PLoS Genetics
Public Library of Science
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Genetic variation of BnaA3.NIP5;1 expressing in the lateral root cap contributes to boron deficiency tolerance in Brassica napus
DOI 10.1371/journal.pgen.1009661 , Volume: 17 , Issue: 7 , Pages: 0-0
Article Type: research-article, Article History
Abstract

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.

Introduction

Boron (B) is an essential micronutrient for all higher plants. Plant roots take up B in the form of boric acid (H3BO3 ). 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 [36]. 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 [1315]. 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.

Results

Differential expression of BnaA3.NIP5;1 is responsible for low-B tolerance

To investigate the relationship between BnaA3.NIP5;1 expression levels and low-B tolerance, we generated transgenic rapeseed expressing BnaA3.NIP5;1-GFP under control of the 2927 bp QY10 promoter (pQ::BnaA3.NIP5;1-GFP) in W10. Three independent pQ::BnaA3.NIP5;1-GFP transgenic lines were analyzed in the T2 generation. qRT-PCR analysis revealed that the expression of BnaA3.NIP5;1 in the roots of the three lines was significantly increased compared with that of the wild-type (WT) W10 (Fig 1B). Consequently, compared with W10, the transgenic lines presented longer roots and greater biomass under low-B (0.25 μM) conditions (Fig 1A and 1C–1E). In comparison, no significant difference was observed among these plants under normal-B (100 μM) conditions (S1A–S1C Fig). We further employed RNA interference (RNAi) to suppress the expression of BnaA3.NIP5;1 in both QY10 (QRNAi) and W10 (WRNAi). With decreased BnaA3.NIP5;1 expression level (Fig 1G), the QRNAi transgenic plants exhibited severe growth defects compared with those of the QY10 plants under 0.25 μM B conditions (Fig 1F), with markedly reduced primary root length and biomass (Fig 1H–1J). No noticeable difference was found under 100 μM B conditions (S1D–S1F Fig). We also noticed the expression of BnaA3.NIP5;1 in the WRNAi transgenic plants was similar to that in W10, and both the WRNAi transgenic plants and W10 showed typical B-deficiency symptoms (S1G–S1J Fig), which is probably due to the low expression level of BnaA3.NIP5;1 in W10.

Response of WT and transgenic plants to B deficiency.
Fig 1
(A) Phenotype of 15-d-old WT (W10) and pQ::BnaA3.NIP5;1-GFP transgenic plants grown under low-B (0.25 μM) conditions. Scale bar, 2 cm. (B-E) Relative expression of BnaA3.NIP5;1 (B), primary root length (C), root dry weight (D) and shoot dry weight (E) of W10 and pQ::BnaA3.NIP5;1-GFP transgenic plants under low-B (0.25 μM) conditions. (F) Phenotype of 15-d-old WT (QY10) and QY10-RNAi lines grown under low-B (0.25 μM) conditions. Scale bar, 2 cm. (G-J) Relative expression of BnaA3.NIP5;1 (G), primary root length (H), root dry weight (I) and shoot dry weight (J) of QY10 and QY10-RNAi lines under low-B (0.25 μM) conditions. The data presented are mean values with s.d. (three replicates in (B and G); six replicates in (C, D, E, H, I and H). * P<0.05, ** P<0.01 (Student’s t-test).Response of WT and transgenic plants to B deficiency.

To further investigate the effects of BnaA3.NIP5;1 expression on seed productivity in B. napus, we compared the seed yield of QRNAi transgenic plants and QY10 growing in pots at the maturity stage. The results showed that the per-plant seed yield of the QRNAi transgenic plants was significantly lower than that of QY10 under low-B conditions (S2A Fig). However, there was no difference under high-B conditions (S2B Fig). Taken together, these data suggest increased expression of BnaA3.NIP5;1 contributes to low-B tolerance in rapeseed during both the seedling and maturity stages.

BnaA3.NIP5;1 functions as a boric acid channel and is expressed preferentially in LRCs

BnaA3.NIP5;1 encodes a major intrinsic protein belonging to the aquaporin protein family [27,28]. Phylogenetic analysis revealed that BnaA3.NIP5;1 is a homolog of AtNIP5;1, with 92% amino acid identity (S3A and S3B Fig). To verify that BnaA3.NIP5;1 protein function in transporting H3BO3 across membranes, we expressed BnaA3.NIP5;1-GFP in Xenopus oocytes. The results showed that BnaA3.NIP5;1-GFP was localized on the plasma membrane of the oocytes (Fig 2A) and that oocytes carrying BnaA3.NIP5;1-GFP had a 10B concentration 3-fold higher than that of H2 O-injected oocytes (Fig 2B). These findings demonstrate that BnaA3.NIP5;1 has H3BO3 transport activity.

