Involvement of NGATHA-Like 1 Transcription Factor in Boron Transport under Low and High Boron Conditions

Abstract

NGATHA-Like 1 (NGAL1) transcription factor has been identified as a gene regulated through AUG-stop-mediated boron (B)-dependent translation stall; however, its function in B response remains unknown. Here, we show that NGAL1 plays an important role in the maintenance of B transport under both low- and high-B conditions in Arabidopsis thaliana. NGAL1 mRNA is accumulated predominantly in shoots in response to B stress. Independent ngal1 mutants carrying transferred DNA (T-DNA) and Ds-transposon insertions exhibit reduced B concentrations in aerial tissues and produce shortened and reduced number of siliques when B supply is limited. Furthermore, the expression of B transporter genes including nodulin 26-like intrinsic protein 6; 1 (NIP6;1), NIP5;1, NIP7;1 and borate exporter 1 (BOR1) is significantly decreased in ngal1 mutants under low-B condition, suggesting that NGAL1 is required for the transcript accumulation of B transporter genes to facilitate B transport and distribution under B limitation. Under high-B condition, ngal1 mutants exhibit reduced growth and increased B concentration in their shoots. The accumulation of BOR4 mRNA, a B transporter required for B efflux to soil, is significantly reduced in roots of ngal1 plants under high-B condition, suggesting that NGAL1 is involved in the upregulation of BOR4 in response to excess B. Together, our results indicate that NGAL1 is involved in the transcriptional regulation of B transporter genes to facilitate B transport and distribution under both low- and high-B conditions.

Introduction

Boron (B) is an essential nutrient for plant growth and is required primarily for cell wall integrity for providing borate cross-linking of pectic polysaccharide in the cell wall (Matthes et al. 2020, Wimmer et al. 2020). When grown under B-limited conditions (hereafter referred to as low B), plants undergo major physiological changes, including inhibition of root elongation, leaf expansion and fertility (Matthes et al. 2020, Wimmer et al. 2020). However, in excess, B is also toxic for plants, and B toxicity causes growth disorders, altered cellular processes and DNA damage (Miwa et al. 2007, Sakamoto et al. 2018). To maintain B homeostasis under B-deficient and B-excess conditions (hereafter referred to as high B), the transport system of B plays a critical role (Tanaka et al. 2010, Yoshinari and Takano 2017).

In Arabidopsis thaliana, different types of B transporters including nodulin 26-like intrinsic proteins (NIPs) and borate exporters (BORs) facilitate the maintenance of adequate B concentrations at different tissues by coordinating B uptake, translocation and distribution (Yoshinari and Takano 2017). Under low B, boric acid channels including NIP5;1, NIP6;1 and NIP7;1 expressed predominantly in roots, stems and developing flowers, respectively, transport B to different tissues in coordination with BORs, such as BOR1 and BOR2 (Yoshinari and Takano 2017). When B levels are high, BOR4 expressed in roots facilitates B export from these organs to increase tolerance (Miwa et al. 2007). The expression of these transporters is known to be controlled by B-dependent tight regulation at both post-transcriptional and translational levels (Tanaka et al. 2016, Yoshinari and Takano 2017). NIP5;1 represents one such unique regulation of B involving the ribosome. NIP5;1 mRNA accumulates at a high level under low-B condition, but not under high-B condition (Tanaka et al. 2011, 2016). When plants are exposed to high B, ribosomes stall on the minimum upstream open reading frame (uORF), AUGUAA, located in the 5ʹ-untranslated region (5ʹ-UTR) of NIP5;1’s mRNA, suppressing the translation of the main ORF and leading to mRNA degradation (Tanaka et al. 2016). The role of uORF-mediated translational regulation in the avoidance of B toxicity under high B has recently also been characterized for BOR1 (Aibara et al. 2018).

In addition to NIP5;1, five B-responsive genes carrying the AUGUAA sequence in their 5ʹ-UTRs were identified in the genome of A. thaliana (Tanaka et al. 2016). NGATHA-Like 1 (NGAL1, also known as ABNORMAL SHOOT 2, ABS2) is one of the B-responsive genes whose expression is regulated by B-dependent ribosome stalling at the AUGUAA sequence (Tanaka et al. 2016). However, the function of NGAL1 in plant responses to B remained unknown.

