The administration did not affect serum calcium levels at any points examined. Again, the serum 1,25 OH 2 D was not different between the two groups 24 h after the injection. In contrast, a decrease in the serum phosphate level was observed only at two higher doses 4. These animals were not permitted to eat for 24 h to avoid interference of intestinal phosphate absorption. The kidneys obtained from the same groups in the study described above were pooled and used to prepare the BBM fractions. BBM fractions prepared from pooled kidneys in each group at an indicated point were analyzed by Western blot.
The blotted membrane was subjected to Coomassie brilliant blue staining to visualize the amount of blotted protein in each lane bottom. These alterations lasted more than 9 h after the administration. Statistical analysis was done by Dunnet's method. This change was temporary, but it was reproduced in another experiment. Because injections of lower doses, such as 0. Volume 19 , Issue 3. If you do not receive an email within 10 minutes, your email address may not be registered, and you may need to create a new Wiley Online Library account.
If the address matches an existing account you will receive an email with instructions to retrieve your username. Research Article Free Access. Takeyoshi Yamashita Corresponding Author E-mail address: tyamashita kirin. Tools Request permission Export citation Add to favorites Track citation.
Share Give access Share full text access. Share full text access. Please review our Terms and Conditions of Use and check box below to share full-text version of article. Preparation of brush border membrane and Western blotting The brush border membrane BBM was prepared by the method previously reported.
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However, the mechanism of low Pi promoting root length still remains unknown and it is significant to further elucidate the underlying mechanism. Informed by our previous research results, three following rice cultivars were selected as test materials: TongJing TJ , ZhengHan 6 ZH6 , and ZhenDao 99 ZD99 corresponding to the primary root lengthening type, phosphorus efficient uptake and utilization type, and intermediate type rice cultivar response to low Pi, respectively.
The germinating seeds were selected and placed into well plastic plates. Then, the plates were placed in plastic boxes and complete nutrient solution of the International Rice Research Institute [containing: 1. When the seedlings had grown to the 3-leaf stage, healthy seedlings were chosen and cultured with either normal nutrient solution CK , low Pi LP , or low Fe LFe nutrient solution. Each treatment contained six biological replicates. The solution was changed daily and the pH was adjusted to about 5. DCB-Fe is a general term for both adsorption and precipitation of Fe on the root surface.
Briefly, after low Pi treatment for 15 d, the rice roots were sampled and soaked overnight using tap water. DCB-Fe content or iron plaque thickness was verified via the iron content of the per unit dry weight of roots. Then, 0. After iron plaque removal, 1.
The precipitation at the bottom was collected for the cell wall component. The fragments on the bottom were collected for evaluation of the organelle composition. The supernatant formed the vacuolar component consisting of vacuole and cytoplasm Fe. To measure callose deposition, the method of frozen section with aniline blue fluorescent staining was used [ 36 ].
The sections were dyed in 0.
Iron and callose homeostatic regulation in rice roots under low phosphorus
The deposited callose was observed with an OlympusBX51 fluorescence microscope, excited by ultraviolet light. The supernatant was used as enzyme extraction. The number of PCR cycles was minimized to avoid skewing representation of the library [ 39 ]. The resulting library was qualified via the Agilent bioanalyzer and quantified via both Qubit and qPCR. The produced libraries were sequenced on the HiSeq platform. Analysis of the gene expression profile: sequencing reads were mapped onto the reference gene set via Bowtie1 software Bowtie parameter: —v 3 —all —best —strata.
A Perl script program was utilized to process the mapping results and to generate a gene expression profile.
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According to credibility interval approaches that had been reported for the analysis of SAGE data5 [ 40 ], the edgeR6 program was used to identify differentially expressed mRNAs based on their relative quantities, which were reflected by individual gene reads [ 41 ]. The method used empirical Bayes estimation and exact tests based on negative binomial distribution. According to the cDNA concentration, the volumes of the products of reverse transcription were regulated to ensure identical cDNA concentration in each treatment.
Amplification was done in parallel with the target gene allowing normalization of gene expression, while providing quantification. The datasets generated and analysed during the current study are available from the corresponding author on reasonable request. GCL conceived the study, edited the manuscript, and supervised the work. WYL participated in conceiving the project, provided financial support for the study, and supervised the work.
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DY carried out most experimentation, contributed to the design of the study, and drafted the manuscript. WZG carried out most transcriptome data analysis. RML and ZP prepared the rice seeds, grew rice plants, and performed low pi treatment. All authors reviewed and contributed to draft the manuscript.
