Boron uptake in is mediated by nodulin 26-like intrinsic protein 5;1 (NIP5;1), a boric acid channel that is located preferentially on the soil side of the plasma membrane in root cells. However, the mechanism underlying this polar localization is poorly understood. Here, we show that the polar localization of NIP5;1 in epidermal and endodermal root cells is mediated by the phosphorylation of Thr residues in the conserved TPG (ThrProGly) repeat in the N-terminal region of NIP5;1. Although substitutions of Ala for three Thr residues in the TPG repeat did not affect lateral diffusion in the plasma membrane, these substitutions inhibited endocytosis and strongly compromised the polar localization of GFP-NIP5;1. Consistent with this, the polar localization was compromised in µ subunit mutants of the clathrin adaptor AP2. The Thr-to-Ala substitutions did not affect the boron transport activity of GFP-NIP5;1 in oocytes but did inhibit the ability to complement boron translocation to shoots and rescue growth defects in mutant plants under boron-limited conditions. These results demonstrate that the polar localization of NIP5;1 is maintained by clathrin-mediated endocytosis, is dependent on phosphorylation in the TPG repeat, and is necessary for the efficient transport of boron in roots.
Boron (B) is an essential micronutrient for plants. Efflux-type B transporters, BORs, have been identified in Arabidopsis thaliana and rice. Here we identified BOR1 genes encoding B efflux transporters, from the hexaploid genome of wheat (Triticum aestivum L.). We cloned three genes closely related to OsBOR1 and named them TaBOR1.1, TaBOR1.2 and TaBOR1.3. All three TaBOR1s showed B efflux activities when expressed in tobacco BY-2 cells. TaBOR1-green fluorescent protein (GFP) fusion proteins were expressed in Arabidopsis leaf cells localized in the plasma membrane. The transcript accumulation patterns of the three genes differ in terms of tissue specificity and B nutrition responses. In roots, transcripts for all three genes accumulated abundantly while in shoots, the TaBOR1.2 transcript is the most abundant, followed by those of TaBOR1.1 and TaBOR1.3. Accumulation of TaBOR1.1 transcript is up-regulated under B deficiency conditions in both roots and shoots. In contrast, TaBOR1.2 transcript accumulation significantly increased in roots under excess B conditions. TaBOR1.3 transcript accumulation was reduced under excess B. Taken together, these results demonstrated that TaBOR1s are the B efflux transporters in wheat and, interestingly, the genes on the A, B and D genomes have different expression patterns.
BackgroundBoron (B) deficiency is an agricultural problem that causes significant losses of crop yield in many areas of the world. However, systematic analysis of BOR family genes for B transport in rapeseed is still lacking. We aimed to identify and characterize BOR transporters in Brassica napus and the potential role of these transporters in B homeostasis in response to B deficiency.ResultsHere, we identified 20 BOR transporters from the Brassica napus genome, which were classified into six distinct groups that represent clear orthologous relationships to their family members in Arabidopsis. qRT-PCR revealed distinct expression profiles for BnBORs in different tissues and in response to external B levels. The B-efficient cultivar QY10 accumulated more B in shoots than the B-inefficient cultivar W10, and overexpression of BnaBOR1;1c could alleviate shoot B-deficiency symptoms in W10 by distributing more B from roots to shoots. Additionally, BnBOR1;1c expression was up-regulated by B deficiency, and the induction of BnBOR1;1c was more intense in QY10. Moreover, two conserved InDels were found in the promoter regions of BnBOR1;1c within different B-efficient genotypes.ConclusionsOverall, the molecular characterization of the BnBOR genes of two B-efficient cultivars and their responses to B deficiency highlights the diversity of the family members in B. napus, and BnaC4.BOR1;1c has potential as a candidate gene for improving B nutrition.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1407-1) contains supplementary material, which is available to authorized users.