NGAL1 is a transcription factor, belonging to the plant-specific B3 superfamily (Swaminathan et al. 2008, Shao et al. 2012). Arabidopsis thaliana B3 transcription factors can be divided into four subfamilies including auxin response factor, leafy cotyledone2-abscisic acid insensitive3-Val, RAV (related to ABI3 and VPI) and reproductive meristem (Swaminathan et al. 2008). In A. thaliana, four NGATHAs (NGA1-4) and three NGALs (NGAL1-3) have been identified with B3 DNA binding domains under the RAV subfamily (Swaminathan et al. 2008, Ikeda and Ohme-Takagi 2009). RAV subfamily transcription factors have been reported to be involved in plant growth and developmental processes and plant responses to phytohormones such as abscisic acid, auxin and brassinosteroids (Hu et al. 2004, Okushima et al. 2005, Alvarez et al. 2009).

NGAL1 was initially identified with the gain-of-function mutant, abs2, with an altered shoot development (Shao et al. 2012). The abs2 mutant has smaller leaves with abnormal shapes and exhibits faster leaf initiation and early flowering phenotype (Shao et al. 2012). The overexpression of NGAL1 in A. thaliana resulted in plants with the small-leaf phenotype (similar to the abs2 mutant) and with defective flower development including loss-of-petal (Shao et al. 2012). Based on these phenotypes, NGAL1 has been suggested as a negative regulator involved in shoot and flower development (Shao et al. 2012). Moreover, NGAL1 has also been suggested to represent a transcriptional repressor based on the screening results of transcriptional repressor domains in plants (Ikeda and Ohme-Takagi 2009). In support of these reports, NGAL1 has recently been known to function as a negative regulator in leaf margin development together with NGAL2 and NGAL3 (Shao et al. 2020).

To date, only a few transcription factors including WRKY6 in Arabidopsis (Kasajima et al. 2010) and BnaA9.WRKY47 in Brassica napus (Feng et al. 2020) have been identified as critical for the regulation of plant responses to B stress, particularly low B. WRKY6 seems essential for root growth under low B by regulating a set of genes expressed around the root tip (Kasajima et al. 2010). BnaA9.WRKY47, in turn, was demonstrated to directly activate the expression of BnaA3.NIP5;1 in B. napus to facilitate B uptake under B-limited condition (Feng et al. 2020).

In this study, we investigated the function of NGAL1 in B response in Arabidopsis. We report that NGAL1 is involved in the transcriptional regulation of B transporter genes under both low- and high-B conditions.

Results

Growth phenotype of ngal1 mutants under low B

Previously, our study on AUGUAA-mediated B regulation of NGAL1 showed that NGAL1 mRNA accumulation in the roots of A. thaliana increases under low B (Tanaka et al. 2016). To characterize the function of NGAL1 in B response, we first examined the accumulation of NGAL1 mRNA in both root and shoot tissues of A. thaliana. For this analysis, plants were grown on Molecular Genetics Research Laboratory (MGRL) solid culture medium for 14 d under low-B (−B: 0.1 µM B in culture medium) and control-B (+B: 30 µM B in culture medium) conditions. Consistent with previous results, we observed about 1.7-fold increases in NGAL1 mRNA accumulation in A. thaliana roots under low B compared to control B (Fig. 1A). In shoots, the induction of NGAL1 expression by low B was even more pronounced, increasing approximately 5.3-fold. Histochemical β-glucuronidase (GUS) staining of NGAL1 promoter–GUS fusion transgenic plants also showed that the NGAL1 promoter activity is stimulated in both shoot and root tissues under low B (Fig. 1B). These results suggest that the NGAL1 transcript accumulation increases under low B, especially in shoot tissues.