All authors read and approved the final manuscript. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Skip to main content Skip to sections. Advertisement Hide. Download PDF. Iron and callose homeostatic regulation in rice roots under low phosphorus. Open Access. First Online: 04 December Background Phosphorus Pi deficiency induces root morphological remodeling in plants.
Results Induced expression of LPR1 genes was observed under low Pi, which also caused Fe accumulation, resulting in iron plaque formation on the root surface in rice; however, in contrast to Arabidopsis , low Pi promoted primary root lengthening in rice. Conclusions The obtained results indicate that Low phosphorus induces iron and callose homeostatic regulation in rice roots.
Background Plant root morphology is regulated by numerous factors, such as water and nutrient availability. However, primary root length change in ZhengHan 6 ZH6 was either not significant at 7 d or significantly reduced at 15 d ; primary root length increased significantly when treated at low Pi for 30 d. These results indicate that low Pi stress promoted rice primary root lengthening, which is one of the main strategies of most rice cultivars to achieve acclimation to Pi deficiency.
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Apparently, the response pattern in root lengthening varied among different cultivars. DCB-Fe is the adsorption or precipitation of iron on the root surface. Consequently, a reddish-brown iron plaque on the rice root surface began to form after treatment by low Pi for 1d Fig. Fe deposition on the rice root surface under low Pi treatment was confirmed by our results. Open image in new window. This provides evidence for apoplastic LPR1 ferroxidase activity and suggests that antagonistic interactions of Pi and Fe availability adjust the primary root growth rate via RAM-specific callose deposition, which is likely triggered by LPR1-dependent redox signaling [ 5 ].
Furthermore, the results of proteomic detection showed that the content of the LPR1 protein in low Pi treated rice roots was higher than that in the roots of control data not shown. This suggests that the formation of Fe plaques on rice root surface was promoted by the induction of LPR1 gene expression. Due to Fe deposition on the root surface, the Fe content increased very significantly in the root symplast of the three tested rice cultivars compared to the control Fig.
It is interesting that the increased degree of Fe content in the root symplast was substantially lower than that deposited on the root surface. For example, in ZH6 cultivar Fe content on low Pi treated root surface increased by 7. This result suggests that Fe uptake by the rice root symplasm might be limited under low Pi stress. Differential expression of Fe uptake related genes detected via transcriptome sequencing The results basically clarified the existence of two distinct high affinity iron transport mechanisms in plants [ 30 ].
Table 3 Effect of low Pi on transcriptional level of the iron absorption related genes detected via illumina expression profile sequencing.
To verify the transcriptome sequencing results, nine differentially expressed genes were selected and their transcriptional levels were tested via real-time fluorescent quantitative PCR qRT-PCR after rice seedlings were treated by low Pi for 15 d. The results Fig. Although low Pi promoted Fe accumulation in rice roots Fig. The Fe content in the vacuole of low Pi treated root cells was significantly higher than that in ck Fig. Furthermore, the Fe content in the cell wall was also higher than that in ck Fig. These results suggest that Fe was mainly stored in root vacuoles and cell walls under low Pi treatment, to alleviate the toxic effect of excessive Fe in the cytoplasm.
As shown in Fig. This result indicates that the low Fe content in both medium and rice roots still resulted in the lengthening of the primary root, which was different in Arabidopsis. In Arabidopsis , Pi limitation triggered cell-specific apoplastic Fe and callose depositions in both meristem and elongation zone of primary roots. Here, we showed that Low-Pi promoted a small callose deposition in the elongation zone of primary roots in rice Fig. However, the relative amount of callose deposition was smaller compared to Arabidopsis.
However, evidence for Fe-related toxicity during low Pi conditions is still missing. It has been proposed that the inhibited primary root growth under low Pi condition, might be caused by the toxic effect of excessive Fe [ 1 ]. Therefore, it is important to investigate how to regulate Fe homeostasis and alleviate the toxic effects of excessive accumulated Fe in low Pi treated rice roots. Therefore, Fe homeostasis in the rice root was appropriately controlled by Fe uptake, transport, and intracellular distribution.
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Consequently, Fe accumulation in the rice root symplast was insufficient to inhibit primary root growth under low-Pi stress. Consequently, Fe does not play an important role in rice root morphological remodeling under low Pi.