Phosphate (Pi) transporters play critical roles in Pi acquisition and homeostasis. However, little is known about these transporters in oilseed rape. Therefore, the aim of the present study was to characterize the members of the PHT1 gene family in allotetraploid Brassica napus and to analyze their expression profiles in response to environmental stresses. In total, 49 PHT1 family members were identified in B . napus , including 27 genes in the A subgenome and 22 in the C subgenome. Most of the PHT1 proteins were predicted to localize to the plasma membrane. Phylogenetic analysis suggested that the members of the PHT1 gene family can be divided into seven clades, with the introns/exons and protein motifs conserved in each clade. Collinearity analysis revealed that most of the BnaPHT1 genes shared syntenic relationships with PHT1 members in Arabidopsis thaliana , B . rapa , and B . oleracea , and that whole-genome duplication (polyploidy) played a major driving force for BnaPHT1 evolution in addition to segmental duplication. Transcript abundance analysis showed that a broad range of expression patterns of individual BnaPHT1 genes occurred in response to phosphorus (P) deficiency. In addition, the expression levels of BnaPHT1 genes can be regulated by different nutrient stresses, including nitrogen (N), potassium (K), sulfur (S) and iron (Fe) stresses. Moveover, salt and drought stresses can regulate the transcript abundances of BnaPHT1 s, as well as phytohormones including auxin and cytokinin. Gene coexpression analysis based on the RNA-seq data implied that BnaPHT1 s might cooperate with each other as well as with other genes to regulate nutrient homeostasis in B . napus . Further analysis of the promoters revealed that GT-1, DRE and P1BS elements are widely distributed within the promoter regions of BnaPHT1 genes. Our results indicate that BnaPHT1 s might be involved in cross-talk for sensing the external status of P, N, K, S and Fe, as well as salt and drought stresses. Moreover, these processes might be mediated by phytohormones. Our findings provide the first step in the complex genetic dissection of the Pi transport system in plants and implicate multiple transcriptional regulation, which probably refers to new roles of PHT1 genes in B . napus .
Summary Boron (B) deficiency is one of the major causes of growth inhibition and yield reduction in Brassica napus (B. napus). However, the molecular mechanisms of low B adaptation in B. napus are largely unknown. Here, fifty‐one BnaWRKY transcription factors were identified as responsive to B deficiency in B. napus, in which BnaAn.WRKY26, BnaA9.WRKY47, BnaA1.WKRY53 and BnaCn.WRKY57 were tested in yeast one‐hybrid assays and showed strong binding activity with conserved sequences containing a W box in the promoters of the B transport‐related genes BnaNIP5;1s and BnaBOR1s. Green fluorescent protein fused to the target protein demonstrated the nuclear localization of BnaA9.WRKY47. CRISPR/Cas9‐mediated knockout lines of BnaA9.WRKY47 in B. napus had increased sensitivity to low B and lower contents of B than wild‐type plants. In contrast, overexpression of BnaA9.WRKY47 enhanced the adaptation to low B with higher B contents in tissues than in wild‐type plants. Consistent with the phenotypic response and B accumulation in these transgenic lines, the transcription activity of BnaA3.NIP5;1, a B efficiency candidate gene, was decreased in the knockout lines but was significantly increased in the overexpressing lines under low B conditions. Electrophoretic mobility shift assays, transient expression experiments in tobacco and in situ hybridizations showed that BnaA9.WRKY47 directly activated BnaA3.NIP5;1 expression through binding to the specific cis‐element. Taken together, our findings support BnaWRKYs as new participants in response to low B, and BnaA9.WRKY47 contributes to the adaptation of B. napus to B deficiency through up‐regulating BnaA3.NIP5;1 expression to facilitate efficient B uptake.
Summary Brassinosteroids (BRs) are pivotal phytohormones involved in the control of root development. Boron (B) is an essential micronutrient for plants, and root growth is rapidly inhibited under B deficiency conditions. However, the mechanisms underlying this inhibition are still unclear. Here, we identified BR‐related processes underlying B deficiency at the physiological, genetic, molecular/cell biological and transcriptomic levels and found strong evidence that B deficiency can affect BR biosynthesis and signalling, thereby altering root growth. RNA sequencing analysis revealed strong co‐regulation between BR‐regulated genes and B deficiency‐responsive genes. We found that the BR receptor mutants bri1‐119 and bri1‐301 were more insensitive to decreased B supply, and the gain‐of‐function mutants bes1‐D and pBZR1‐bzr1‐D exhibited insensitivity to low‐B stress. Under B deficiency conditions, exogenous 24‐epibrassinolide rescued the inhibition of root growth, and application of the BR biosynthesis inhibitor brassinazole exacerbated this inhibitory effect. The nuclear‐localised signal of BES1 was reduced under low‐B conditions compared with B sufficiency conditions. We further found that B deficiency hindered the accumulation of brassinolide to downregulate BR signalling and modulate root elongation, which may occur through a reduction in BR6ox1 and BR6ox2 mRNA levels. Taken together, our results reveal a role of BR signalling in root elongation under B deficiency.
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