To further explore the role of NGAL1 under low B, we then used the ngal1 T-DNA insertion mutant alleles, ngal1-1 and ngal1-2. T-DNA insertion sites and NGAL1 mRNA accumulations in ngal1 mutant alleles are shown in Supplementary Fig. S1A, B. For phenotypic analysis, we grew wild-type Col-0 and ngal1 mutant plants on MGRL solid medium using a culture plate. In our control-B condition, we observed slight growth reductions in ngal1 mutant alleles compared with that of wild-type Col-0 (Fig. 1C–E), with only the fresh weight (FW) of roots being significantly reduced (Fig. 1E). However, under low-B condition, both shoot and root growth of ngal1 mutant alleles were significantly decreased compared to wild-type plants (Fig. 1C–E). In addition to T-DNA mutants, we also used the ngal1 Ds-transposon insertion mutant (hereafter referred to as ngal1-3 in our study that has strongly reduced accumulation of NGAL1 mRNA compared with its wild type, No-0, (Supplementary Fig. S1A, C). In phenotypic observations, we observed a slight shortening of main roots and a slight increase of lateral roots in the ngal1-3 transposon mutant compared with that of No-0 wild type under low-B condition. However, there were no significant differences in fresh weights of shoot and root tissues of wild type and the ngal1-3 mutant under low B (Supplementary Fig. S2). In this ecotype, we observed strong growth reductions in No-0 wild type under low-B conditions that might diminish the phenotypic comparisons between wild type and the ngal1-3 mutant.

Phenotypical analysis of ngal1 mutants in hydroponics

Because B is critical for undisturbed pollen development and growth of young leaves (Tanaka et al. 2008, Routray et al. 2018), ngal1 mutants were then further assessed under low B using an open hydroponic culture system that allows growing plants until they reach the reproductive stages. Thus, we grew wild-type Col-0 and ngal1 mutant alleles in hydroponic culture solution supplemented with 0.1 µM B or 30 µM B for low-B and control-B treatment, respectively, for 30 days. B deficiency symptoms in plants are generally first visible in young tissues and characterized by the inhibition of root elongation and expansion of young leaves (Noguchi et al. 1997, Tanaka et al. 2008). In wild-type plants, we observed the inhibition of young rosette leaf growth under low B (Fig. 2A). To estimate the severity of the symptoms, we counted individual plants displaying inhibited young rosette growth and calculated their percentage in relation to all cultured plants of each genotype (Supplementary Fig. S3). Under control B, no signs of leaf growth inhibition were detected in both wild-type and ngal1 mutant alleles (Fig. 2A). In our low-B condition, from a total of 63 cultured wild-type plants, about 53% of them exhibited inhibited young rosette growth (Fig. 2A, bottom rows). Interestingly, under these conditions, these symptoms were less pronounced in ngal1-1 and ngal1-2 mutant plants as only about 30% and 20% of them, respectively, showed clearly inhibited young rosette leaf growth (Fig. 2A, bottom rows). Despite these differences in young rosette leaf growth, the FW of the whole rosette leaves of both wild-type and ngal1 mutant plants was not significantly different (Fig. 2B–C).

To further examine the reproductive growth of ngal1 mutant plants under low B, we grew wild-type and ngal1 mutant alleles in a hydroponic culture until seed production was completed. Under control-B condition, there were no differences in silique growth and production between wild-type and ngal1 mutant alleles. However, under low B, we observed that the siliques of ngal1-1 and ngal1-2 mutant alleles were reduced and shortened compared with wild-type plants (Fig. 3). Of note, the more pronounced defects in silique growth of nga1-2 plants might be due to a stronger reduction of NGAL1 mRNA accumulation in the ngal1-2 allele than in the ngal1-1 allele (Supplementary Fig. S1B). Taken together, these results suggest that the growth of young rosette leaves and siliques are inversely correlated under low B and that NGAL1 could be critical to sustaining silique development at the expense of leaf growth under these conditions.

Next, we also assessed the growth phenotypes of plants under conditions of severe B limitation. Strong B limitation was obtained by removing residual trace B levels from hydroponic culture boxes by washing them three to four times with hydrochloric acid before exchanging the culture solutions of low B (0.1 µM B). Under these conditions, we observed that ngal1 mutant plants grew less and had shorter and less branched stems and smaller leaves compared to wild type (Supplementary Fig. S4). This indicates that ngal1 mutant plants are indeed impaired in vegetative growth when B is strongly limited. Importantly, under severe B limitation, both wild-type and ngal1 mutant plants were not able to produce siliques. Together, these results show that NGAL1 is critical for silique growth under low B.

