,39]. A ThrProGly (TPG) repeat in the N-terminus of AtNIP5;1 is essential for AtNIP5;1 polar localization [40]. Two TPG repeats were found in the N-terminus of BnaA3.NIP5;1 (S3B Fig), and our results show that BnaA3.NIP5;1 localized in the plasma membrane in a polar manner, like AtNIP5;1 is. The upstream open reading frames (uORFs) within the 5’UTR of AtNIP5;1 induce B-dependent AtNIP5;1 mRNA degradation is important for plant growth under high-B conditions [41,42]. Two conserved uORFs were found in the 5’UTR of BnaA3.NIP5;1 (Figs 5F and S10), which indicates that BnaA3.NIP5;1 mRNA degradation under high-B conditions is similar to AtNIP5;1 mRNA degradation. Transposons have been reported to produce a wide variety of changes in plant gene expression [43]. However, we found that the different TE insertion sequences do not alter the gene expression levels or the expression patterns in QY10 and W10. The 849 bp promoter upstream of ATG is responsible for gene expression, and a CTTTC deletion increased BnaA3.NIP5;1 expression levels, which improved low-B tolerance in QY10. No CTTTC element was found within the promoter of AtNIP5;1 (S10 Fig), which suggests that CTTTC is unique to BnaA3.NIP5;1 regulation in B. napus. However, the activities of pQ::BnaA3.NIP5;1-GFP and pW::BnaA3.NIP5;1-GFP differed when they were expressed in nip5;1–1 (Fig 4), it is possible that Brassica napus and Arabidopsis show the same trans-factor or transcript factor to regulate the CTTTC element, and further study can be carried out in Arabidopsis to found the upstream regelation element of BnaA3.NIP5;1. Overall, BnaA3.NIP5;1 and its elite allele can serve as direct targets for genetic improvement of low-B tolerance in rapeseed breeding. BnaA3.NIP5;1 expressing in LRC cells for B uptake into root tips affords greatly increased seed yields under B deficit, highlighting the importance of nutrient uptake in root tips. The results in this study may provide a new way for improving rapeseed low-B tolerance and improving the efficiency of other nutrients.

Methods Plant materials and growth conditions The following plants were used in this study: the rapeseed (B. napus) cultivars W10 and QY10 and a CMS restorer line, L-135R; Arabidopsis (Arabidopsis thaliana) Col-0; and the Arabidopsis nip5;1–1 mutant. For long-term hydroponic cultivation, seeds of rapeseed were germinated on a piece of moist gauze submerged in ultrapure water (18.25 MΩ·cm) in a black plastic tray. After they germinated, uniform seedlings were transplanted into 10 L black plastic containers filled with Hoagland’s solution [44] consisting of 5 mM KNO 3 , 5 mM Ca(NO 3 ) 2 , 2 mM MgSO 4 , 1 mM KHPO 4 , 50 μM FeEDTA, 9 μM MnCl 2 , 0.8 μM CuSO 4 , 0.8 μM ZnSO 4 and 0.1 μM Na 2 MO 4 . H 3 BO 3 (100 μM) was used for normal-B treatment, and H 3 BO 3 (0.25 μM) was used for low-B treatment. The nutrient solutions were refreshed every 3 d. The seedlings were grown in a greenhouse under a 16 h light (24°C)/8 h dark (22°C) photoperiod with an approximately 300 μM m-2 s-1 photon density. For plate culture, seeds of B. napus and Arabidopsis were surface-sterilized for 15 min with 1% NaClO (w/v), rinsed with ultrapure water (18.25 MΩ·cm), chilled at 4°C for 2 d in the dark, and then sown onto solid media for plate culture. The plant growth medium was MGRL media [45], consisting of 1.75 mM sodium phosphate buffer (pH 5.8), 1.5 mM MgSO 4 , 2 mM Ca(NO 3 ) 2 , 3 mM KNO 3 , 67 μM Na 2 EDTA, 8.6 μM FeSO 4 , 10.3 μM MnSO4, 1 μM ZnSO 4 , 24 nM (NH 4 ) 6 Mo 7 O 24 , 130 nM CoCl 2 , 1 μM CuSO 4 , 1% sucrose and 1% gellan gum. H 3 BO 3 (100 μM) was used for normal-B treatment, H 3 BO 3 (0.1 μM ) was used for rapeseed low-B treatment, and H 3 BO 3 (0.3 μM) was used for Arabidopsis low-B treatment. The seedlings were grown in a growth chamber at 22°C under a 16 h light/8 h dark photoperiod. RNA extraction and qRT-PCR analyses Total RNA was extracted using an RNA extraction kit (Promega). The concentration of RNA was subsequently determined by a NanoDrop 2000 (Thermo Fisher). cDNA was prepared using Rever Tra Ace qPCR RT Master Mix with gDNA Remover