Functional characterization and tissue and subcellular localization of BnaA3.NIP5;1.
Fig 2
(A) Subcellular localization of BnaA3.NIP5;1-GFP within the oocyte membrane. (B) Boric acid uptake in Xenopus oocytes injected with BnaA3.NIP5;1-GFP composed of 10B. The data presented are mean values with s.d. of four replicates. ** P<0.01 (Student’s t-test). (C and D) RNA in situ RT-PCR of the cell-specific expression pattern of BnaA3.NIP5;1 in root tip cross-sections. The dark blue signal indicated the presence of BnaA3.NIP5;1 mRNA; no reverse transcription (no RT) conditions were used as negative controls. Similar results were observed in each of three independent experiments. (E-G) GUS staining of roots and cross-sections of root tips of pQ::BnaA3.NIP5;1-GUS transgenic plants. Similar results were observed in each of the three independent transgenic lines. (H-J) Confocal images of pQ::BnaA3.NIP5;1-GFP transgenic plants with vertical sections and cross-sections of the root tips; FM4-64 was used as a membrane-selective tracer. Similar results were observed in each of the three independent transgenic lines. All the rapeseed plants analysed were grown in solid media consisting of 0.1 μM B. Scale bars as shown.Functional characterization and tissue and subcellular localization of BnaA3.NIP5;1.

To investigate the expression pattern of BnaA3.NIP5;1 , we generated pQ::BnaA3.NIP5;1-β-glucuronidase (GUS) transgenic plants in the W10 background. GUS activity was distinctly detected in the root tips (Fig 2E and 2F); specifically, cross-sections of the root tips revealed high GUS activity in the peripheral tissues (Fig 2G). The subcellular localization of BnaA3NIP5;1 was examined using pQ::BnaA3.NIP5;1-GFP transgenic plants. Confocal imaging showed that BnaA3.NIP5;1-GFP was polarly located in the distal plasma membrane of LRC cells (Fig 2H–2J). Such an expression pattern was confirmed by an in situ RT-PCR assay (Fig 2C and 2D), as well as in Arabidopsis (S4A–S4C Fig). Collectively, these findings provide strong evidence that BnaA3.NIP5;1 was distinctly polar-localized in the distal plasma membrane of LRC cells and functions directly in H3BO3 uptake.

BnaA3.NIP5;1 transports B into the root tips to promote root growth

To investigate the role of BnaA3.NIP5;1 in B. napus, we performed short-term 10B accumulation assays of QY10, W10 and NILQ-W10 (NIL carrying the homozygous allele from QY10 in the W10 background). The results showed that the 10B concentration in both the shoots and roots of QY10 was lower than that in both the shoots and roots of W10 and NILQ -W10 (Fig 3A and 3B), while the 10B concentration was significantly increased in the root tips of QY10 and NILQ -W10 compared with those in W10 (Fig 3C). No significant difference was observed in 11 B concentrations (S5A and S5B Fig). We further performed an in situ laser ablation-inductivity coupled plasma-mass spectrometry (LA-ICP-MS) assay to visualize the distribution of B in the root tips. Consistent with the results of the 10B accumulation assay in the root tip, the results showed that QY10 and NILQ -W10 had higher B concentrations in the root tips than did W10 at 0.1 μM B, especially in the cells in meristem region (Fig 3E); however, there was no distinct difference under 100 μM B (Fig 3F). On the basis of these results, we hypothesized that BnaA3.NIP5;1 might facilitate root growth in B. napus. As expected, QY10 and NILQ -W10 had longer primary roots than did W10 at 0.1 μM B (Fig 3D and 3G), while there was no difference under 100 μM B (Figs 3H and S5C).