B concentration in aerial tissues of ngal1 decreases under low B

It has been suggested that the B requirement during the reproductive stage is higher than that during the vegetative growth (Takano et al. 2002, Matthes et al. 2020). Since ngal1 mutants showed defects in silique growth under low B, we measured B concentrations in different aerial tissues of plants to further determine whether B accumulation is affected in ngal1 mutants. Under control conditions, B concentrations in old and young rosette leaves, cauline leaves and stems showed no significant differences between wild-type and ngal1 mutants (Fig. 4A). However, under low B, ngal1 mutants showed significantly decreased B concentrations in young rosette leaves, cauline leaves and especially stems as compared to wild-type plants (Fig. 4B). Consistent with the NGAL1 mRNA levels still detected in the two mutant alleles (Supplementary Fig. S1B), the reductions of B concentrations were stronger in tissues of ngal1-2 than ngal1-1 plants (Fig. 4B).

Considering the apparent tissue-specific impact of NGAL1 function, we examined whether NGAL1 mRNA accumulation differs in different tissues under low B. Samples were collected from plants grown hydroponically under low-B and control-B conditions for 35 d. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) results showed that rosette leaf, especially the young rosette leaf, accumulates higher level of NGAL1 mRNA compared with other tissues under control B (Fig. 4C). Notably, NGAL1 mRNA accumulation increased in both young and old rosette leaves with about 1.7–2.1-fold increases under low B. The NGAL1 expression was also induced by 1.5–1.8-fold in roots. However, the most prominent upregulation was detected in stems, where NGAL1 mRNA accumulation increased about 6.5-fold in response to low B, suggesting that the NGAL1 expression is critical in this tissue under B limitation. Taken together, these results suggest that the defects in silique production of ngal1 mutant plants under low B could have resulted from a B shortage in aerial tissues of these plants.

mRNA accumulation of B transporter genes is reduced in ngal1 mutants under low B

Since B accumulation in aerial tissues of B transporter mutants such as bor1-1 and nip6;1 is reduced due to insufficient B transport into young tissues (Noguchi et al. 1997, Tanaka et al. 2008), we hypothesized that components of the B transport system might be affected in ngal1 mutants. To validate this result, we performed RT-qPCR on the expression level of B transporter genes in wild-type and ngal1 mutant plants grown hydroponically for 4 weeks (Fig. 5). As expected, NIP6;1 mRNA accumulation was significantly reduced in roots of ngal1 mutant alleles under both control- and low-B conditions (Fig. 5B). Interestingly, also the expression of NIP7;1 was significantly decreased in roots of ngal1 mutants irrespective of B, whereas the mRNA accumulation of NIP5;1 was decreased in the mutants only under low B (Fig. 5A, C). Since NIP6;1 is expressed predominantly in the stem (Tanaka et al. 2008), we also checked its transcript accumulation in stem tissues. Different from what was observed in roots, NIP6;1 mRNA accumulation in the stem of ngal1 mutant alleles was only significantly reduced under B limitation (Fig. 5D). Furthermore, the mRNA accumulation of BOR1 was reduced in roots and stems of ngal1 mutant alleles compared to wild type under low B but not under sufficient B (Fig. 5E–F). The reductions of mRNA accumulations of tested B transporter genes including NIP5;1, NIP6;1, NIP7;1 and BOR1 were confirmed in ngal1-3 transposon mutant roots under low B (Supplementary Fig. S5). Of note, we observed increased BOR1 mRNA levels in wild-type plants under low B in our experiments with the hydroponic culture. Since BOR1 has so far been shown to be regulated by low B at the protein level and not at the mRNA level (Takano et al. 2005), we will further examine the mRNA level of BOR1 to confirm whether the increased expression detected in the present study resulted only from low B or whether additional factors such as different tissue samples and/or plant growth conditions were involved. Together, our RT-qPCR results suggested that NGAL1 positively regulates the expression of B transporter genes in response to low B.

ngal1 mutants are sensitive to high B

For our curiosity, we also tested the expression of NGAL1 in wild-type Col-0 under high-B condition (3,000 µM B in culture medium). Surprisingly, we observed that NGAL1 mRNA accumulation also increased in both root and shoot tissues of wild type under high B (Fig. 6A). As observed under low B, the increased level of NGAL1 mRNA was higher in shoots than in roots. Histochemical GUS staining of NGAL1 promoter–GUS fusion transgenic lines further showed that the NGAL1 promoter is induced in shoot and root tissues by high B (Fig. 6B).