kit (Toyobo). Quantitative real-time PCR assays were performed on a Real-time PCR Detection System (Applied Biosystems) in a 384-well plate via SYBR Green PCR (Toyobo). The 2-ΔΔct quantification method was used, and the variation in expression was estimated for three biological replicates. Transgenic rapeseed construction and phenotypic analyses An approximately 3 kb upstream regulatory sequence was amplified from the genomic DNA template of BnaA3.NIP5;1W and BnaA3.NIP5;1Q, and the coding sequence of BnaA3.NIP5;1 was amplified from a complementary DNA (cDNA) template fused in frame with GFP and then cloned into a pBI121 binary vector to generate pQ::BnaA3.NIP5;1-GFP constructs. The resulting vector was introduced into the B-inefficient variety W10 via Agrobacterium-mediated hypocotyl transgenic transformation. Additionally, BnaA3.NIP5;1-specific 312 bp sense and antisense fragments were amplified from the cDNA template and cloned into a pFGC5941binary vector to generate an RNAi construct. The resulting vector was transformed into QY10 and W10, yielding QRNAi and WRNAi transgenic seedlings. Positive transgenic seedlings were identified in each generation via insertion-specific PCR analyses. The sequences of the primers used for vector construction and transgenic plant identification are listed in S1 Table. Independent homozygous transgenic T 2 lines were grown as described above. The roots of both transgenic and WT seedlings were subsequently washed. After measuring the primary root length, the samples were dried at 65°C for 72 h to obtain the shoot and root dry weights. The BnaA3.NIP5;1 expression level was determined via qRT-PCR from root samples obtained from hydroponically cultivated 15-d-old rapeseed seedlings and normalized to the rapeseed EF1α and Tubulin internal control gene expression levels. 10B uptake in Xenopus laevis oocytes The coding DNA sequence (CDS) of BnaA3.NIP5;1-GFP was amplified from a pQ::BnaA3.NIP5;1-GFP vector, cloned into a pT7Ts X. laevis oocyte expression vector between the restriction sites BglII and SpeI and then linearized with BamHI. Capped mRNA was synthesized in vitro using an mMESSAGE mMACHINE kit (Ambion, AM1340). X. laevis oocytes were injected with 46 ng of BnaA3.NIP5;1-GFP cRNA and then cultured in ND96 media for 2 d for GFP observations via confocal microscopy (TCS SP8, Leica). GFP signal-positive oocytes or water-injected negative controls were collected from six-well plates filled with 5 ml of ND96 media, and then the ND96 media was removed and replaced with B-ND96 media consisting of 5 mM 10B. After a 30 min incubation at 18°C, each sample was rinsed five times with ice-cold ND96, and 8–11 oocytes were collected in a 2 ml tube and frozen at -20°C until sampled for elemental analysis. The oocytes were digested with HNO 3 at a maximum temperature of 110°C in plastic tubes, and the 10B concentration was analyzed via inductively coupled plasma-mass spectrometry (ICP-MS, 7700X; Agilent Technologies). Four replicates of oocytes were used for 10B uptake assays. The sequences of the primers used are listed in S1 Table. In situ RT-PCR and GUS staining Rapeseed root samples from 5-d-old seedlings grown under B-deficiency conditions were fixed with a solution consisting of 63% (v/v) ethanol, 5% (v/v) acetic acid and 2% (v/v) formaldehyde for 4 h, embedded into 5% (w/v) agarose and then sectioned to 50 μm. BnaA3.NIP5;1 in situ RT-PCR flowed method with the modifications of Athman et al. (2014) [46]. The samples were stained using BM purple AP substrate (Roche) for 30 min, washed in an orderly manner with washing buffer, mounted in 40% (v/v) glycerol and then observed under a microscope (Nikon DS-Ri 2). The pQ::BnaA3.NIP5;1 fragment was amplified from the pQ::BnaA3.NIP5;1-GFP vector and cloned into pBI121 to generate pQ::BnaA3.NIP5;1-GUS constructs. The resulting vectors were transformed into W10 rapeseed. The pQ::BnaA3.NIP5;1-GUS transgenic seedlings were incubated in a solution of 1 mg ml-1 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-gluc), 100 mM sodium phosphate (pH 7.0), 0.5 mM K 3 Fe(CN) 6 , 0.5 mM K 4 