BnaA3.NIP5;1 transports B into the root tips to promote root growth.
Fig 3
(A-C) 10B concentrations in the shoots (A), roots (B) and root tips (C) of QY10, W10 and NILQ-W10. In (A and B), QY10, W10 and NILQ-W10 seedlings were pre-cultured with 25 μM 11B for 15 d and then exposed to a solution without B for 1 d. The seedlings were subsequently exposed to 10 μM 10B for 1 h. In (C), the seedlings were pre-cultured in solid media without B for 5 d, after which the root tips were covered by a piece of solid media consisting of 10 μM 10B for 1 h, with 50 to 60 root tips constituting one replicate. After HNO3 digestion, 10B and 11B were determined by ICP-MS. (D) Primary root length of QY10, W10 and NILQ-W10 seedlings grown on solid media consisting of 0.1 μM B for 10 d. (E and F) 10B distribution in the root tips of 5-d-old QY10, W10 and NILQ-W10 under 0.1 μM (E) and 100 μM (F) B according to LA-ICP-MS. Roots of QY10, W10 and NILQ-W10 seedlings grown on solid media consisting of 0.1 μM 10B and 100 μM 10B conditions for 5 d. The root tips were put into the laser ablation chamber and scanned together with the standard reference materials; 10B was determined by ICP-MS, and similar results were observed in each of three independent experiments. Scale bars, 100 μm. (G and H) Morphology of QY10, W10 and NILQ-W10 root growth. The rapeseed seedlings were grown on solid media consisting of 0.1 μM B for 10 d (G) and 100 μM B for 5 d (H). Scale bars, 1 cm. The data presented are mean values with s.d. Three replicates in (A-C); sixteen replicates in (D). * P<0.05, ** P<0.01 (Student’s t-test).BnaA3.NIP5;1 transports B into the root tips to promote root growth.

To further validate the role of BnaA3.NIP5;1 in root growth, we generated transgenic plants expressing pQ::BnaA3.NIP5;1-GFP or pW::BnaA3.NIP5;1-GFP in the background of Arabidopsis mutant nip5;1–1. The primary roots of all the transgenic plants were longer than those of the nip5;1–1 plants and compared with the pW::BnaA3.NIP5;1-GFP#n transgenic plants, the pQ::BnaA3.NIP5;1-GFP#n lines had higher BnaA3.NIP5;1 expression levels and longer primary roots (Fig 4A–4C). By contrast, there was no significant difference in shoot growth among all these genotypes (Fig 4D). These results demonstrate that BnaA3.NIP5;1 transports B into root tips and specifically promotes root growth and development rather than facilitates B uptake in root vasculature tissues.

BnaA3.NIP5;1 promotes Arabidopsis root growth.
Fig 4
(A) Phenotype of 10-d-old nip5;1–1, pQ::BnaA3.NIP5;1-GFP#n (pQ#n) and pW::BnaA3.NIP5;1-GFP#n (pW#n) transgenic plants under 0.3 μM B conditions. Scale bar, 1 cm. (B-D) Relative expression level of BnaA3.NIP5;1 (B), primary root length (C) and shoot fresh weight (D) of nip5;1–1, pQ::BnaA3.NIP5;1-GFP#n and pW::BnaA3.NIP5;1-GFP#n transgenic plants grown under 0.3 μM B conditions. The data presented are mean values with s.d. Three replicates in (B), twelve replicates in (C), and six replicates in (D). ** P<0.01 (Student’s t-test).BnaA3.NIP5;1 promotes Arabidopsis root growth.

A CTTTC tandem repeat in the 5’UTR alters the expression of BnaA3.NIP5;1

An approximately 3 kb promoter fragment containing the 5’UTR of BnaA3.NIP5;1 was compared between QY10 and W10. Fifty-one single-nucleotide polymorphisms (SNPs), 4 InDels and one transposable element (TE) (differed in length between the two) were found between the two promoter sequences (Figs 5A and 6). To identify the functional allelic variations in the promoter sequence, we generated a series of GUS constructs whose promoter sequence was truncated and transformed them into Columbia-0 (Col-0) plants (Fig 5B). GUS activity was subsequently detected in the tips of both primary roots and lateral roots of G1 (pQ2727::GUS) and G2 (pW3175::GUS) transgenic plants (S7 Fig), consistent with the expression pattern of BnaA3.NIP5;1 in rapeseed (Fig 2E), and increased GUS activity was observed in the G1 transgenic plants compared with the G2 transgenic plants (Figs 5C and S7). For each construct, the GUS activity was measured in the roots of three independent lines. GUS activity in the G1 transgenic plants was significantly higher than that in the G2 transgenic plants, and transgenic lines carrying G3 (pQ2468::GUS) and G5 (pQ849::GUS) had significantly higher GUS activity compared with that of G4 (pW2706::GUS) and G6 (pW852::GUS) (Fig 5C and 5D). GUS activity in the G3 and G5 transgenic plants was similar to that in the transgenic lines carrying G1, while the GUS activity in G4 and G6 was similar to that in the G2 transgenic plants (Fig 5C and 5D). These results revealed that the polymorphisms from -849 to -1 were responsible for the different expression of BnaA3.NIP5;1 between QY10 and W10.