To understand the function of NGAL1 under high B, we next examined the growth phenotype of ngal1 mutant alleles under high-B condition. For the analysis, wild-type and ngal1 mutant alleles were grown on solid media supplemented with control B (30 µM B) and high B (3,000 µM B) for 14 d. We observed that the growths of ngal1-1 and ngal1-2 mutant alleles were reduced compared with that of wild type under high B. Root and shoot FWs of ngal1 mutant alleles were significantly lower than in wild type when B was present in excess (Fig. 6C, D). We performed the phenotypic analyses in three independent experiment replicates and obtained similar results. Moreover, we confirmed the phenotypic results in ngal1-3 transposon mutant and wild-type No-0 plants (Supplementary Fig. S6). Together, these results show that the NGAL1 expression is induced by high-B condition and that NGAL1 plays a role in tolerance to excess B.

Shoot B concentration of ngal1 increases under high B

Reduced growth of plants under high concentrations of B could generally result from increased B concentrations in plant tissues (Miwa and Fujiwara 2011, Miwa et al. 2014). To assess whether altered B accumulation was responsible for the inhibited growth of ngal1 mutants under high B, we measured B concentrations in wild-type and ngal1 mutant alleles. We only used shoot samples because the contact of roots with excess B in the growth media results in high levels of apoplastic B, which cannot be efficiently eliminated from the root samples. As expected, inductively coupled plasma mass spectrometry (ICP-MS) results showed that B concentrations in shoots were increased in plants grown under high B (Fig. 7A). More importantly, B concentrations in shoots of ngal1-1 and ngal1-2 mutant alleles were significantly increased compared with wild-type plants. Elevated shoot B under high B was further confirmed in the ngal1-3 transposon mutant (Supplementary Fig. S7A). These results, therefore, indicated that the reduced growth of ngal1 mutant plants under high B could possibly have resulted from the increased accumulation of B in shoots.

BOR4 expression in roots is decreased in ngal1 under high B

Under high-B condition, the B exporter BOR4 plays a critical role in B tolerance by exporting B from roots to the rhizosphere (Yoshinari and Takano 2017). Consequently, the lack of BOR4 activity in a bor4 mutant results in increased B accumulation in shoots and enhanced sensitivity to high B (Miwa and Fujiwara 2011, Miwa et al. 2014). Since we observed similar phenotypes in ngal1 mutants under high B, we next examined the accumulation of BOR4 mRNA in ngal1 mutants. The results showed that BOR4 mRNA accumulation is significantly lower in roots of ngal1-1 and ngal1-2 mutant alleles compared with wild-type roots under both control- and high-B conditions (Fig. 7B). Especially, under high B, BOR4 mRNA accumulation levels in ngal1 mutant roots were approximately half of those recorded in roots of wild-type plants. In ngal1-3 transposon mutant plants, BOR4 was also significantly downregulated but irrespective of the B condition (Supplementary Fig. S7B). Based on these results, we concluded that the reduced accumulation of BOR4 mRNA in ngal1 mutant roots could have caused the increased shoot B accumulation in ngal1 mutant plants under high B. The results also indicate that NGAL1 positively regulates the expression of BOR4 in response to high B.

Discussion

Regulation of B transport enables plants to adapt to conditions in which B is limited or presented in excess. In this study, we show that the transcription factor NGAL1 is involved critically in the maintenance of B transport under both low- and high-B conditions.