Fe(CN) 6 , 10 mM Na 2 EDTA, 0.1% (v/v) Triton X-100 and 20% (v/v) methanol at 37°C in the dark for 1 h. After incubation, the chlorophyll was removed using 75% ethanol, and images were taken using stereomicroscope (Olympus SZX18). Tissue, subcellular localization assay and fluorescence intensity measurements The T 2 generation of pQ::BnaA3.NIP5;1-GFP transgenic plants was used for tissue and subcellular assays. The transgenic plants were grown on 0.1 μM B MGRL solid media for 4–6 d. N-(3-Triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) (FM4-64) was used as an endocytic tracer. Confocal imaging was performed via confocal microscopy (TCS SP8, Leica). The pQ::BnaA3.NIP5;1-GFP and pW::BnaA3.NIP5;1-GFP constructs were transformed into Arabidopsis ecotype Col-0 plants and nip5;1–1 mutants via the floral-dip method used for tissue, subcellular localization assays and fluorescence intensity measurements. Transgenic Arabidopsis plants were grown on 0.3 μM B MGRL solid media for 4–6 d, and confocal imaging was performed as described above. 10B boric acid uptake activity in the roots and root tips Fifteen-day-old QY10, W10 and NILQ-W10 seedlings pre-cultured with 25 μM 11B (Cambridge Isotope Laboratories) were exposed to a solution containing 0 B for 1 d. The seedlings were subsequently exposed to 10 μM 10B (Cambridge Isotope Laboratories) for 1 h, after which the roots were washed with ultrapure water three times. Shoot and root samples were collected, separated and then dried at 65°C for 72 h. The samples were then digested with HNO 3 at a maximum temperature of 110°C in plastic tubes and the resulting digestions were analyzed via ICP-MS (7700X; Agilent Technologies). To investigate B uptake in root tips, rapeseed seedlings were pre-cultured in 0 B MGRL solid media for 5 d. Afterward, 1×1 cm pieces of MGRL solid media consisting of 10 μM 10B were applied such that the root tips were covered for 1 h. The roots were then washed three times with ultrapure water, after which the root tips (5 mm) were excised by the use of a razor. The fresh weight was immediately recorded for 50 to 60 root tips, which constituted one replicate. After digestion, the 10B concentration was determined as described above. Phenotyping the primary root growth of rapeseed and Arabidopsis under B deficiency Seeds of QY10, W10, NILQ-W10 and the representative varieties selected from the natural population were put atop solid media, and the seedlings were grown on vertically oriented solid media in a growth chamber. Under the 100 μM B conditions, 5 d was enough for the primary roots of the rapeseed seedlings to reach the bottom of the plastic dish, while under the 0.1 μM B conditions, the seedlings were allowed to grow for 10 d before images were collected and the primary root length was measured. Seedlings of the Arabidopsis pQ::BnaA3.NIP5;1-GFP#n and pW::BnaA3.NIP5;1-GFP#n T 3 transgenic lines were grown on MGRL solid media consisting of 0.3 μM B. After 10 d of growth in the growth chamber, the primary root length was measured, and the expression of BnaA3 was measured. NIP5;1 expression in the roots was determined via qRT-PCR and normalized to the Arabidopsis EF1α and Actin internal control gene expression. Analysis of B distribution using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) Seedlings of rapeseed were grown on B-deficient solid media for 10 d, after which the plant material was collected and ground into powder after drying. Afterward, 0, 20, 40, 100, 200, 400, or 1000 μl of 10 mg L-1 10B were added to the 200 mg powder, respectively, and were mixed together well. After absorption for 48 h, the mixture was re-dried and ground to a powder. After digestion, the 10B concentration was determined as described above. Fifty milligrams of powder were then compressed under 8 atm to generate standard reference material (S11 Fig). Seedlings of QY10, W10 and NILQ-W10 were grown in solid media under 0.1 μM 10B and 100 μM 10B conditions for 5 d. The roots were washed three times with ultrapure water, and the root tips (1.5 to 2 cm in length) were excised and then affixed to