A CTTTC tandem repeat in the 5’UTR confers the different expression of BnaA3.NIP5;1.
Fig 5
(A) Schematic diagram of BnaA3.NIP5;1 in QY10 and W10. The white boxes represent 5’UTRs and 3’UTRs, grey boxes represent exons, the lines between grey boxes represents introns, and the brown and red boxes represent different TEs from QY10 and W10, respectively. (B) Constructs used for Arabidopsis transformation. G1 pQ(2927)::GUS contains the 2927 bp promoter of QY10. G2 pW(3175)::GUS contains the 3175 bp promoter of W10, and G3-G6 have a 5’ series deletion as indicated. (C and D) GUS staining and activity of transgenic plants harbouring G1-G6 constructs. Seedlings of the G1-G6 transgenic plants were grown on solid media consisting of 0.3 μM B for 10 d. Scale bar, 100 μm. (E) Variations in the 849 bp fragment in the promoters of QY10 and W10. CTTTC tandem repeats are indicated by the red box in the BnaA3.NIP5;1Q promoter and the green box in the BnaA3.NIP5;1W promoter, respectively. The red bars indicate uORFs, and minus signs indicate nucleotide deletions and polymorphisms. (F) Constructs used for tobacco transformation. The 5’UTR and CTTTC repeat in G5/G6 were exchanged to generate G7/G8 and mutated to generate G10/G9 constructs. (G) Relative GUS activity of different constructs (G5-G10), (CaMV) 35S-luciferase was co-transfected and used as an internal control. The data presented are mean values with s.d. Three replicates were used in (D), and six replicates were used in (G). ** P<0.01 (Student’s t-test).A CTTTC tandem repeat in the 5’UTR confers the different expression of BnaA3.NIP5;1.

Compared to W10, ten SNPs and 1 InDel (one CTTTC copy in QY10 and two tandem CTTTCs in W10) in the -464 to -1 5’UTR and 5 SNPs and 1 InDel in the -849 to -465 region were detected in the promoter region of QY10 compared with W10 (Fig 5E). We first verified whether the -849 to -1 sequence of BnaA3.NIP5;1Q (G5) could transcribe GUS in tobacco, which presented higher GUS activity than did the -852 to -1 sequence of BnaA3.NIP5;1W (G6) (Fig 5F and 5G). We exchanged the -465 to -849 promoter region between QY10 and W10 to generate G7 and G8 constructs (Fig 5F). Still, no effects on GUS activity were observed (Fig 5G), suggesting that polymorphisms from -464 to -1 are responsible for the different expression level of BnaA3.NIP5;1 between QY10 and W10. We then deleted one CTTTC copy from the 5’UTRW (G9) and inserted one CTTTC copy into the 5’UTRQ (G10) to generate G9 and G10 constructs, respectively, and compared the GUS activity of G9 and G10 (Fig 5F). The deletion of one CTTTC copy from the 5’UTRW (G9) significantly enhanced GUS activity, and the insertion of one copy of CTTTC into the 5’UTRQ (G10) largely decreased GUS activity (Fig 5G). Taken together, these data indicate that a tandem repeat of CTTTC in the 5’UTR is responsible for the different expression of BnaA3.NIP5;1.