Under low B, NGAL1 mRNA accumulation increases mainly in aerial tissues (Fig. 1 and 2) and facilitates the maintenance of B concentrations in these tissues (Fig. 4) by regulating positively the expression of B transporter genes (Fig. 5 and Supplementary Fig. S5). Under high B, the accumulation of NGAL1 mRNA in shoot and root tissues also increases (Fig. 6) and facilitates the upregulation of the B efflux transporter gene to prevent excess B accumulation in the shoot (Fig. 7 and Supplementary Fig. S7). Based on these data, we proposed a conceptual diagram describing the role of NGAL1 under opposing B-induced stress conditions (Fig. 8). We hypothesize that under low B, NGAL1 is involved in the positive regulation of B transporter genes such as NIP5;1, NIP6;1, NIP7;1 and BOR1 in root and stem tissues and thus facilitates B transport and distribution into aerial tissues with high B demand. Under high B, NGAL1 is also involved in the positive regulation of BOR4 expression to prevent excess B translocation to shoots.

In agreement with our hypothesis, ngal1 mutants have defective silique production as their low-B phenotypes are likely due to impaired B transport and distribution in different aerial tissues when B supply is limited (Fig. 3). Previously, NIP6;1 was shown to transport B preferentially to the growing young tissues under low B (Tanaka et al. 2008). In our study, we observed strongly reduced NIP6;1 mRNA accumulation in ngal1 mutants. In line with this, we observed the significant increase of NGAL1 mRNA accumulation in the stem of B-deficient wild-type plants (Fig. 4), which could potentially be necessary for the activation of NIP6;1 expression in this tissue. Therefore, it is possible that the reduced NIP6;1 expression could have caused the defects in silique growth of ngal1 mutants. Our results also suggest a more general role for NGAL1 in the low-B-signaling pathway since the expression of other B transporter genes is also reduced in ngal1 mutants. However, future studies will be necessary to validate whether the genes differentially regulated in ngal1 mutants are indeed the direct targets of NGAL1.

It was surprising to determine that in addition to its role under B-limited condition, NGAL1 also affects B transport under excess-B condition. In A. thaliana, high-B-induced BOR4 transporter is critical to confer tolerance to excess B (Miwa and Fujiwara 2011, Miwa et al. 2014). We observed that the transcript accumulation of BOR4 reduced significantly in ngal1 roots under both control- and excess-B conditions (Fig. 7B and Supplementary Fig. S7B). The severe growth reduction of ngal1 under high-B supply (Fig. 6 and Supplementary Fig. S6A) and the increased shoot B concentration (Fig. 7A and Supplementary Fig. S7A) could, likely, have been a direct consequence of the reduced expression of BOR4 in this mutant.

Similar regulations have recently been observed for two WKRY47 homologs under different B conditions (Feng et al. 2020, 2021). Whereas BnaA9.WKRY47 transcription factor in B. napus was found as a positive regulator of BnaA3.NIP5;1 under low B to confer tolerance to B limitation (Feng et al. 2020), its homolog AtWRKY47 in A. thaliana has been identified as a negative regulator of plant tolerance to high B by altering the B accumulation in plants (Feng et al. 2021). Therefore, it is possible that NGAL1 has a dual role in regulating B transport under low- and excess-B conditions. It is intriguing that the upregulation of NGAL1 results in the upregulation of different sets of genes, depending on B conditions. One possibility is that B is involved in the regulation of NGAL1 activity/specificity. However, future studies will be necessary to uncover how NGAL1 regulates the expression of B transporter genes in these two different B conditions. In addition, it would also be interesting to study the NGAL1-regulated genes at the translational level in future.

In A. thaliana, NGAL1-3 transcription factors have been reported to function as negative regulators in leaf development (NGAL1-3, Shao et al. 2020 and NGAL1, Shao et al. 2012) and the control of seed size (NGAL2 and NGAL3, Zhang et al. 2015). We observed the low-B-induced inhibition of young rosette leaf in wild-type plants, whereas such inhibitions were reduced in ngal1 mutants (Fig. 2A). Previously, it has been shown that the NGAL1 overexpression in A. thaliana results in reduced shoot growth, abnormal leaf shapes (Ikeda and Ohme-Takagi 2009, Shao et al. 2012) and altered development of leaf margins (Shao et al. 2020). Our results here showed that NGAL1 mRNA accumulation is high in young rosette leaf under low B and the inhibition of young rosette growth is reduced in ngal1 mutant alleles. Since NGAL1 is reported as a negative regulator of leaf development (Shao et al. 2020), by correlating with the NGAL1 overexpression phenotypes, we could, therefore, assume that NGAL1 might be involved in negative regulation of tissues, especially the rosette leaves, under low B.