slides, which were then dried overnight at -20°C. LA-ICP-MS analysis was subsequently performed with a laser ablation system (New Wave Research UP 213) equipped with a Nd: YAG laser (wavelength, 213 nm; repetition frequency, 20 Hz; spot size, 50 μm; scan speed, 20 μm s-1; energy output: 50%; He carrier flow rate, 900 ml min-1). The root tips were put into the laser ablation chamber and scanned together with the standards. Element image transformation was performed by Surfer 11 software. Three biological replications of each sample were analyzed, each of which showed similar results. GUS activity and transient gene expression assays The BnaA3.NIP5;1 promoter regions from W10 and QY10 were serially deleted and cloned into pBI121 vectors to generate G1-G6 constructs, and the resulting vectors were transformed into Arabidopsis ecotype Col-0 by the floral-dip method. Independent homozygous T 3 transgenic lines were grown in MGRL solid media consisting of 0.3 μM B for 10 d. Total protein extraction and quantitative GUS activity assays were conducted as described by Jefferson et al. (1987) [47]. The protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad), and the fluorescence intensity was measured on a Spark 20M multimode plate reader (Tecan). Addition mutations in G5 and G6 were investigated to generate G7-G10 constructs, and the resulting vectors were transformed into tobacco by Agrobacterium-mediated transformation for transient gene expression assays. After transfection, GUS activity was determined as described above. Luciferase was co-transfected and used as an internal control to normalize the data. NIL construction The homozygous NILQ-W10 line was previously derived from a QY10 donor parent and a W10 recurrent parent (25, 26). With respect to NILQ-L135R, F 1 seeds were obtained by crossing L-135R with QY10. BnaA3.NIP5;1 in each generation was genotyped, and the heterozygous plants were backcrossed with L-135R to the BC 5 F 1 generation. The BC 5 F 1 plants were then self-pollinated to obtain BC 5 F 2 plants, which were self-pollinated to get BC 5 F 3 plants for further analysis. Morphological differences and B-deficiency tolerance were compared between NILs homozygous for BnaA3.NIP5;1Q and recurrent parents for BnaA3.NIP5;1W. Evaluation of B-deficiency tolerance under pot and field conditions For pot cultivation, each pot contained 7 kg of grey purple sandy soil. The basic agrochemical characteristics of the soil were as follows: pH (1:1 soil: H 2 O (w/v)), 7.7; organic matter, 1.33 g kg-1; total nitrogen (N), 0.25 g kg-1; total phosphorus (P), 72 mg kg-1; and hot water-soluble B, 0.10 mg kg-1. Two B treatments, 1 mg B kg-1 soil (HB) and 0.25 mg B kg-1 soil (LB), were applied, with four replicates per treatment. The plants were irrigated with ultrapure water. For field trials, the B-deficiency tolerance of QY10, W10, NILQ-W10, NILQ-L135R and the representative varieties were compared under field conditions during the regular rapeseed growing season in 2019 at Guotan village (30°18′ N, 115°60′ E, Wuxue, Hubei Province, China). The basic agrochemical characteristics of the soil were as follows: pH (1:1 soil: H 2 O (w/v)), 5.18; organic matter, 37.16 g kg-1; total N, 1.86 g kg-1; Olsen-P, 21.70 mg kg-1; and hot water-soluble B, 0.10 mg kg-1. The application rate of B fertilizer was 15 kg borax ha-1 in the normal-B treatment, and no B fertilizer application was applied as the B-deficiency treatment. Rapeseed plants were cultivated at a distance of 15×25 cm in a 1.2×15 m plot, each variety was planted in four lines, and each treatment included three replicates. After harvest, all the seeds were allowed to dry naturally before determining the seed yield. Statistical analysis The data were analyzed using Student’s t-test, and significance was defined as P<0.05 or P<0.01. r and p values of the correlation analysis were determined by Pearson correlation analysis.

Acknowledgments We thank Professor Junpei Takano (Osaka Prefecture University, Japan) for kindly providing the Arabidopsis mutant nip5;1–1.

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