BnaA3.NIP5;1Q allele improves seed yield under low-B conditions

To verify the contribution of the BnaA3.NIP5;1Q allele to seed yield in rapeseed, we further introgressed the tolerant allele of BnaA3.NIP5;1 from QY10 into the cytoplasmic male-sterile (CMS) restorer line L-135R through five generations backcrossing with L-135R. After two additional generations of self-pollination, we ultimately obtained BC5F3 plants (NILQ-L135R) carrying homozygous BnaA3.NIP5;1Q alleles. Furthermore, W10, NILQ-W10, L-135R and NILQ -L135R were used to evaluate the low-B tolerance in field trials with and without B fertilizer. W10 and L-135R showed severe reproductive development defects with no B supply, reflected by fewer and seedless siliques (Fig 6B, 6D and 6E), while no B-deficiency symptoms were observed for NILQ-W10 and NILQ -L135R (Fig 6A and 6B). The per-plant seed yield of NILQ-W10 and NILQ -L135R was significantly higher than that of W10 and L-135R, respectively, in the no B supply treatment (Fig 6C and 6F). Taken together, these results indicate the selection of BnaA3.NIP5;1Q can effectively improve seed yield under low-B conditions.

The BnaA3.NIP3;1Q allele improves low-B tolerance and seed yields of B. napus.
Fig 6
(A and B) Phenotypes of W10, NILQ-W10, L-135R and NILQ-L135R with B fertilization (+B) (A) and without B fertilization (-B) (B). Scale bar, 20 cm. (C) Total seeds per plant of W10, NILQ-W10, L-135R and NILQ-L135R with B fertilization (+B) and without B fertilization (-B). Scale bar, 1 cm. (D-F) Statistical comparisons of pod number per plant (D), seed number per pod (E), and seed yield per plant (F). The data presented are mean values with s.d. Twelve biological replicates in (D-F). ** P<0.01 (Student’s t-test).The BnaA3.NIP3;1Q allele improves low-B tolerance and seed yields of B. napus.

BnaA3.NIP5;1Q haplotype confers high BnaA3.NIP5;1 expression and low-B tolerance to the natural population

To investigate the association between the variation in BnaA3.NIP5;1 and B-deficiency tolerance, 10 B-efficient and 19 B-inefficient rapeseed varieties were selected from a natural population comprising 210 rapeseed accessions [29]. We re-sequenced the -849 to -1 fragment of the BnaA3.NIP5;1 promoter from the 29 varieties together with QY10, W10 and NILQ-W10. Based on the CTTTC tandem repeat, these 32 lines were classified into 2 groups, 13 of which presented the QY10 haplotype, with a single CTTTC copy (BnaA3.NIP5;1Q), and 19 of which showed the W10 haplotype, with two tandem CTTTCs (BnaA3.NIP5;1W ) (S8 Fig). The expression levels of BnaA3.NIP5;1 in these plants were subsequently examined and compared with the BnaA3.NIP5;1W haplotype, the BnaA3.NIP5;1Q haplotype presented increased expression under low-B conditions (Fig 7A and 7B). In agreement with these results, the primary roots of these varieties carrying BnaA3.NIP5;1Q were longer than those of the varieties presented the BnaA3.NIP5;1W haplotype (Figs 7C, 7D and S9A). Furthermore, the varieties presented BnaA3.NIP5;1W haplotype showed severe reproductive development defects, and the per-plant seed yield of the varieties presenting the BnaA3.NIP5;1Q haplotype were significantly higher than those presenting the BnaA3.NIP5;1W haplotype (Figs 7F and 7G and S9B). The increased BnaA3.NIP5;1 expression levels in the representative varieties were positively correlated with primary root length and seed yield under low-B conditions (Fig 7E and 7H). Overall, the genetic variations of BnaA3.NIP5;1 in the different rapeseed varieties further confirmed that the CTTTC tandem repeats within the BnaA3.NIP5;1 5’UTR directly regulates its expression, ultimately affecting primary root length and productivity under B-deficiency conditions.