Apart from its functional analyses in B response, our study has also revealed that the transcript accumulation of NGAL1 represents the unique regulation that can be activated both under low- and high-B conditions. The results indicated that NGAL1 mRNA regulation is different than the case of NIP5;1 mRNA although NGAL1 has initially identified with B-triggered AUGUAA-mediated ribosomal regulation under mild-high B (30–100 µM B in culture medium) following up the NIP5;1 mRNA regulation (Tanaka et al. 2016). However, unlike the case of NIP5;1 (Tanaka et al. 2016), NGAL1 mRNA accumulation increases again when very high B (3,000 µM B) is present in the growth medium. This shows that NGAL1 is regulated in a B-dependent manner and that the downregulation of its mRNA under sufficient or mild-high B conditions is possibly under the AUGUAA-mediated regulation. The upregulation of NGAL1 mRNA under excess- or high-B conditions, in turn, is not mediated by AUGUAA regulation but by another yet unknown mechanism. Future studies to identify the regulatory mechanisms of NGAL1 mRNA expression in response to high B could also be important for improving plant tolerance to excess B. Moreover, as depicted in our summarizing model (Fig. 8), we propose that NGAL1 regulates NIP5;1 mRNA and other B transporter genes under low- and high-B conditions, but not under sufficient-B conditions. Under normal-B conditions, the NGAL1-dependent regulation seems to be less effective since we observed no significant differences in mRNA accumulations of B transporter genes in ngal1 mutants (Fig. 5 and Supplementary Fig. S5). Instead, under sufficient B, the AUGUAA-mediated regulation might effectively function on NIP5;1 mRNA level rather than NGAL1 regulation.

In conclusion, we report that NGAL1 functions as a positive regulator of plant responses to a range of B conditions. Both under B limitation and B excess, NGAL1 is involved in activating and sustaining the expression of several B transporter genes to facilitate the transport and distribution of B in different tissues. Our study, therefore, has the potential to contribute to improving plant tolerance to both B limitation and B excess.

Materials and Methods

Plant materials and growth conditions

Arabidopsis thaliana accession Columbia-0 (Col-0) was used as wild type if not indicated otherwise. Two T-DNA insertion mutants for NGAL1 (At3g36080), SALK_146872C (ngal1-1) and SALK_069174C (ngal1-2) were obtained from the Arabidopsis Biological Resource Center. Accession Nossen-0 (No-0) was used as wild type for the Ds-transposon insertion mutant ngal1-3 (RATM 12-1962-1). In plate culture, MGRL medium (Fujiwara et al. 1992) supplemented with 1% (w/v) sucrose and solidified with 1.5% (w/v) gellan gum (Wako Pure Chemicals, Chuo-ku, Osaka, Japan) was used to grow plants. For the culture, surface-sterilized seeds were first sown on MGRL solid medium and kept at 4°C for 1–2 d for vernalization treatment. Culture plates were further placed in a growth chamber (LPH-350S, Nippon Medical and Chemical Instruments Co. Ltd, Tennoji-ku, Osaka, Japan) under long-day conditions (16/8 h light/dark cycle) at 22°C. For phenotypic analysis and quantification of mRNA transcript accumulation, plants were grown on MGRL solid media for 10–15 d. For hydroponic culture, MGRL hydroponic culture solution in a 1.5-l plastic box with no supplementation of sucrose and gellan gum was used to grow plants in a greenhouse. Culture solutions were exchanged every week and maintained under long-day conditions (16/8 h light/dark cycle). B concentration in the culture medium and the hydroponic culture solution were adjusted by the addition of the desired concentrations of boric acid from a stock solution. In hydroponic culture, plants were grown for 3–4 weeks before collecting the tissue samples for quantifications of mRNA accumulation and the elemental analysis of B concentration in tissue.

RT-qPCR for transcript accumulation

Total RNA was prepared from plant tissues using the RNeasy Plant Mini Kit (Qiagen, Germantown, Maryland, USA) according to the manufacturer’s instructions. Five-hundred nanograms of total RNA was used for RT with the Prime Script RT Master Mix (Takara Kusatsu, Shiga, Japan) in 10 µl reaction mixture; 5-fold diluted cDNA was further amplified by RT-qPCR using a Thermal Cycler Dice Real Time System TP800 (Takara) with SYBR Premix Ex Taq™ kit (Tli RNaseH Plus, Takara). The primers used for RT-qPCR are listed in Supplementary Table S1. Eukaryotic Elongation Factor 1α (eEF1α) was used as an internal control, and Actin10 was used to confirm the results. The experiment for transcript analysis was repeated independently two to three times, and each independent experiment was used for three sample replicates.

ICP-MS for the quantification of B concentration

For ICP-MS analysis, plants were grown for 15 and 30 d on MGRL solid media and hydroponic culture, respectively. Different tissue samples collected from five to eight plants were pooled as one sample replicate. Samples were further washed, digested and prepared for measurement on ICP-MS as described previously (Bian et al. 2018). The experiment for ICP-MS analysis was repeated twice with n = 3 sample replicates in each experiment.

Generation of pNGAL1::GUS lines and histochemical GUS staining

To generate transcriptional NGAL1 promoter–GUS fusion lines, the 2,025-bp promoter sequence of NGAL1 upstream from ATG was amplified from gDNA by PCR using specific primers (Supplementary Table S1) and cloned into pENTR/D/TOPO entry vector, following the manufacturer’s instructions (Invitrogen). The fragment was then sub-cloned into pGWB3 (no promoter, C-GUS) by the recombination of attL and attR sites. The final binary vector was then transferred to Agrobacterium tumefaciens (GV3101). Arabidopsis thaliana plants (accession Col-0) were transformed according to the floral dip method (Clough and Bent 1998). One representative line was selected, and homozygous T3 seedlings were grown on MGRL solid media for 5–7 d. For GUS staining, seedlings were vacuum-infiltrated with GUS staining solution containing 100 mM Na₂HPO4 buffer pH 7.0, 0.1% Triton X-100, 2 mM K₃Fe[CN]₆, K₄Fe[CN]₆ and 0.5 mg ml⁻¹ X-GlcA (5-bromo-4chloro-3-indolyl-β-d-glucuronide cyclohexyl ammonium salt, Wako Pure Chemicals, Chuo-ku, Osaka, Japan) for 20 min at room temperature and incubated overnight at 37°C. After removing the staining solution, the seedlings were incubated in 70% ethanol at 4°C for 3–4 h and photographed using Canon Eos Kiss digital camera.

Statistical analysis

Statistical analyses were performed using a Student’s t-test to compare two groups of samples and analysis of variance with Dunnett’s test to compare the multiple groups of samples. All data and error bars in graphs are represented as means and standard deviations of separate experiments and/or culture replicates, respectively. Statistical significance is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001 in Student’s t-test; different letters, P < 0.05 in Dunnett’s test.

Supplementary Data

Supplementary data are available at PCP online.

Data Availability

Data from current article will be shared on request to the corresponding author.

Arabidopsis sequence data from this article can be found in Arabidopsis Genome Initiative or GeneBank/European Molecular Biology Laboratory (EMBL) data libraries under following accession numbers: AtNGAL1, At2g36080; AtNIP5;1, At4g10380; AtNIP6;1, At1g80760; AtNIP7;1, At3g06100; AtBOR1, At2g47160; AtBOR4, At1g15460.

Funding

Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (19H05637 and 18H05490 to T.F. and 18K06278 to M.Ts.); Deutsche Forschungsgemeinschaf (DFGt) Eigene Stelle from the Deutsche Forschungsgemeinschaft (HE 8362/1-2 to R.F.H.G.).

Acknowledgements

We thank Yuko Kawara (The University of Tokyo) for excellent technical assistance.

Author Contribution

M.Ts., M.T. and T.F. conceived project and designed experiments; M.Ts. performed most of the experiments with the assistance from M.T.; R.F.H.G. and Nv.W. provided ngal1-3 allele and generated the transcriptional reporter line; M.Ts., M.T. and T.F. analyzed data and wrote the manuscript; all the authors read and approved the contents of the manuscript.

Disclosures

The authors have no conflicts of interest to declare.

References

Author notes