A tandem CTTTC repeat modulates BnaA3.NIP5;1 expression and low-B tolerance.
Fig 7
(A) Analysis of BnaA3.NIP5;1 expression in the roots of representative varieties. Seedlings of the representative varieties were grown on solid media consisting 0.1 B for 10 d; afterward, the roots were sampled, and the expression level was determined by qRT-PCR. (B) The expression levels of BnaA3.NIP5;1 of the different varieties were grouped according to the BnaA3.NIP5;1Q and BnaA3.NIP5;1W haplotypes. (C) Primary root length of representative varieties. Seedlings of the representative varieties were grown on solid media consisting of 0.1 B for 10 d. (D) The primary root lengths of different varieties were grouped according to the BnaA3.NIP5;1Q and BnaA3.NIP5;1W haplotypes. (E) Correlations between primary root length and BnaA3.NIP5;1 expression level among different varieties under low-B conditions. (F) Per-plant seed yield of representative varieties grown in a B-deficient plot in the field. (G) Per-plant seed yield of representative varieties with the BnaA3.NIP5;1W or BnaA3.NIP5;1Q haplotype. (H) Correlations between per-plant seed yield and BnaA3.NIP5;1 expression level among the different varieties under low-B conditions. In (B, D and G), the data represented are mean values of three replicates with s.d., the box shows the median and the lower and upper quartiles. The numbers (n) of each haplotype and associated P value (Student’s t-test) are shown above the graph. In (E and H), the r and p values were determined by Pearson correlation analysis. Six replicates were used for the primary root length data, and twelve replicates were used for the seed yield data.A tandem CTTTC repeat modulates BnaA3.NIP5;1 expression and low-B tolerance.

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 [21,3032]. 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 KNO3, 5 mM Ca(NO3)2, 2 mM MgSO4, 1 mM KHPO4, 50 μM FeEDTA, 9 μM MnCl2, 0.8 μM CuSO4, 0.8 μM ZnSO4 and 0.1 μM Na2MO4. H3BO3 (100 μM) was used for normal-B treatment, and H3BO3 (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 MgSO4, 2 mM Ca(NO3)2, 3 mM KNO3, 67 μM Na2EDTA, 8.6 μM FeSO4, 10.3 μM MnSO4, 1 μM ZnSO4, 24 nM (NH4)6Mo7O24, 130 nM CoCl2, 1 μM CuSO4, 1% sucrose and 1% gellan gum. H3BO3 (100 μM) was used for normal-B treatment, H3BO3 (0.1 μM) was used for rapeseed low-B treatment, and H3BO3 (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 T2 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 HNO3 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 10 B 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 K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 10 mM Na2EDTA, 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 T2 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 HNO3 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 T3 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 10 B 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 T3 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, F1 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 BC5F1 generation. The BC5F1 plants were then self-pollinated to obtain BC5F2 plants, which were self-pollinated to get BC5F3 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: H2O (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: H2O (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.

Acknowledgements

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

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2 May 2021

Dear Dr Xu,

Thank you very much for submitting your Research Article entitled 'Genetic variation of BnaA3.NIP5;1 expressing in the lateral root cap contributes to boron deficiency tolerance in Brassica napus' to PLOS Genetics.

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Zhixi Tian, Ph.D

Associate Editor

PLOS Genetics

Li-Jia Qu

Section Editor: Plant Genetics

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: In the manuscript entitled “Genetic variation of BnaA3.NIP5;1 expressing in the lateral root cap contributes to boron deficiency tolerance in Brassica napus”, the authors investigated the natural genetic variation in BnaA3.NIP5; 1 gene, which they identified but has not functionally characterized as a key determinant of low-B tolerance in B. napus in their previous studies. It was found that that a CTTTC tandem repeat in the 5’UTR of BnaA3.NIP5;1 altered its expression level, and associated with plant growth and seed yield. Though the orthologue of this gene in Arabidopsis has been well characterized, novel data and strong evidence were provided in this study to tell a novel story on the contribution of a superior allele to low-B tolerance of B. napus that requires high B supplies. Overall, the results are clear and the manuscript is easy to follow. Here I list my concerns for suggestion.

1 The CTTTC tandem repeat in the 5’UTR underlies the difference in expression levels in parental lines, NILs and natural populations. Then how does the CTTTC tandem repeat works? Does this repeat hinder the gene translation? Typically, the sequence variation in UTRs play roles on mRNA stability and translation, but not on transcription. More data should be provided to make the story clear.

2 Besides the phenotypic data on primary root length and productivity, the data on B uptake and/or accumulation are necessary to solidify the allelic difference in natural population (line 215-235) .

3 some typos:

L58 Under low-B conditions, B can be taken up plants via two different mechanisms.

L59 “Channel-mediated facilitated transport via NOD26-LIKE MAJOR INTRINSIC PROTEIN5;1 (NIP5;1), which encodes a channel protein belonging to the aquaporin family; this protein has been shown to facilitate B uptake from the soil in Arabidopsis roots.

Reviewer #2: Boron is essential for plant development and reproduction. In this study, the authors characterized the molecular mechanism of BnaA3.NIP5;1 in boron deficiency response. They found that higher expression of BnaA3.NIP5;1 in B. napus varieties conferred low-boron tolerance. Further analysis revealed that a CTTTC tandem repeat in the 5’UTR of BnaA3.NIP5;1 determined its expression level. They also convincingly demonstrated that BnaA3.NIP5;1 transport boron into root tips and promote root growth under B-deficiency condition. Overall, this study provides new insights into the low-B tolerance and a novel perspective on the genetic improvement of boron efficiency in B. napus.

However, there are still a few issues need to be addressed.

1. The authors claimed that B taken up by BnaA3.NIP5;1 was used only for local root growth, however changing the expression of BnaA3.NIP5;1 dramatically influence the shoot growth and yield. Therefore, the tissue specific expression of BnaA3.NIP5;1 should be present. Is there any possibility that the BnaA3.NIP5;1 also has function for boron transport at SAM cells, which contribute to the shoot growth. If BnaA3.NIP5;1 only expressed in lateral root cap and affected root tip boron uptake and distribution, the authors should propose a possible mechanism to explain the impact of BnaA3.NIP5;1 for shoot growth.

2. In fig3E, it's hard to distinguish the QC from other regions in root tips, it seems that high content of boron was accumulated in root meristem region.

3. Given the presence of other boron related transporters in plant, how could the higher expression of BnaA3.NIP5;1 in lateral root cap to maintain higher boron distribution in root tip under boron deficient condition and promote root growth? Possible explanation should be discussed.

Reviewer #3: Boron (B) is an essential element for plant growth and development. B nutrition is of great important for productivity of crops like rapeseed. In this manuscript He and her/his coworkers characterized a previously identified B transporter gene NIP5;1 for B-dependent root growth. They further discovered a CTTTC tandem repeat in the 5’-UTR of NIP5;1 that was capable to regulate the gene expression levels, and the corresponding natural variations were associated with root growth and seed yield under limited B conditions. This story hightlights a novel function of B transporters in root development, and provides the favorite gene alleles for breeding low-B tolerant crops. The manuscript is well written with a proper language. Some minor points are needed to be considered for improving the quality of manuscript.

Minor points:

1) Line 161-162: this sentence can be “ By contrast, the shoots development did not differ among all genotypes”.

2) Line 172-173: this sentence can be moved to the materials and methods part.

3) Line 285-285: As shown in Figure 4, the activities of pQ and pW differed when they were expressed in Arabidopsis. Thus, this type of regulation may also exist in Arabidopsis even through the absence of CTTTC element in AtNIP5;1.

4) Line 287-291: few words on the further research can be added.

Reviewer #4: Boron (B) is an essential micronutrient. But different plant species have different demand for B, among which allotetraploid rapeseed has a special high B demand. Defficiency in B supply usually caused serious yield loss in rapeseed. However, there is also a very large genetice difference in rapeseed resistance to B deficiecny, which lies the base of the current study by He et al. This is a followed study from the same lab. In their previous studies, they identified a major QTL qBED-A3a and the possible candidate gene BnaA3.NIP5;1, whose expression is higher in B effecient varity QY10, but there is no difference in amion acid sequence with that in the B inefficient varity W10. In this study, the authors tried to elucidate why BnaA3.NIP5;1 expression in QY10 is higher, which also contributes to its better growth in B deficient soil. They found that BnaA3.NIP5;1 is polarly localized in the distal plasma membrane of LRC cells to facilitate B uptake into root tips, which also clarify why QC region has a higher B concentration. The interesting finding is that A CTTTC copy deletion within the 5’UTR increased BnaA3.NIP5;1 expression levels in QY10, whereas addition of the CTTTC into QY10 decreased the expression, so this tandem repeat is closely related with plant growth and seed yield. Finally, the authors presented solid evidence in the field test with natural populations and near-isogenic lines that the varities carrying BnaA3.NIP5;1Q allete produced higher seed yield under low-B condition. Overall speaking, this is a excellent work to provide novel insight into low-B tolerance in rapeseed, and more important is that the elite allele of BnaA3.NIP5;1 could be used as a direct target for breeding low-B tolerant cultivars in future practice. All the experiments were well designed and the contents were presented logically and easy to follow.

minor suggestions:

1. Line 157-158: it is better to rephase the sentence into : we generated transgenic plants expresseing pQ::BnaA3.NIP5;1-GFP or pW::BnaA3.NIP5;1-GFP in the background of Arabidospsis mutant nip5;1-1.

2. It is interesting that B taken up by LRC sells is used only for local root growth. Is there any effect of these transformation on the seed production of the transgenic arabidospsis plants?

3. In the discussion, is it possible to give proposes how the tandem repeat of CTTTC functioning in regulating the expression of BnaA3.NIP5;1?

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No


26 May 2021


10 Jun 2021

Dear Dr Xu,

We are pleased to inform you that your manuscript entitled "Genetic variation of BnaA3.NIP5;1 expressing in the lateral root cap contributes to boron deficiency tolerance in Brassica napus" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Zhixi Tian, Ph.D

Associate Editor

PLOS Genetics

Li-Jia Qu

Section Editor: Plant Genetics

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: All of my concerns have been well adressed. it is a nice story and I recommend this manuscript for publication.

Reviewer #2: I'm happy with the current version.

Reviewer #3: The revised version had addressed all the question I have rasied.

Reviewer #4: I am satisfactory with the revisions and have no further comments

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: None

Reviewer #3: None

Reviewer #4: Yes

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

----------------------------------------------------

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28 Jun 2021

PGENETICS-D-21-00377R1

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

Dear Dr Xu,

We are pleased to inform you that your manuscript entitled "Genetic variation of BnaA3.NIP5;1 expressing in the lateral root cap contributes to boron deficiency tolerance in Brassica napus" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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Thank you again for supporting PLOS Genetics and open-access publishing. We are looking forward to publishing your work!

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PLOS Genetics

On behalf of:

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https://creativecommons.org/licenses/by/4.0/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.
https://www.researchpad.co/tools/openurl?pubtype=article&doi=10.1371/journal.pgen.1009661&title=Genetic variation of <i>BnaA3</i>.<i>NIP5;1</i> expressing in the lateral root cap contributes to boron deficiency tolerance in <i>Brassica napus</i>&author=&keyword=&subject=Research Article,Biology and Life Sciences,Bioengineering,Biotechnology,Genetic Engineering,Genetically Modified Organisms,Genetically Modified Plants,Engineering and Technology,Bioengineering,Biotechnology,Genetic Engineering,Genetically Modified Organisms,Genetically Modified Plants,Biology and Life Sciences,Bioengineering,Biotechnology,Plant Biotechnology,Genetically Modified Plants,Engineering and Technology,Bioengineering,Biotechnology,Plant Biotechnology,Genetically Modified Plants,Biology and Life Sciences,Plant Science,Plant Biotechnology,Genetically Modified Plants,Biology and Life Sciences,Organisms,Eukaryota,Plants,Genetically Modified Plants,Biology and Life Sciences,Organisms,Eukaryota,Plants,Flowering Plants,Rapeseed,Biology and Life Sciences,Organisms,Eukaryota,Plants,Seedlings,Research and Analysis Methods,Animal Studies,Experimental Organism Systems,Model Organisms,Arabidopsis Thaliana,Research and Analysis Methods,Model Organisms,Arabidopsis Thaliana,Biology and Life Sciences,Organisms,Eukaryota,Plants,Brassica,Arabidopsis Thaliana,Research and Analysis Methods,Animal Studies,Experimental Organism Systems,Plant and Algal Models,Arabidopsis Thaliana,Biology and Life Sciences,Developmental Biology,Plant Growth and Development,Root Growth,Biology and Life Sciences,Plant Science,Plant Growth and Development,Root Growth,Biology and Life Sciences,Plant Science,Plant Anatomy,Seeds,Biology and Life Sciences,Genetics,Heredity,Genetic Mapping,Haplotypes,Research and Analysis Methods,Animal Studies,Experimental Organism Systems,Model Organisms,Xenopus,Xenopus Oocytes,Research and Analysis Methods,Model Organisms,Xenopus,Xenopus Oocytes,Research and Analysis Methods,Animal Studies,Experimental Organism Systems,Animal Models,Xenopus,Xenopus Oocytes,Biology and Life Sciences,Organisms,Eukaryota,Animals,Vertebrates,Amphibians,Frogs,Xenopus,Xenopus Oocytes,Biology and Life Sciences,Zoology,Animals,Vertebrates,Amphibians,Frogs,Xenopus,Xenopus Oocytes,